EP0714308A1 - Methode, zusammensetzungen und vorrichtungen zur verabreichung von nackten polynukleotiden, welche für biologisch aktive peptide kodieren - Google Patents

Methode, zusammensetzungen und vorrichtungen zur verabreichung von nackten polynukleotiden, welche für biologisch aktive peptide kodieren

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Publication number
EP0714308A1
EP0714308A1 EP94926603A EP94926603A EP0714308A1 EP 0714308 A1 EP0714308 A1 EP 0714308A1 EP 94926603 A EP94926603 A EP 94926603A EP 94926603 A EP94926603 A EP 94926603A EP 0714308 A1 EP0714308 A1 EP 0714308A1
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Prior art keywords
naked
host
polynucleotides
naked polynucleotide
polynucleotide
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EP94926603A
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English (en)
French (fr)
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EP0714308A4 (de
Inventor
Dennis A. Carson
Eyal Raz
Meredith L. Howell
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University of California
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University of California
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Publication of EP0714308A4 publication Critical patent/EP0714308A4/de
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
    • A61B17/20—Surgical instruments, devices or methods, e.g. tourniquets for vaccinating or cleaning the skin previous to the vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
    • A61B17/20—Surgical instruments, devices or methods, e.g. tourniquets for vaccinating or cleaning the skin previous to the vaccination
    • A61B17/205—Vaccinating by means of needles or other puncturing devices
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/35—Allergens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
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    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5406—IL-4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/77—Ovalbumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56977—HLA or MHC typing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to methods for administering biologically active peptides to a mammalian host by the introduction thereto of one or more polynucleotid- es to operatively encode for the peptides, preferably by non-invasive means. It also relates to the administration of said polynucleotides to prevent and treat illnesses and loss of immune function associated with aging in mammals.
  • Feigner et al., Proc.Nat'IAcad.Sci USA, 81 :5849-5852, 1984.
  • Feigner et al., reported that they obtained expression of protein from "naked" polynucleotides (i.e., DNA or RNA not associated with liposomes or a viral expression vector) injected into skeletal muscle tissue (Feigner, et al., Science, 24Z-1465, 1990; see also. PCT application WO 90/11092). Feigner, et al.
  • muscle cells efficiently take up and express polynucleotides because of the unique structure of muscle tissue, which is comprised of multinucleated cells, sarcoplasmic reticulum and a transverse tubular system which extends deep into the muscle cell.
  • intramuscular injections may be effective on at least a short term basis in therapies directed to disease in the muscle tissue itself, it is likely to be less effective in stimulating a tissue specific immune or other biological response to the expressed peptide elsewhere in the patient's body.
  • intramuscular injection is not a particularly viable route for achieving expression of peptides at the primary entry points for many infections; i.e., skin and mucosa.
  • cationic liposomes or a biolistic device i.e., a vaccine "gun” which "shoots" polynucleotides coupled to beads into tissue
  • any invasive means of introducing nucleotides poses problems of tissue trauma (particularly in long-term therapies) and presents limited access to certain target tissues, such as organs.
  • Means for non-invasive delivery of pharmaceutical preparations of peptides have at least the advantage of minimizing tissue trauma.
  • bioavailability of peptides following transdermal or mucosal transmission is limited by the relatively high concentration of proteases in these tissues.
  • reliable means of delivering peptides by transdermal or mucosal transmission of genes encoding for them has been unavailable.
  • cytokine proteins such as interleukin-2, hereafter "IL-2"
  • IL-2 interleukin-2
  • Certain diseases occur as part of the aging process in virtually all mammalian species, despite differing life styles and life spans among those species. These diseases include cancer, hypertension, vascular diseases and insulin resistance that can result in diabetes. Because the timing of the onset of these diseases cannot be solely attributed to environmental factors, it has been assumed that their onset is genetically programmed. However, the processes which actually control the aging process and the incidence of age-associated illnesses are not known.
  • aging is associated with a reduced ability to mount an immune response to exogenous antigens, a decreased functional reserve and response to stress, as well as an increased tendency toward fibrosis. All of these states are contributed to or controlled in part in vivo by circulating cytokines.
  • interleukin-1 (IL-1 ) proteins affect glucose homeostasis and can act as a hypoglycemic agent in insulin resistant C57BL-/KS db mice and C57BIJ6G ob/ob mice (Del Rey, et al., Proc. Natl. Acad. ScL, USA, 86:5943, 1989).
  • IL-1 interleukin-1
  • IL-1 gene therapy (administered exactly as described for IL-2) may, therefore, prevent the onset of diabetes in mice and humans.
  • High blood pressure is another concomitant of aging that is frequently associated with diabetes.
  • nitric oxide a major regulator of blood pressure is the endothelium-derived relaxing factor, nitric oxide.
  • the production of nitric oxide is regulated by IL-1.
  • IL-1 protein induces fever and even shock when administered acutely to animals.
  • the continuous production of low levels of IL-1 following somatic gene therapy will avoid these side effects.
  • cytokine proteins have been explored recently by researchers seeking to stimulate the immune system to augment its response to certain pathogens and to maintain immune function in immunodeficient patients, such as those infected with human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • Toxicity of the IL-2 protein has been a major impediment to its use as an effective therapeutic.
  • lL-2 therapy generally requires that the administered protein be present in serum in sufficient quantity to saturate high affinity IL-2 receptors and induce marked expansion of circulating natural killer (NK) cells.
  • NK natural killer
  • a need therefore, exists for an efficient means of introducing a protein (including but not limited to cytokines and antigens) into a host in a manner which will minimize the toxicity of the protein. More specifically, a need also exists for a means for introducing a protein into a host in a manner which will produce a consistent but subtherapeutic level of protein expression over a long period of time. In the latter respect, a need particularly exists for a means of administering cytokines to prevent as well as treat age-associated illnesses.
  • the above discussion also illustrates the need for an effective means of introducing naked nucleotides which will express in vivo a peptide which can induce local immunity in skin and mucosa to vaccinate a host against, for example, sexually transmitted diseases and respiratory illnesses. It also suggests a need for a means of introducing a gene encoding for a biologically active peptide to a host in a tissue-specific manner without significant tissue trauma.
  • the present invention addresses all of these needs.
  • naked polvnucleotidefsY' refers to DNA or RNA and can include sense and antisense strands as appropriate to the goals of the therapy practiced according to the invention.
  • Polynucleotide in this context may include oligonucleotides. Naked in this context means polynucleotides which are not complexed to colloidal materials (including liposomal preparations), or contained within a vector which would cause integra ⁇ tion of the polynucleotide into the host genome.
  • "Qperativelv encoding” refers to a polynucleotide which has been modified to include promoter and other sequences necessary for expression and, where desired, secretion of the desired translation product; e.g., a peptide or protein. All the embodiments of the invention can be practiced using known plasmid expression vectors. Preferably, these vectors will include cDNA('s) which encode for the desired translation product. Therefore, unless context otherwise requires, it will be assumed that "polynucleotide” or “naked polynucleotide” refers to operatively encoding sequences contained in a suitable plasmid expression vector, examples of which are provided herein.
  • Matture of polynucleotides shall refer to more than one and up to 200 polynucleotide species which are under the control of the same promoter.
  • Synthesis refers to well-known means of synthesizing polynucleotide sequences and may include isolation and purification of native poly ⁇ nucleotides.
  • Peptide refers to small peptides, polypeptides, oligopeptides and proteins which have a desired biological effect in vivo.
  • Iontophoresis refers to a known means of transdermal transmission presently used to deliver peptides continuously to a host. More specifically, it is a process that facilitates the transport of ionic species by the application of a physiologically acceptable electrical current. This process and other transdermal transmission means are described in Chien, et al. Transdermal Drug Delivery, “Novel Drug Delivery Systems", Ch. 7, part C, (Marcel Dekker, 1992), the relevant disclosures of which are incorporated herein by this reference for the purpose of illustrating the state of knowledge in the art concerning techniques for drug delivery.
  • Detergents/Absorption Promoters refers to chemical agents which are presently known in the art to facilitate absorption and transfection of certain small molecules, as well as peptides.
  • Anti ⁇ en Presenting Cells or “APC's” include known APC's such as Langerhans cells, veiled cells of afferent lymphatics, dendritic cells and interdigitating cells of lymphoid organs.
  • the definition also includes mononuclear cells such as (1) lymphocytes and macrophages which take up and express polynucleotides according to the invention in skin and (2) mononuclear cells depicted on histological photographs contained herein. These cells are not tissue cells but are likely to be antigen presenting cells.
  • APC's which are known to be present in high numbers in epithelia and thymus dependent areas of the lymphoid tissues, including epidermis and the squamous mucosal epithelia of the buccal mucosa, vagina, cervix and esophagus (areas with "relatively high” concentrations of APC's).
  • skin and “mucosa” as used herein particularly refer to these sites of concentration of APC's.
  • Host refers to the recipient of the therapy to be practiced according to the invention.
  • the host may be any vertebrate, but will preferably be a mammal. If a mammal, the host will prefera ⁇ bly be a human, but may also be a domestic livestock or pet animal.
  • Target tissue refers to the tissue of the host in which expression of the naked polynucleotide is sought.
  • Skin refers to the epidermal, dermal and subcutaneous tissues of a host.
  • Mucosa refers to mucosal tissues of a host wherever they may be located in the body including, but not limited to, respiratory passages
  • bronchial passages including bronchial passages, lung epithelia and nasal epithelia
  • genital passages including vaginal, penile and anal mucosa
  • urinary passages e.g., urethra, bladder
  • m. "Point of Entry” refers to the site of introduction of the naked polynucleo ⁇ tide into a host, including immediately adjacent tissue.
  • “Surrogate End Point” refers to a biological state of the host occurring just prior to the onset of disease. Examples include loss of glucose tolerance (diabetes), increased cholesterol levels (heart disease) and the presence of free radical amino acids in blood and urine (indicates loss of free radical detoxifying enzymes in the central nervous system).
  • Bio Impairment refers to a loss of immune function or wellness (including disease and impairment of the host's resistance to disease) which is associated with aging in mammals.
  • Subtherapeutic Levels and “Subtherapeutic Dosage” refer to expression of a peptide by a naked polynucleotide in a host in a quantity which is not sufficient to invoke an acute, detectable response by the host to the expressed peptide following a single noncumulative administration to the host of the naked polynucleotide.
  • Dermal and “Epidermal Administration” mean routes of administration which apply the naked polynucleotide(s) to or through skin. Dermal routes include intradermal and subcutaneous injections as well as transdermal transmission. Epidermal routes include any means of irritating the outermost layers of skin sufficiently to provoke an immune response to the irritant.
  • the irritant may be a mechanical or chemical (preferably topical) agent.
  • Epidermal Administration involves essentially the same method as chem ⁇ ical epidermal administration, except that the chemical irritant is applied to mucosal epithelium.
  • s. ]1L ⁇ refers to interleukin.
  • TH1 Responsefs refers to a humoral immune response that is induced preferentially by antigens that bind to and activate certain APC's; i.e., macrophages and dendritic cells.
  • Bioly Active Peptide(s) refers to a peptide which, when adminis ⁇ tered to a host, exerts a therapeutic benefit or induces an immune response therein.
  • the invention consists of means of inducing local immunity to an antigen or a systemic response to a therapeutic peptide or polynucleotide by delivering a naked polynucleotide to a host's cells which operatively encodes the antigen or peptide. More particularly, the naked polynucleotide is preferably delivered to a tissue which contains a relatively high concentration of antigen presenting cells as compared to other tissues of the body.
  • the invention will be entirely limited by a particular theory as to the mechanism of expression involved, it is believed that a biological response in these tissues following administration of the naked polynucleotide is achieved because the polynucleotide is expressed by mononuclear cells, most likely the host's antigen presenting cells. It is also believed that the mononuclear cells are involved in an inflammatory immune response to the introduction of the naked polynucleotide.
  • Example 9 and FIGURE 15 Based on histological studies, the naked polynucleotides do not appear to be taken up directly by fibroblasts or other tissue cells in significant quantities (see. Example 9 and FIGURE 15). This conclusion is borne out by studies showing that (1) intradermal administration of even minute amounts of naked polynucleotides into mice induced a prominent TH1 response (indicative of antigen presentation by macrophages and dendritic cells; see. Example 17 and FIGURES 24-25); (2) intradermal administration of naked polynucleotide to mice induced the formation of cytotoxic T cells without stimulating production of detectable levels of antibody (see. Example 15 and FIGURE 21); and, (3) induction of prolonged immunological memory with respect to the polynucleo ⁇ tide as an antigen (Example 16 and FIGURES 22-23).
  • the target tissue will be skin or mucosa. These tissues are particularly preferred when the therapy is directed to infections or diseases where it is desirable to induce a localized therapeutic or immune response.
  • a mucosal route of administration would be preferred for treatment of sexually transmitted diseases, where the therapy was directed to boosting the immune response to antigens in infected tissues.
  • a nasal route of administration via inhalation or insufflation
  • a mucosal or dermal route would be useful in immunizing against allergens.
  • These tissue are also preferred for their regenerative ability, which limits the length of time that introduced materials will remain at the point of entry.
  • the method of the invention may not be as useful for inducing systemic responses to the expressed peptide as it is for inducing a localized response. However, at sufficient dosage levels a transitory systemic effect can be induced.
  • a useful application of this aspect of the invention for induction of systemic responses to the expressed peptide may, therefore, be as an adjuvant for other systemic therapies.
  • the APC's serve as vehicles to deliver the naked polynucelotide to lymphatic organs and to mucosal tissues other than those at the point of entry.
  • This embodiment is illustrated by reference to the following hypothesis; the mechanism described should not, however, be construed as limiting the invention.
  • the APC's take up the naked polynucelo ⁇ tide at or near the point of entry then carry them into lymphatic circulation. Once at a lymph node, the APC will present the expressed protein as an antigen, thereby stimulating an immune response. From there, ' those APC's which carry "homing" receptors for, e.g., mucosa, may reenter lymphatic circulation until they settle in a target tissue other than the tissue at the point of entry. Where desired, homing receptors (specific membrane proteins which bind to target cell ligands) may be sequenced and incorporated into the naked polynucleotide.
  • this embodiment also provides a means of enhancing the host's immune responsiveness by delivering cytokines to increase the concentration of specific cytokines present in the host.
  • increases in the host's levels of circulating cytokines can boost the host's immune response to pathogenic antigens and (1) serve as an adjuvant for vaccines, (2) decrease the immune response to self-antigens in autoimmune diseases, or (3) decrease the immune response to alloantigens (produced, for example, following tissue or organ transplantation).
  • the gene can be administered at an accessible point of entry for expression at a less convenient or accessible site.
  • a naked polynucelotide delivered intranasally may, under appropriate conditions, be expressed in the genital mucosa.
  • Another use for the invention would be in moderating an allergic response to an antigen.
  • the nasal route of administration is of particular use in this regard.
  • genes for IL-2, gamma interferon and/or transforming growth factor (TGF ⁇ ) could be administered to suppress production of IgE molecules.
  • TGF ⁇ transforming growth factor
  • This approach is of particular interest because, in recent clinical trials, IL-2 and gamma interferon have proved toxic at dosages sufficient to interfere with production of IgE.
  • IgE molecules are predominately present in skin and mucosa, use of these routes as points of entry according to the invention can be expected to be particularly effective in moderating allergic responses in these tissues.
  • Examples where it would be useful to induce a localized response in skin or mucosa are extant.
  • a mucosal route of administration would be preferred for treatment of sexually transmitted diseases.
  • the therapy can be directed toward modulating the local immune response to an infectious agent such as HIV, human papillomae viruses (such as those responsible for causing genital warts), or to cutanaceous viral infections.
  • an infectious agent such as HIV, human papillomae viruses (such as those responsible for causing genital warts), or to cutanaceous viral infections.
  • gene(s) operatively encoding for immunosuppres ⁇ sive agents such as TGF/3 could also be supplied according to the method of the invention.
  • TGF/3 an example where this approach would be useful is in the treatment of inflammatory bowel disease.
  • One particularly useful aspect of the invention is its use to supply cytokines and biochemicals relevant to the incidence of illnesses associated with aging to the host at subtherapeutic levels for a prolonged period of time.
  • the naked polynucleotide will operatively encode for a protein such as a cytokine or related growth factor whose presence or absence in mammalian circulation impacts the immune system in ways that facilitate or retard the development of illnesses associated with aging.
  • a protein such as a cytokine or related growth factor whose presence or absence in mammalian circulation impacts the immune system in ways that facilitate or retard the development of illnesses associated with aging.
  • Such illnesses may be retarded by enhancing levels of proteins whose concentrations in sera normally decrease with age, such as cytokines, growth hormones and enzymes (e.g., human ⁇ -Lfucosidase, which can mediate inflammatory responses to antigens).
  • Another particular advantage of the invention is that it involves the administra- tion of relatively minute doses of antigen. More specifically, because a polynucleotide that will operatively encode for an antigen is administered in lieu of the antigen itself, the quantity of foreign material being introduced to the host is relatively minimal. Moreover, routes of administration of naked polynucleotides through skin or mucosa require a lower concentration of DNA to produce the same magnitude of immune response than does the intramus ⁇ cular route of administration (e.g., about 10-50 fold lower; see, e.g., Example 16 and FIGURES 22-23). As a result, the invention lends itself well to the administration of naked polynucleotides which encode for up to several hundred different antigens for use, as an example, as a polyvalent vaccine.
  • Another particular advantage of the invention will be its use in antisense therapy.
  • a nucleotide sequence that interferes with the specific expression of the mutated gene at the transcriptional or translational level can be used.
  • This approach utilizes, for example, antisense oligonucleotides and/or ribozymes to block transcription or translation of a specific mutated mRNA, either by masking that mRNA with an antisense nucleic acid or by cleaving it 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).
  • genes and oncogenes have been targeted for suppression or down-regulation including, but not limited to, p53 (V. S. Prasolov et al., Mol. Biol. (Moscow) 22:1105-1112, 1988); ras (S.K. Anderson et al., Mol. /mmut7 ⁇ /._26:985-991 , 1989; D. Brown et al., Oncogene ftes._4:243- 249, 1989); fos (B. Levi et al., Cell.
  • Antisense therapy can also be used to block production of mutant proteins that act directly to increase the probability of producing neoplastic cells, such as in the Type III, Type IV and Type V mutations that mimic Type III described by Levine, et al., supra.
  • Antisense polynucleotides are also therapeutically effective when mutation is not dominant, for instance when a non-mutant allele remains that encodes the proper protein. However, when the mutation is dominant, as in Type I mutations, and in cases wherein either both alleles are deleted or one is deleted and the other is mutant, as in certain Type III mutations, antisense therapy is preferably accompanied by replacement therapy.
  • a wild type gene is introduced into the target cells identified as having a mutant gene or protooncogene which results in production of the wild type protein necessary to forestall development of the disorder or neoplasia associated with the identified mutant gene(s).
  • tumor suppressor genes In the case of tumor suppressor genes, it is known that introducing a suppressor gene into cultured cells either causes cell death or causes no discernible changes, however, the cells may no longer be tumorigenic in animals. Thus, in cases where ribozyme and/or antisense therapy is accompanied by gene replacement therapy, the chances are increased that the cell population containing the mutant gene for which the ribozyme or antisense oligonucleotide is specific will no longer contribute to development of neoplasia in the subject being treated.
  • the present invention also provides gene therapy for the treatment of cancer conditions; i.e., cell proliferative disorders that are mediated by a deletion of, or polymorphism in, a particular gene.
  • Such therapy would achieve its effect by introduction of the specific antisense polynucleotide and/or replacement wild type gene into cells identified by the methods of this invention as having the proliferative disorder caused by mutated genes.
  • Whether the cell will require replacement of the wild type gene as well as antisense therapy to prevent replication of a gene bearing a polymorphism must be determined on a case by case basis and will depend upon whether the mutation has a dominant effect, ie., whether both alleles of the wild type gene have been destroyed so that total absence of the gene has a cell proliferative effect.
  • the preferred routes of administration for inducing local immunity in or near the skin will be by transdermal transmission, intradermal injection or superficially scratching or irritating the outermost layer of epidermal cells (i.e., epidermal administration), although subcutaneous injection may also be of use in certain applications.
  • the preferred routes of administration for inducing local immunity in the respiratory tract will be by inhalation or insufflation; routes of administra ⁇ tion to other mucosal tissues will vary according to their location.
  • the naked polynucleotides are to be introduced into skin or mucosa
  • delivery of the polynucleotide is preferably facilitated without need for injection by use of detergents, absorption promoters, chemical irritants (such as keratinolytic agents), or mechanical irritants.
  • Detergents and absorption promoters which facilitate uptake of small molecules other than genes are well known in the art and may, without undue experimentation, be adapted for use in facilitating uptake of genes.
  • Another substantially noninvasive approach to introducing the naked polynucleotides is by transdermal transmission (preferably iontophoresis) which has been used with success for transdermal transmission of peptides.
  • any parenteral route of administration is possible, although use of routes involving little or no invasion of host tissues are greatly preferred.
  • intramuscular injections are not preferred. Instead, introduction of the naked polynucleotide (s) to an area of the body which is regenerative, such as skin and mucosa, is preferred for their ability to replace cells which have been directly affected by trauma associated with each dosage.
  • sequences controlling secretion known to those skilled in the art will be included in the administered naked polynucleotide, if not already present in the full-length gene.
  • the polynucleotide may be conjugated to a liposome or delivered in cells which have been transfected in vitro with the polynucleotide. Nonetheless, for the reasons discussed above, use of a naked polynucleotide and a mucosal or dermal route of administration in these embodiments is still preferred. In particular, use of liposomes is likely to result in reduced levels of expression. This phenomenon is likely to be the result of impaired recognition by APC's of a liposome as an antigenic material.
  • FIGURE 1 depicts sections of muscle tissue demonstrating chronic inflamma ⁇ tion (panel A) and myonecrosis (panel B) following intra-muscular injections of pREVk3 and pRSVIL-2.
  • Panel C depicts sections of similar muscle tissue following subcutaneous injections of pREVK3 or pRSVIL-2.
  • FIGURE 2A depicts the results of an ELISA for anti-NP IgG in serum following intradermal injection of naked pCMVRNP
  • FIGURE 2B depicts the results of an ELISA for anti-NP IgG in serum following intramuscular injection of naked pCMVRNP.
  • FIGURE 3 depicts the results of an ELISA for anti-NP IgG before intranasal introduction of naked pCMVRNP to Balb/c mice.
  • FIGURE 4 depicts the results of an ELISA for anti-NP IgG in an unanesthesized group of Balb/c mice.
  • FIGURE 5 depicts the results of an ELISA for anti-NP IgG in an anesthesized group of Balb/c mice.
  • FIGURE 6 depicts the results of ELISA's for anti-KLH levels in sera of mice injected intramuscularly with pRSVIL-2 and pRSVIL-4 (panel A) as well as pRSVTGF ?1 (panel B).
  • FIGURE 7 depicts the results of ELISA's for anti-transferrin levels in sera of mice following intramuscular injection of pRSVIL2 (panel A), pRSVTGF ⁇ l (panel A).
  • FIGURE 8 depicts the results of a foot pad swelling assay after antigen challenge (in pRSVIL2 and pRSVTGF ⁇ injected mice).
  • FIGURE 9 depicts the results of ELISA's for anti-chromatin serum levels in pRSVIL-2 and pRSVTGFyS injected MRL/lpr/lpr mice.
  • FIGURE 10 depicts the results of ELISA's for IL-2 expression over time in individual AKR/J mice.
  • FIGURE 11 depicts longevity data for the mice described with respect to FIGURE 10.
  • FIGURE 12 depicts the results of a 51 Cr-release assay for NK cell cytolytic activity in AKR/J mice (mouse lymphoma model) following introduction of a naked polynucleotide at a dosage sufficent to produce IL-2 gene expression at subtherapeutic levels.
  • FIGURE 13 (a)-(b) depict the levels of IL-2 expression in following administra ⁇ tion of naked pRSVIL-2 in different dosages to young Balb/c mice and older Balb/c mice.
  • FIGURE 14 depicts the levels of IL-2 expression detected in sera following pRSVIL-2 administration at different points of entry.
  • FIGURE 15 is a photograph of the results of histological studies of skin at the point of entry for pCMVRNP in Balb/c mice showing uptake of the plasmid by mononuclear cells (APC's).
  • An APC is indicated by an arrows; a tissue cell (not containing the plasmid) is indicated by a slashed line.
  • FIGURE 16 depicts the results of an ELISA for anti-NP IgG following mechani ⁇ cal epidermal administration of naked pCMVRNP to Balb/c mice.
  • FIGURE 17 depicts the results of an ELISA for anti-NP IgG following chemical epidermal administration of naked pCMVRNP to Balb/c mice.
  • FIGURE 18 depicts the stability of IL-2 expression detected in sera over time after administration of a naked polynucleotide encoding IL-2.
  • FIGURE 19 contains a Kaplan-Meyer survival curve depicting the length of time that Balb/c mice injected intradermally with naked pCMVRNP survived following viral challenge.
  • FIGURE 20 graphically compares NP gene expression following separate intradermal injections of naked plasmids containing either a CMV or an RSV promoter sequence.
  • FIGURE 21 depicts the levels of cytotoxic T cells detected in mice after injection of various naked plasmids administered by intradermal injection.
  • FIGURE 22 depicts the results of an ELISA for anti- ⁇ -galactosidase antibodies after administration of (1 ) a polynucleotide encoding the enzyme by intramus ⁇ cular or intradermal injection, and (2) the enzyme by intradermal injection.
  • FIGURE 23 depicts the results of an ELISA for anti- ⁇ -galactosidase antibodies in sera from the mice described with respect to FIGURE 22 after a booster injection of antigen.
  • FIGURE 24 depicts the results of an ELISA for IgG 2A type antibodies in sera for mice (1) injected intradermally or intramuscularly with a polynucleotide encoding -galactosidase, or (2) the enzyme by intradermal injection.
  • FIGURE 25 depicts the results of an ELISA for IgG 1 type antibodies in sera for mice (1) injected intradermally or intramuscularly with a polynucleotide encoding 3-galactosidase, or (2) the enzyme by intradermal injection.
  • FIGURE 26 depicts the results of an ELISA for IgG 2A type antibodies in sera of the mice described with respect to FIGURE 25 after a booster injection of antigen.
  • FIGURE 27 depicts the results of an ELISA for IgG 1 type antibodies in sera of the mice described with respect to FIGURE 24 after a booster injection of antigen.
  • FIGURE 28 depicts the results of an ELISA for IgG 2A type antibodies in sera for mice (1) introduced by scratching the skin with tynes coated with a polynucleotide encoding ⁇ -galactosidase, or (2) the enzyme by intradermal injection.
  • FIGURE 29 depicts the results of an ELISA for IgG 1 type antibodies in sera for mice (1) introduced by scratching the skin with tynes coated with a polynucleotide encoding ⁇ -galactosidase, or (2) the enzyme by intradermal injection.
  • the polynucleotides to be used in the invention may be DNA or RNA, but will preferably be a complementary DNA (cDNA) sequence.
  • the polynucleotide sequences used in the invention must be (a) expressible and (b) either non- replicating or engineered by means well known in the art so as not to replicate into the host genome. Illustrations of the preparation of polynucleotides suitable for use in the invention follow and specific examples showing how particular polynucleotide compositions were made are provided infra. It will, however, be apparent to those skilled in the art that other known means of preparing nonreplicating polynucleotides may also be suitable.
  • Polynucleotides for use in the invention can be obtained using hybridization methods well known in the art.
  • DNA and RNA may also be synthesized using automated nucleic acid synthesis equipment well known in the art.
  • Use of the well-known polymerase chain reaction (PCR) is particularly preferred for generating mixtures of polynucleotides.
  • Genomic nucleic acids may be prepared by means well-known in the art such as the protocols described in Ausubel, et al., Current Protocols in Molecular Biology, Chs. 2 and 4 (Wiley Interscience, 1989).
  • cDNA can be synthesized according to means well known in the art (see, e.g., Maniatis, et al., Molecular Cloning; A Laboratory Manual (Cold Spring Harbor Lab, New York, 1982).
  • a cDNA expression library containing polynucleotides of interest can also be screened by means well known in the art. For reference, examples of such means are illustrated by the discussion below.
  • the naked polynucleotides may operatively encode for therapeutic peptides, but will preferably encode for immunogenic peptides which can act as antigens to provoke a humoral and/or cellular response.
  • the naked polynucleotides can also operatively encode for an antibody.
  • the term "antibody” encompasses whole immunoglobulin of any class, chimeric antibodies, hybrid antibodies with dual or multiple antigen specificities and fragments including hybrid fragments. Also included within the meaning of "antibody” are conjugates of such fragments, and so-called antigen binding proteins (single chain antibodies) as described, for example, in U.S. Patent No. 4,704,692.
  • the encoded antibodies can be anti-idiotypic antibodies (antibodies that bind other antibodies) as described, for example, in U.S. Patent No. 4,699,880.
  • the methods of the invention may be adapted for use in administering any polynucleotide or mixture thereof which operatively encode therapeutic and/or immunogenic peptides of interest.
  • the invention is therefore not limited to use with any particular polynucleotide (s).
  • polynucleotide refers to a polymer of deoxyribonucleotides or ribonucleotides, in the form of a separate fragment or as a component of a larger construct.
  • DNA encoding an therapeutic and/or immunogenic peptide of the invention can be assembled from cDNA fragments or from oligonucleotides which provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit.
  • Polynucleotide sequences of the invention include DNA, RNA and cDNA sequences. A polynucleotide sequence can be deduced from the genetic code, however, the degeneracy of the code must be taken into account.
  • Polynucleotides of the invention include sequences which are degenerate as a result of the genetic code, which sequences may be readily determined by those of ordinary skill in the art.
  • Polynucleotide sequences encoding a desired therapeutic and/or immunogenic peptide can be expressed in either eukaryotes or prokaryotes.
  • Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokary- otes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are also well known in the art. Such vectors are used to incorporate DNA of the invention.
  • Polynucleotides of the invention include functional derivatives of known polynucleotides which operatively encode for a therapeutic and/or immuno- genie peptide of interest.
  • functional derivative is meant the “fragments,” “variants,” “analogs,” or “chemical derivatives” of a molecule.
  • a “fragment” of a molecule, such as any of the DNA sequences of the present invention includes any nucleotide subset of the molecule.
  • a “variant” of such molecule refers to a naturally occurring molecule substantially similar to either the entire molecule, or a fragment thereof.
  • An “analog” of a molecule refers to a non- natural molecule substantially similar to either the entire molecule or a fragment thereof.
  • a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half-life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties which are known in the art to be capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th Ed., Mack Publishing Co., Easton, Penn. (1980).
  • polynucleotide includes any functional derivative, fragments, variants, analogs, and chemical derivatives which may be substan ⁇ tially similar to the polynucleotides described herein and which possess similar activity.
  • DNA sequences for use in producing therapeutic and/or immunogenic peptides of the invention can also be obtained by several methods. For example, the
  • DNA can be isolated using hybridization procedures which are well known in the art. These include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect shared nucleotide sequences; 2) antibody screening of expression libraries to detect shared structural features and 3) synthesis by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the development of specific DNA sequences encoding or fragments thereof, can also be obtained by: 1) isolation of double-stranded DNA sequences from the genomic DNA: 2) chemical manufacture of a DNA sequence to provide the necessary codons for the polypeptide of interest;and 3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell. In the latter case, a double-stranded DNA complement of mRNA is eventually formed which is generally referred to as cDNA.
  • Hybridization procedures are useful for the screening of recombinant clones by using labeled mixed synthetic oligonucleotide probes where each probe is potentially the complete complement of a specific DNA sequence in the hybridization sample which includes a heterogeneous mixture of denatured double-stranded DNA.
  • hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA.
  • Hybridization is particularly useful in the detection of cDNA clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present. In other words, by using stringent hybridization conditions directed to avoid non-specific binding, it is possible, for example, to allow the autoradiographic visualization of a specific cDNA clone by the hybridization of the target DNA to that single probe in the mixture.
  • a cDNA library believed to contain a polynucleotide of interest can be screened by injecting various mRNA derived from cDNAs into oocytes, allowing sufficient time for expression of the cDNA gene products to occur, and testing for the presence of the desired cDNA expression product, for example, by using antibody specific for a peptide encoded by the polynucleotide of interest or by using probes for the repeat motifs and a tissue expression pattern characteris ⁇ tic of a peptide encoded by the polynucelotide of interest.
  • a cDNA library can be screened indirectly for expression of therapeutic and/or immunogenic peptides having at least one epitope using antibodies specific for the peptides. Such antibodies can be either polyclonally or monoclonally derived and used to detect expression product indicative of the presence of cDNA of interest.
  • the naked polynucleotides may be conjugated to or used in association with other polynucleotides which operatively code for regulatory proteins that control the expression of these polypeptides or may contain recognition, promoter and secretion sequences.
  • Those of ordinary skill in the art will be able to select regulatory polynucleotides and incorporate them into the naked polynucleotides of the invention (if not already present therein) without undue experimentation.
  • suitable promoters for use in murine or human systems and their use are described in Current Protocols in Molecular Biology, supra at Ch. 1.
  • a particularly preferred form of a naked polynucleotide for use in the invention will be one which has been incorporated into a plasmid vector.
  • Use of a plasmid vector, particularly one which comprises a replicator, will prolong expression of the gene in target tissues.
  • Certain plasmid vectors are also good mediators of immune responses to immunogenic peptides because high levels of expression are achieved when the gene encoding the peptides is incorporated into the vector.
  • Suitable plasmid vectors are well-known in the art and include the vectors described in Current Protocols in Molecular Biology, supra at Ch. 1.
  • Two particularly preferred plasmid vectors are the pRSV (Rous sarcoma virus) and pCMV (cytomegalovirus) promoter vectors.
  • pRSV Raster sarcoma virus
  • pCMV cytomegalovirus
  • CMV CMV is preferred for polynucleotides to be introduced into tissue other than muscle. This preference is based on observations that higher levels of expression are achieved in this context when the CMV promoter is employed.
  • plasmid vector A suitable protocol for isolation of the RSV promotor and its use in construction of a plasmid vector is described in 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 of San Diego, California.
  • a particularly suitable plasmid for production of mRNA is the pSP64T cloning vector described by Kreig, et al., Nucleic Acids Res., 12:7057- 7070, (1984). Any cDNA containing an initiation codon can be introduced into this plasmid and mRNA prepared from the expressed DNA templates using conventional techniques.
  • RNA virus such as a retrovirus
  • retroviral vector is a derivative of a murine or avian retrovirus.
  • retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • a number of additional retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.
  • Retroviral vectors can be made target specific by inserting, for example, a polynucleotide encoding a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody to target the retroviral vector.
  • a polynucleotide encoding a sugar, a glycolipid, or a protein Preferred targeting is accomplished by using an antibody to target the retroviral vector.
  • a separate vector can be utilized for targeted delivery of a replace ⁇ ment gene to the cell(s), if needed.
  • an antisense oligonucleotide and the replacement gene may also be delivered via the same vector since the antisense oligonucleotide is specific only for target gene containing a polymorphism.
  • helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence that enables the packaging mechanism to recognize an RNA transcript for encapsidation.
  • Helper cell lines that have deletions of the packaging signal include, but are not limited to, ⁇ 2, PA317 and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such helper cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion can be produced.
  • these vectors may be modified to include known reporter genes.
  • the pRSV lac-Z DNA vector described in Norton, et al., Mol. Cell. Biol., 5:281, (1985) may produce ⁇ - galactosidase with protein expression.
  • Luciferase and chloramphenicol acetyl transferase (“CAT 1 ; see, e.g., Gorman, et al., supra, re construction of a pRSV- CAT plasmid) may also be used.
  • Convenient plasmid propogation may be obtained in E. coli (see, e.g., Molecular Cloning: A Laboratory Manual, supra.)
  • a mixture of polynucleotides or separately coadministered group of polynucleotides may include a gene operatively encoding for an immunosuppressive cytokine (such as TGF ⁇ ) and a separate gene operatively encoding for a relevant histocompatibility protein.
  • an immunosuppressive cytokine such as TGF ⁇
  • a separate gene operatively encoding for a relevant histocompatibility protein may be used as a tolerizing vaccine.
  • synthetic antisense oligonucleotides are generally between 15 and 25 bases in length. Assuming random organization of the human genome, statistics suggest that a 17-mer defines a unique sequence in the cellular mRNA in human DNA; a 15-mer defines a unique sequence in the cellular mRNA component. Thus, substantial specificity for a selected genetic target is easily obtained using the synthetic oligomers of this invention.
  • the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule.
  • the antisense nucleic acids interfere with the translation of the mRNA, since the cell will not translate a mRNA that is double-stranded.
  • Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target nucleotide mutant producing cell.
  • the use of antisense methods to inhibit the in vitro translation of genes is well known in the art (Marcus-Sakura, Anal.Biochem., 172:289, 1988). Less commonly, antisense molecules which bind directly to the DNA may also be used.
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences that encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences associated with production of a mutated proto oncogene or tumor suppressor gene in an RNA molecule and cleave it (Cech, J ⁇ mer.Me- d. Assn., 260:3030. 1988). A major advantage of this approach is that, because they are sequence-specific, only target mRNAs with particular mutant sequences are inactivated.
  • ribozymes There are two basic types of ribozymes, namely, tetrahymena-type (Hasselhoff, Nature, 334:585, 1988) and "hammerhead"-type. Tetrahymena-type ribozymes recognize sequences which are four bases in length, while “hammerhead”-type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species, and 18-based recognition sequences are preferable to shorter recognition sequences.
  • oligodeoxyribonucleotides are readily degraded by serum and cellular nucleases. Therefore, as is well known in the art, certain modifications of the phosphate backbone have conferred nuclease resistance to antisense DNA. For instance phosphorothioate, methylphosphonate, and ⁇ -anomeric sugar-phosphate, backbone-modified oligomers have increased resistance to serum and cellular nucleases. In addition, methylphosphonates are nonionic and offer increased lipophilicity to improve uptake through cellular membranes. The use of modified oligonucleotides as antisense agents may require slightly longer or shorter sequences because chemical changes in molecular structure can affect hybridization (L A.
  • compositions of naked polynucleotides and mixtures of polynucleotides may be placed into a pharmaceutically acceptable suspension, solution or emulsion.
  • Suitable mediums include saline and may, for those embodiments which do not rely on antigen presenting cells for delivery of the polynucleotides into target tissue, liposomal preparations.
  • pharmaceutically acceptable carriers may include sterile aqueous of non-aqueous solutions, suspensions, and emulsions.
  • 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/aqueous 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 replen- ishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. Further, a composition of naked polynucleotides may be lyophilized using means well known in the art, for subsequent reconstitution and use according to the invention.
  • a colloidal dispersion system may also be used for targeted delivery.
  • the advantages of employing the method of the invention to administer naked nucleotides, and of administering those nucleotides to tissues having relatively high concentrations of antigen presenting cells are such that the use of collodidal dispersion systems for delivery of polynucleotides will not be a preferred method. The discussion below regarding such systems is therefore provided principally for reference in the event that the preferred method of the invention is determined to be unavailable for use with respect to a particular indication.
  • Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • the preferred colloidal system of this invention is a liposome.
  • Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 ⁇ m can encapsulate a substantial percentage of an aqueous buffer containing large macromoiecules. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci., 6:77,
  • LUV large unilamellar vesicles
  • liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells.
  • a liposome In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes encoding the antisense polynucleotides at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques, 6:682, 1988).
  • the composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used.
  • the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
  • lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidyls- erine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
  • Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated.
  • Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
  • the targeting of liposomes can be classified based on anatomical and mechanistic factors.
  • Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific.
  • Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries.
  • RES reticulo-endothelial system
  • Active targeting involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein
  • the surface of the targeted delivery system may be modified in a variety of ways.
  • lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer.
  • Various linking groups can be used for joining the lipid chains to the targeting ligand.
  • liposomal preparations substantially limit uptake of the naked polynucleotides in vivo and should not be used. Instead, isotonic buffered solution is the preferred medium for maximal uptake of the naked polynucleotides in such embodiments. Further, use of absorption promoters, detergents, chemical irritants or mechanical irritation means is also preferred to enhance transmis ⁇ sion of the naked polynucleotide composition through the point of entry. For reference concerning general principles regarding promoters and detergents which have been used with success in mucosal delivery of organic and peptide-based drugs, see Chien, Novel Drug Delivery Systems, Ch. 4 (Marcel Dekker, 1992).
  • the means of introduction may be by epidermal administration, subcutaneous or intradermal injection. Of these means, epidermal administration is preferred for the greater concentrations of APC's expected to be in intradermal tissue.
  • the means of introduction for dermal routes of administration which are most preferred, however, are those which are least invasive. Preferred among these means are transdermal transmission and epidermal administration.
  • iontophoresis is a suitable method
  • lontophoretic transmission may be accomplished using commercially available "patches" which deliver their product continuously through unbroken skin for periods of several days or more.
  • Use of this method allows for controlled transmission of pharmaceutical compositions in relatively great concentrations, permits infusion of combination drugs and allows for contemporaneous use of an absorption promoter.
  • An exemplary patch product for use in this method is the LECTRO PATCH trademarked product of General Medical Company of Los Angeles, CA. This product electronically maintains reservoir electrodes at neutral pH and can be adapted to provide dosages of differing concentrations, to dose continuously and/or to dose periodically. Preparation and use of the patch should be performed according to the manufacturer's printed instructions which accompany the LECTRO PATCH product; those instructions are incorporated herein by this reference.
  • Epidermal administration essentially involves mechanically or chemically irritating the outermost layer of the epidermis sufficiently to provoke an immune response to the irritant. Specifically, the irritation should be sufficient to attract APC's to the site of irritation. As discussed previously, it is believed that the APC's then take up and express the administered naked polynucleotide.
  • An exemplary mechanical irritant means employs a multiplicity of very narrow diameter, short tynes which can be used to irritate the skin and attract APC's to the site of irritation, to take up naked polynucleotides transferred from the end of the tynes.
  • a multiplicity of very narrow diameter, short tynes which can be used to irritate the skin and attract APC's to the site of irritation, to take up naked polynucleotides transferred from the end of the tynes.
  • MONO-VACC old tuberculin test manufac ⁇ tured by Pastuer Merieux of Lyon, France contains a device suitable for introduction of naked polynucleotides.
  • the device (which is distributed in the U.S. by Connaught Laboratories, Inc. of Swiftwater, PA) consists of a plastic container having a syringe plunger at one end and a tyne disk at the other.
  • the tyne disk supports a multiplicity of narrow diameter tynes of a length which will just scratch the outermost layer of epidermal cells.
  • Each of the tynes in the MONO-VACC kit is coated with old tuberculin; in the present invention, each needle is coated with a pharmaceuti ⁇ cal composition of naked polynucleotide or a mixture thereof.
  • Use of the device is according to the manufacturer's written instructions included with the device product; these instructions regarding use and administration are incorporated herein by this reference to illustrate conventional use of the device. Similar devices which may also be used in this embodiment are those which are currently used to perform allergy tests.
  • Another suitable approach to epidermal administration of naked polynucleo ⁇ tides is by use of a chemical which irritates the outermost cells of the epidermis, thus provoking a sufficient immune response to attract APC's to the area.
  • a chemical which irritates the outermost cells of the epidermis thus provoking a sufficient immune response to attract APC's to the area.
  • An example is a keratinolytic agent, such as the salicylic acid used in the commercially available topical depilatory creme sold by Noxema Corporation under the trademark NAIR.
  • This approach may also be used to achieve epithelial administration in the mucosa.
  • the chemical irritant may also be applied in conjunction with the mechanical irritant (as, for example, would occur if the MONO-VACC type tyne were also coated with the chemical irritant).
  • the naked polynucleotide may be suspended in a carrier which also contains the chemical irritant or coadministered
  • the means of introduction will vary according to the location of the point of entry. Particularly for immunization to and treatment of respiratory infections, intranasal administration means are most preferred. These means include inhalation of aerosol suspensions or insufflation of the naked polynucleotide or mixtures thereof. Suppositories and topical prepara ⁇ tions will also be suitable for introduction to certain mucosa, such as genital and ocular sites. Also of particular interest with respect to vaginal delivery of naked polynucleotides are vaginal sandwich-type rings and pessaries. Examples of these devices and their use are described in Chien, supra at Ch.9.
  • each naked polynucleotide or mixture thereof to be supplied using the method of the invention will vary depending on the desired response by the host and the polynucleotide used. Generally, it is expected that up to 100-200 ⁇ g of DNA can be administered in a single dosage, although as little as about 0.3 ⁇ g of DNA administered through skin or mucosa can induce long lasting immune responses.
  • the naked poly ⁇ nucleotides be supplied at a dosage sufficient to cause expression of the biologically active peptide encoded by the polynucleotide.
  • Dosages suitable for particular indications e.g., supplying a subtherapeutic dosage of cytokine
  • These dosages may be modified to achieve therapeutic, subtherapeutic or immunogenic levels of expression.
  • Means to confirm the presence and quantity of expressed peptides are well-known to those skilled in the art and will not, therefore, be described in detail.
  • the methods described above for preparation and introduction of naked polynucleotides are suitable for use in this embodiment of the invention.
  • the methods described are employed to produce subtherapeutic levels of circulating cytokines and related proteins.
  • the quantity of naked polynucleotide necessary to produce subtherapeutic levels of expressed protein can be readily determined and adjusted as necessary by skilled clinicians.
  • This aspect of the invention is practiced by administering a polynucleotide or recombinant mixture thereof to a mammal which operatively encodes for the desired protein, preferably prior to the onset of biological impairment associated with aging.
  • a polynucleotide or recombinant mixture thereof operatively encodes for the desired protein, preferably prior to the onset of biological impairment associated with aging.
  • an immunostimulatory interleukin such as IL-2
  • the therapy may be continued throughout the patient's life span.
  • polynucleotide and resulting levels of protein expression must be low (i.e., subtherapeutic).
  • the polynucleotides can be expected to degenerate far more slowly than would the protein (i.e., a difference of months) so the doses may be administered less frequently, thus minimizing the risk of toxicity and trauma to the patient.
  • administration of the desired polynucleotides will begin prior to or concurrent with onset of a disease state associated with aging or another biological event which results in the compromise of the immune system, such as the loss of CD4 T cells during the course of an HIV-1 infection.
  • Prolonged, continuous therapy beginning prior to the onset of a disease state or loss of immune function is preferred.
  • Therapy is accomplished by subtherapeutic levels of expression of the genes of interest, in particular of genes encoding for cytokines (e.g., interleukins and lymphotoxins).
  • At least one of the genes of interest will be those which encode for IL-1 , IL-2, growth hormone (GH), somatomedins (such as insulin-like growth factor [IGF-1]), and/or TGF .
  • GH growth hormone
  • IGF-1 insulin-like growth factor
  • TGF TGF
  • this particular method of the invention is, in part, a subset of the method disclosed in Section I, supra.
  • those of skill in the art will understand that it is possible for this embodiment of the invention to be practiced using polynucleotides that are not "naked”; i.e., polynucleotides that are conjoined to a colloidal dispersion system. Nonetheless, the advantages of the method disclosed in Section I, supra, are such that its practice (i.e., using "naked" polynucleotides introduced into a tissue having a relatively high proportion of APC's) will be much preferred.
  • Another aspect of the invention is the administration of a peptide cocktail (i.e., mixture of polynucleotides) via expression of gene constructs containing, for example, up to 200 polynucleotide sequences under the control of a single promoter.
  • a cocktail vaccine could be administered according to the method of the invention which is capable of stimulating an immune response to many different rhinoviruses.
  • This approach also allows for the construction of a vaccine to various strains of HIV, using pooled isolates of envelope genes from different patients (which genes may, if necessary, then be amplified).
  • a naked polynucleotide operatively encoding for an immunogenic peptide may be coupled to or administered with a naked polynucleotide operatively encoding an antibody in such a way that both peptide and antibody will be expressed.
  • genes which will jointly express IL-2 and anti- gp71 may (based on results obtained with the IL-2 protein) result in localization of the antibody in tumor tissue developed in response to murine leukemia virus (MuLV) in mice (see, re results obtained with concurrent administration of IL- 2/anti-gp71 mAb's, Schultz, et al., Cancer Res., 50:5421-5425, 1990).
  • MuLV murine leukemia virus
  • pREVk3 and pRSVIL2 were prepared as follows. Preparation of Plasmids. A rearranged kappa light gene from a human patient with chronic lymphocytic leukemia was isolated which contains a Humkv 325 (which encodes the 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, ef al J. Exp. Med., 175:983, (1992), which article is incorporated herein by this reference.
  • a 1040 bp Hindlll-Xhol fragment containing the V-J region of this gene was excised and inserted into the polycloning site of the mammalian expression vector pREP7 (Invitrogen, San Diego, CA), downstream of the Rous sarcoma virus (RSV) long terminal repeat (LTR) to produce a vector designated pREVk3. Downstream of the rearranged JK1 segment, there is a natural stop codon, which terminates translation.
  • pREP7 Invitrogen, San Diego, CA
  • RSV Rous sarcoma virus
  • LTR long terminal repeat
  • pRSVIL-2 an IL-2 expression vector, designated pRSVIL-2
  • the luciferase cDNA in the vector pRSVL (Wolff, et al., Science, 247_:1465. 1990) was replaced with a 680 bp Hindlll-BamHI fragment of pBC12/HIV/IL-2 (American Type Culture Collection, No. 67618) according to the method taught in Cullen, Cell, 46:937, (1986).
  • the Wolff, et al., and Cullen references are incorporated herein to illustrate knowledge in the art concerning construction of these expression vectors.
  • mice with plasmid cDNA Intramuscular injection of mice with plasmid cDNA. Eight week old BALB/c mice were anesthetized with methoxyflurane. Plasmid cDNA (100 ⁇ g per injection) was suspended in 100 ⁇ l of saline, and then was injected four times into the quadricep muscles through a 28-gauge needle at weekly intervals. One group of six mice received 100 ⁇ g of pREVk3. Another group of six mice received 100 ⁇ g each of pREVk3 and pRSVIL-2 while a third group received 100 ⁇ g of saline alone. Just before every injection, blood samples were collected from the orbital arteries. ELISA To Verify In Vivo Gene Expression by the Plasmids.
  • Antibodies against Humkv325 products were measured by ELISA (enzyme-linked immunosorbent assay).
  • the IgM rheumatoid factor Glo is encoded by the Humkv325 gene and has 17.109 idiotype positive kappa light chains.
  • the purified protein was dissolved at 10 ⁇ g/ml in 0.1 M borate, 0.2 M NaCI, pH8.2 (i.e., buffered borate saline or BBS), and then 100 ⁇ l aliquots were added to the wells of plastic microtiter plates.
  • BBS/Tween BBS supplemented with 1% bovine serum albumin
  • mice were injected with a naked cDNA plasmid intradermally. Gene expression was observed and measured.
  • RNP influenza ribonucleoprotein
  • mice Four eight week old Balb/c mice were injected three times with 15 ⁇ g of pCMV ⁇ RNP suspended in 100 ⁇ l of HBSS. Injections were made intradermally at the base of the tails at two week intervals. Cytotoxic T lymphocytes (CTL) recognize antigens presented by class I MHC molecules and play an important role in the elimination of virally infected cells. Intramuscular (i.m.) immunization by means of cDNA expression vectors should be an effective method to introduce antigen into class I MHC molecules and thus stimulate CTL responses.
  • CTL Cytotoxic T lymphocytes
  • Plasmid DNA was purified by CsCI banding in the presence of ethidium bromide and was stored frozen in 10 mM Tris-HCL, 0.1 mM EDTA, pH 8.0. Before injection, the plasmid was precipitated in ethanol and dissolved in normal saline containing 0.1 mM EDTA. The presence of anti-NP IgG in serum was measured by ELISA substantially as described in Viera, et al., Int. Immunl., 2:487, (1990). The results of this assay are shown in FIGURE 2A; all of the animals developed high titer anti-NP antibodies, which persisted for more than 20 weeks. As shown in FIGURE 2B, the intradermal injections appeared to give about four fold higher antibody titers than intramuscular injections (made as described in Example I) of equivalent amounts of plasmid DNA.
  • the axes of FIGURE 2 represent, respectively, the ELISA titer (mean, 1 ounce) against time. Serum dilution for all graph points is 2560.
  • naked polynucleotide encoding for influenza ribonucleoprotein was introduced to Balb/c mice in 3 groups of 6 intranasally.
  • Levels of anti-NP IgG in peripheral blood before and after introduction of the plasmid at various serum dilutions were measured by ELISA as described in Example II. Blood was drawn from each mouse after intranasal introduction after 6 weeks.
  • FIGURE 3 graphically depicts the results of the ELISA assays before and after intranasal introduction of the plasmid.
  • the graphs plot ELISA titer against serum dilution.
  • values are shown for individual mice from each group (#1-3) and an average value from all mice in each group (#G1-G3).
  • mice in a second group which received 3x7.5 ⁇ g of plasmid showed enhanced titers of antibody as compared to background (FIGURE 3). These data are shown in FIGURE 4.
  • a third group of mice received the same gravity of plasmid under anesthesia. Expression of RNP as indicated by titers of anti-NP IgG in these mice was substantially similar to the expression achieved in the unanethesized mice. The data for the anethesized mice are shown in FIGURE 5.
  • Expression can be enhanced by additional use of absorption promoters, and prolonged by time-released promoters whose identity and use are known in the art such as those suggested in Chien, supra, at Ch. 5.
  • IL-2 Construction of Expression Vectors for IL-2, TGF--61, and IL-4.
  • the cDNAs for human (h) IL-2 (ATCC 67618), TGF- ⁇ 1 (ATCC 59954), and mouse IL-4 (ATCC 37561) were subcloned into the vector pBSII SK (from Invitrogen, San Diego, CA) to generate 5'-Hindlll and 3'-Sma1 sites. These were used to replace the Hindlll-BamH1 luciferase cDNA fragment in the expression vector pRSVL described in Example I.
  • the resulting expression vectors (pRSVIL2, pRSVTGF ⁇ l , and pRSVIL4) contain the Rous Sarcoma Virus (RSV) long terminal repeat (LTR) promoter, and an SV40 polyadenylation site. Plasmid DNA was purified from transformed DH5 E coli using QIAGEN kits from
  • mice Five-week old Balb/c mice were purchased from Jackson Laboratory (Bar Harbor, ME). At 6 weeks of age (day 0) animals were divided into 4 groups of four mice. At days 0, 7 and 14, groups 2-4 were injected with a 28-gauge needle intramuscularly (i.m.) at 5 different sites in the right thigh with a total of 100 ⁇ g of plasmid DNA (pRSVIL-2, pRSVTGFSl or pRSVIL4) dissolved in 100 ⁇ l of normal saline. At days 3, 10, and 17 (3 days after the cytokine gene injections) all animals were immunized with 100 ⁇ g of keyhole limpet hemocyanin (KLH) (Sigma, St. Louis, MO), dispersed in 100 ⁇ l of IMJECT ALUM (aluminum hydroxide, Pierce, Rockford, IL) intramuscularly in the same thigh.
  • KLH keyhole limpet hemocyanin
  • mice Five groups of eight mice each were injected in the right thigh with 100 ⁇ g of plasmid DNA (pRSVIL-2, pRSVTGFSl or pRSVIL4, groups 2-4 respectively). Group 1 was injected with normal saline, while group 5 was injected with 100 ⁇ g of pRSVIL2 and 100 ⁇ g of pRSVTGF ⁇ .
  • mice were boosted subcutaneously with 50 ⁇ g of antigen (KLH or transferrin, respectively) suspended in 50 ⁇ l normal saline. All animals were bled weekly from the retroorbital plexus for measurement of antibody levels.
  • mice Six-week-old MRL/lpr/lpr mice (Jackson Laboratory; 10 mice/group) were injected three times at four-week intervals following the same method with
  • mice 100 ⁇ g of pRSVIL2, pRSVTGF ⁇ I or pRSVnull (i.e., no introduced gene). Mice were bled at 16 weeks, two weeks after the last injection for measurement of antichromatin antibody levels. In the transferrin experiment, three mice from the pRSVTGF ⁇ l group, one mouse from the control group, and one mouse from the pRSVIL2 groups, died during bleeding or anesthesia.
  • Total IgG and lgG1 concentrations were determined in the transferrin experiment with radial immunodiffusion kits (The Binding Site, San Diego, CA), according to the manufacturer's instructions.
  • Antibodies to chromatin were assayed by ELISA, as described in the preceding examples.
  • the ELISA OD values are referred to a standard curve that was established with a strongly positive reference serum.
  • the results are the dilution of the standard curve which gave the same OD as the test sera x10 6 , and have been shown to represent a linear measure of the amount of antibody present.
  • the absolute units are arbitrary and are expressed in FIGURES 6 and 7 as equivalent dilution factors.
  • mice were bled at week 6, four weeks after the 1st gene injection, and the plasma samples were assayed for TGF- ⁇ 1 activity using the CCL64 mink lung cell proliferation assay, slightly modified as described in Latz, et al., J. Immunol., 144:4189, which is incorporated herein by this reference.
  • DTH delayed type hypersensitivity
  • TGF/3 can antagonize the effects of IL-2. It is unknown whether TGF ⁇ can have similar activities in vivo. Experiments were thus performed where plasmids encoding IL-2 or TGF- ⁇ 1 were injected simultaneously. Anti-transferrin antibody levels in the group that received both pRSVIL2 and pRSVTGF ⁇ l were indistinguishable from the pRSVTGF ⁇ group (FIGURE 10 panel C), demonstrating that TGF-91 expression completely neutralized the IL-2 effect. Mean anti-transferrin antibody levels in the pRSVIL4 group were higher but not significantly different from the control group (2.3 ⁇ 0.4 vs 1.7 ⁇ 0.36 mg/L in the 11th week).
  • Levels of Total IgG and lgG were measured in the sera from the transferrin injected mice. The highest IgG levels were observed in the pRSVIL2 and the pRSVIL4 groups after 10 and 11 weeks; the lowest levels were detected in the pRSVTGF ⁇ l group (Table I). TGF- ⁇ 1 plasmid injections completely inhibited the IL-2 mediated increase. The levels of total IgG in the pRSVIL2/pRSVTGF ⁇ 1 group were similar to the pRSVTGF ⁇ l group. The mice injected with pRSVIL4 had significantly higher concentrations of lgG1 than the other groups.
  • lgG1 levels were determined by radial immunodiffusion. Values are means ⁇ SEM. The numbers in parentheses denote the ratio of lgG1/IGG at the same time point. (*p ⁇ 0.05 vs. IL-4).
  • DTH Delayed Type Hypersensitivity
  • TGF- ⁇ 1 The mean plasma levels of TGF- ⁇ 1 at week 6, four weeks after the last pRSVTGF/91 injection, were 2.6 ng/ml as compared to only 0.32 ng/ml in the pRSVIL2 injected or untreated animals, which represents an 8 fold difference.
  • the TGF- ⁇ activity was neutralized by specific antibodies to TGF-3l (TABLE II).
  • Plasma samples from mice injected with either pRSVTGF- ⁇ 1 or pRSV IL-2 were collected 4 weeks after the last plasmid injection.
  • the samples were diluted 1 :10, acidified to pH 4, neutralized and tested in triplicate in the CCL64 assay for TGF-/3 activity. Aliquots of the samples were also incubated with neutraliz ⁇ ing rabbit antibody specific for TGF- ⁇ 1 (10ng/ml) prior to their addition to the CCL64 assay. Preimmune rabbit IgG did not change the levels of TGF ⁇ activity.
  • TGF ⁇ levels were derived by comparison with a standard curve containing recombinant TGF- / 91 (R&D Systems), are shown in ng/ml, and represent means ⁇ SE from 3 samples per group.
  • AKR/J retired breeder female mice 16 month old retired breeder female ICR outbred mice, 16 month old BALB/c old retired breeder female mice, and six-week-old BALB/c female mice were purchased from The Jackson Laboratory (Bar Harbor, ME).
  • AKR female mice have a 90% incidence of lymphoma by 9 months of age.
  • the AKR/J mice were pre-screened for the presence of circulating lymphoblastic cells by analysis of blood smear slides.
  • the lymphoma-free animals were divided into two groups of twenty-three mice for DNA injection. AKR and ICR mice were given three weekly injections of DNA and then were subsequently injected biweekly throughout the experiments.
  • the cDNA for IL-2 was subcloned into the appropriate expression vectors as described in the preceding example.
  • the vector referred to as pRSVIL-2 contains the IL-2 coding sequence (ATCC CRL #67618, Rockville, MD) sandwiched between a Rous sarcoma virus long terminal repeat promoter sequence and a SV40 polyadonylation sequence.
  • the control vector, pRSV contains no IL-2 coding sequence.
  • Plasmid DNAs were purified in large quantity using Promega MEGAPREP kits (Madison, Wl). Purified plasmid DNAs (25 ⁇ g per injection) were suspended in 0.9% NaCI (100 ⁇ l per injection) and then were directly injected into the right quadricep muscles of each mouse with the use of a 28-gauge needle.
  • Serum samples were assayed for human IL-2 using an IL-2 ELISA kit from Advanced Magnetics, Inc. (Cambridge, MA). Each sample was assayed in duplicate and was compared to a standard curve using sera levels of recombinant human IL-2. The lower limit of detection in this assay is 75 pg/ml. Minimal cross-reactivity with mouse IL-2 was observed.
  • NK Natural Killer
  • YAC-1 an NK-sensitive Moloney murine leukemia virus-induced mouse lymphoma cell line
  • P815 an NK-resistant murine mastocytoma cell line
  • 51 Cr-labeled target cells were added to wells of round-bottom 96-well microtiter plates, and effector cells were plated in triplicate to yield various effector to target (E/T) ratios.
  • Nonadherent mouse PBL's (effector cells) were isolated by Lymphocyte M
  • Results are expressed in FIGURE 12 as percent specific 51 Cr release as calculated from the following formula: 100 X [(mean cpm experimental - mean cpm spontaneous release) (mean cpm total release - mean cpm spontaneous release)]. Values shown are mean +. SEM for all samples at a specified E/T ratio, and at the time point indicated. Spontaneous release was measured after addition of 2% SDS. The results of this assay confirm natural killer activity against lymphoblastic cells in AKR/J mice which received the injections of plasmid. This activity would normally be substantially absent in these mice. EXAMPLE VI
  • mice Using Balb/c mice and the plasmids described in Example V, pRSVIL2 in 0.9% saline was injected into separate mice weekly at dosages of, respectively, 0 (i.e., pRSV only), 5, 12.5, 25, 50 and 100 ⁇ g per injection. For each dosage, three younger (about 16 weeks) and three older (16 months old or older) mice are injected in the right hind leg muscle.
  • FIGURES 13 (a)-(b) These data are shown in FIGURES 13 (a)-(b). The weight and physical appearance of the animals were also monitored for signs of toxicity. Some toxicity was apparent at the 50 and 100 ⁇ g dosage levels tested.
  • mice Groups of young Balb/c mice were injected at weekly intervals with naked pRSVIL2 either (1) subcutaneously (back), (2) intramuscularly (right hind leg), (3) intradermally (base of the tail), or (4) intranasally.
  • the plasmids used were as described in Example V.
  • the data regarding the levels of systemic expression achieved from administra ⁇ tion of naked pRSVIL2 via each of these routes are shown in FIGURE 14.
  • IL-2 levels were therefore correlated with natural killer activity. Both values increased significantly compared with the values from the control-injected group, which increase was experienced during the same time interval (0 to 3 months of injection). At the same time, no changes in the levels of peripheral lymphocytes or granulocytes were detected by analysis of blood smear slides in the treated animals. This finding is significant because it indicates that no inflammation occurred after injections and that subsets of the immune system other than NK cell population were unaffected by the levels of IL-2 expression after injections.
  • mice contained ten aged ICR (an outbred mouse strain without inherent pathology) mice. Animals were injected with pRSV or pRSV-IL- 2 using the same protocol as the AKR animals in the preceding examples. The relative white blood cell counts in these animals (0 to 4 months after injection) was measured. The results are shown in Table II below: TABLE III Relative White Blood Cell Count
  • NK cell activity was measured with a 51 Cr-release assay as described in Example V.
  • Target cells were either 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:
  • mice Three days after intradermal injection of the tails of naked pCMV/acz into Balb/c mice, the mice were sacrificed. Tissue cultures were obtained at the point of entry for the plasmid and stained for E. coli ⁇ -galactosidase activity. A photograph (40x magnification) of a slide from the histological examination of these cultures is contained in FIGURE 15.
  • uptake of the plasmid is shown (in blue) to be by mononuclear cells.
  • the fibroblasts in the tissue samples are not stained, thus indicating that the plasmid was not taken up by these cells.
  • the rounded, mononuclear cells which did take up the plasmid appear to be macrophages and/or other antigen presenting cells, which would indicate that uptake of the plasmid is by phagocytosis.
  • FIGURE 16 depicts the results of an ELISA performed as described in Example I for serum levels of anti-NP IgG following epidermal administration of pCMVRNP via mechanical means.
  • the plasmid was coated onto the tynes of an uncoated MONO-VACC device as described supra. (It should be noted that it is alternatively possible for the naked polynucleotides to be lyophilized onto the tynes of the device for longer storage stability).
  • Total plasmid concentration on all of the device tynes was approximately 50 ⁇ g in an isotonic normal saline carrier (approximately 150 ⁇ g plasmid per milliliter).
  • the back of a Balb/c mouse was shaved and the shaved skin gently scratched with the tyne device. As shown in FIGURE 16, anti-NP IgG were subsequently detected in serum (e.g., at day 42, the serum from this mouse contained antibodies at a titer of 1 :10240).
  • FIGURE 17 depicts the results of an ELISA performed as described in Example I for serum levels of anti-NP IgG following epidermal administration of pCMVRNP in conjunction with the application of a chemical agent.
  • the plasmid was suspended in 40 ⁇ g of an isotonic normal saline solution containing approximately 150 ⁇ g of plasmid per milliliter. This solution was absorbed onto the nonadhesive pad of a BAND-AID brand bandage (Johnson & Johnson).
  • Example X A Balb/c mouse was shaved as described in Example X and a commercially available keratinolytic agent (here, the previously described depilatory creme sold under the tradename NAIR) was applied to the shaved skin. After several minutes, the keratinolytic agent was washed off of the skin and the plasmid- containing bandage applied thereto. As shown in FIGURE 17, the treated animal developed serum anti-NP IgG at a titer of 1 :640. EXAMPLE XII
  • Example VII Stability of IL-2 expression was measured in the mice described with respect to Example VII. After two weekly injections of pRSV-IL2, serum levels of IL-2 were measured as described in Example V. In older mice, serum levels of IL-2 declined more slowly than in younger mice. These data are shown in FIGURE 18 and demonstrate that relatively stable gene expression can be achieved via introduction of naked polynucleotides to tissues having a relatively high concentration of APC's therein, particularly as compared to muscle tissue.
  • mice were injected intradermally 3 times with 15 ⁇ g of a pCMVRNP plasmid which contained the NP gene from an H1 N1 strain of influenza virus (A/PR/8/34; provided by Dr. Inocent N. Mbawvike at the Baylor College of Medicine, U.S.)
  • Control groups included uninjected animals as well as animals injected with an irrelevant plasmid (pnBL3).
  • pCMVRNP cytomegalovirus immediate early promoter, enhancer and intron region.
  • the other plasmid contained the promoter from the Rous sarcoma virus LTR region (pRSVRNP). As shown in
  • FIGURE 20 antibody responses to the NP protein expressed by the plasmids were consistently higher with the CMV promoter after intradermal injections. This contrast with the responses seen after intramuscular injection of the NP gene, where antibody levels produced by the two plasmids are essentially equivalent (data not shown).
  • mice of the C57/B6 strain were injected intradermally in the tail at two week intervals with 100 ⁇ g naked DNA purified from a CDM8 ova plasmid (described in detail in Shastri, et al., J.lmmunol., 150:2724-2736, 1993).
  • the CDM8 ova plasmid contains the full length (1.8 kb) cDNA for ovalbumin.
  • mice 2 weeks after the second gene adminstration, the spleens of the mice wre removed and cultured in vitro with lethally irradiated (3000 rad) syngeneic splenocytes that had been pulsed with a synthetic ovalbumin peptide.
  • This peptide is a class I restricted target for cytotoxic T cells in mice with the histocompatibility haplotype K described by Shastri, et al..
  • the cells were incubated with targets of 2 types to test for the generation of cytotoxic T cells by the mice who had received the gene encoding ovalbumin.
  • the targets were mouse EL-4 lymphocytes pulsed with the synthetic ovalbumin peptide, or EL-4 cells that had been stably transfected with the cDNA for ovalbumin (see. FIGURE 21 ; the cDNA for ovalbumin is designated as "EG7" in the FIGURE).
  • the percent lysis of the 2 targets was determined for different effector-to-target ratios (designated as "E:T ratio" in FIGURE 21).
  • the animals that received the naked CDM8 ova plasmid had produced cytotoxic T cells that were specific for the ovalbumin targets (i.e., for EL-4 with the ovalbumin peptide and for EG7), but were not specific for the control EL-4 cells (i.e., those without the ovalbumin peptide).
  • IM intramuscularly
  • ID intradermally
  • PR /3-galactosidase protein
  • Serum samples were serially diluted in BBS starting at a 1 :40 dilution for the first 8 weeks, them a 1 :320 dilution thereafter. These samples were added to the plates and stored overnight at room temperature. Plates were washed in BBS+0.05% polysorbate 20, then reacted with a 1 :2000 dilution of alkaline phosphatase labeled goat anti-mouse IgG antibody (Jackson Immunoresearch Labs., West Grove, PA) for 1 hour at room temperature, or were reacted with a 1:2000 dilution of alkaline phosphatase labeled goat anti-mouse IgG 1 antibody (Southern Biotech of AL), or were reacted with a 1 :500 dilution of alkaline phosphatase labled rat anti-mouse IgG 2A antibody (Pharmingen, of CA), under the same conditions.
  • antibody responses of equivalent magnitude were induced in the animals who had received the pCMV Lac-Z plasmids by ID injection and the amimals who had received the PR, while lesser antibody responses were measured in the animals who had received the pCMV Lac-Z plasmids by IM injection.
  • the animals were then boosted with 0.5 ⁇ g of PR at a separate site by ID injection. If these animals had developed memory T cells to control production of antibody to ⁇ -galactosidase, they would be expected to mount a more vigorous immune response after boosting with soluble protein antigen than had been demonstrated in response to the priming dose of antigen.
  • IgG 2A antibodies are serological markers for a TH1 type immune response, whereas IgG 1 antibodies are indicative of a TH2 type immune response.
  • TH2 responses include the allergy-associated IgE antibody class; soluble protein antigens tend to stimulate relatively strong TH2 responses.
  • TH1 responses are induced by antigen binding to macrophages and dendritic cells. TH1 responses are to be of particular importance in the treatment of allergies and AIDS.
  • mice were vaccinated with pCMV Lac-Z or protein as described in the preceding example.
  • any IgG 2a and IgG 1 to ⁇ -galactosidase were measured by enzyme-linked immunoabsorbent assay (using antibodies specific for the IgG 1 and IgG 2A subclasses) on microtiter plates coated with the enzyme.
  • mice who received the plasmid by ID injection produced high titers of IgG 2A antibodies.
  • immunization of the mice with the enzyme itself (“PR") induced production of relatively high titers of IgG 1 antibodies.
  • PR enzyme itself
  • low titers of both IgG 2A and IgG 1 antibodies were produced without apparent selectivity.
  • the data shown in the FIGURES comprise averages of the values obtained from each group of 4 mice.
  • Example XVII The experiments described in Example XVII were repeated in separate groups of mice, except that (1) only a priming dose was tested, and (2) the pCMV Lac-Z plasmid was administered to one group of 4 mice using the tyne device described in Example X, while s-galactosidase protein (10 ⁇ g) was adminis- tered to another group of 4 mice by intradermal (ID) injection.
  • ID intradermal
  • mice who received plasmid produced relatively low titers of IgG 1 antibody compared to the mice who received the protein.
  • the mice who received plasmid produced substantially higher titers of IgG 2A antibody as compared to the mice who received the protein.
  • mice who received the plasmid via scratching of their skin with the tyne device produced even higher titers of IgG 2A antibody than did the mice who received the same plasmid via ID injection (both of which groups produced higher titers of IgG 2A antibody than did the mice who received the plasmid via IM injection).
  • scratching of skin with the tyne device attracts greater number of APC's to the "injured" point of entry for the naked polynucleotides and are consistent with the theory that APC's are more efficient targets for gene administration and expression than are muscle or other somatic cells.
  • the data shown in the FIGURES comprise averages of the values obtained from each group of 4 mice.
EP94926603A 1993-08-26 1994-08-25 Methode, zusammensetzungen und vorrichtungen zur verabreichung von nackten polynukleotiden, welche für biologisch aktive peptide kodieren Withdrawn EP0714308A4 (de)

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