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WO1996020731A1 - Administration directe en plusieurs sites de vehicules distributeurs de genes - Google Patents

Administration directe en plusieurs sites de vehicules distributeurs de genes Download PDF

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Publication number
WO1996020731A1
WO1996020731A1 PCT/US1995/016471 US9516471W WO9620731A1 WO 1996020731 A1 WO1996020731 A1 WO 1996020731A1 US 9516471 W US9516471 W US 9516471W WO 9620731 A1 WO9620731 A1 WO 9620731A1
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Prior art keywords
gene delivery
virus
antigen
substance
immune response
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PCT/US1995/016471
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English (en)
Inventor
Michael J. Irwin
John F. Warner
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Chiron Viagene, Inc.
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Priority to AU47400/96A priority Critical patent/AU4740096A/en
Publication of WO1996020731A1 publication Critical patent/WO1996020731A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • 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
    • 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
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates generally to the administration of gene delivery vehicles to warm-blooded animals at multiple sites, in order to stimulate an immune response in the animal.
  • bacterial diseases are, in general, easily treatable with antibiotics, very few effective treatments or prophylactic measures exist for many viral, cancerous and other nonbacterial diseases.
  • Traditional attempts to treat these diseases have employed the use of chemical drugs. In general, these drugs have lacked specificity and have exhibited high overall toxicity.
  • Another classic technique for treating a number of nonbacterial diseases involves the elicitation of an immune response to a pathogenic agent, such as a virus, through the administration of a nonpathogenic form of the agent, such as a killed virus, thereby providing antigens from the pathogenic agent which would confer specific immunity.
  • a more recent approach for treating viral diseases involves blocking receptors on cells susceptible to infection by HTV from receiving or forming a complex with viral envelope proteins.
  • AIDS acquired immunodeficiency syndrome
  • Lifson et al. Science 232:1123-1121, 1986
  • T4 CD4
  • Syncytia cell fusion
  • McDougal et al. Science 257:382-385, 1986
  • Pert et al. Proc. Natl. Acad Sci.
  • Another recently suggested technique for inhibiting a pathogenic agent, such as a virus, which transcribes RNA is to provide antisense RNA, which is complementary to and binds at least a portion of the transcribed RNA, so as to inhibit translation (To et al., Mol. Cell. Biol. 6:758, 1986).
  • antisense RNA which is complementary to and binds at least a portion of the transcribed RNA, so as to inhibit translation
  • an immunostimulant expressed in increased amounts immediately following infection by the pathogenic agent may be desirable to have an immunostimulant expressed in increased amounts immediately following infection by the pathogenic agent.
  • antisense RNA large amounts would be required for useful therapy in an animal, which, given current techniques, is extremely difficult to administer to the location at which it is actually needed, that is, at the cells infected by the pathogenic agent.
  • proteins have been expressed from viral vectors based on DNA viruses, such as adenovirus, simian virus 40, bovine papilloma virus, and vaccinia virus.
  • DNA viruses such as adenovirus, simian virus 40, bovine papilloma virus, and vaccinia virus.
  • Panicali et al. Proc. Natl. Acad Sci. USA #0:5364, 1983
  • Each of the commonly used vector systems based on DNA viruses have also presented particular problems, which limit their usefulness as gene delivery vehicles. For example, because of the ubiquitous nature of adenoviruses, rescue of a wild-type adenovirus by recombination is a possibility. As well, prevalent preexisting immunity potentially renders an individual refractory to infection or may lead to a seriously damaging immune response. In this regard, a recent cystic fibrosis patient receiving therapy using an adenovirus vector presented with a damaging inflammatory response. Likewise, vaccinia virus is a highly immunogenic virus, making repeated treatments with a vaccinia virus vector difficult.
  • vectors include naked DNA, liposome-assisted gene transfer, and polycation-assisted gene transfer. Although obviating some of the problems outlined above associated with DNA virus vectors, these physical gene transfer vectors suffer from their own set of difficulties. Limitations affecting their usefulness include transient expression of introduced genes, a low efficiency of transfection or the inability to transfect a large number of cells, manufacturing complexity making large-scale production problematic, and the elicitation of an immune response to various components of the complexes.
  • retroviruses Due to the nontransitory nature of their expression in infected target cells, retroviruses have been suggested as a useful vehicle for the treatment of genetic diseases (for example, see F. Ledley, The Journal of Pediatrics 110:1, 1987).
  • long- term expression from retroviral vectors which is necessary for the treatment of genetic diseases, has been demonstrated for factor DC (Dai et al., Proc. Natl. Acad Sci. USA 59:10892, 1992) adenosine deaminase (Van Beusechem et al., Proc. Natl. Acad. Sci. USA 59:7640, 1992; Blaese, Pediatr. Res.
  • the present invention is directed toward methods of stimulating an immune response, either humoral or cell-mediated, through the administration to a warm-blooded animal at multiple sites of one or more gene delivery vehicles, each gene delivery vehicle directing the expression of at least one substance in host cells modified (i.e., transduced, transfected or transformed) with the vehicle, such that an immune response is generated.
  • the present invention provides gene delivery vehicles which direct the expression of at least one antigen or modified form thereof in host cells infected or transfected with the gene delivery vehicle, the antigen or modified form thereof being capable of stimulating an immune response within an animal.
  • the expressed substance elicits a cell-mediated immune response, preferably an HLA class I-restricted immune response.
  • the gene delivery vehicle preferably directs the expression of a substance which will elicit both an HLA class I- and class II- restricted immune response.
  • the antigen is preferably selected from gp 160, gp 120 and gp 41, which have been modified to reduce their pathogenicity.
  • the antigen may be modified to reduce the possibility of syncytia, to avoid expression of epitopes leading to a disease enhancing immune response, and to remove immunodominant, but strain-specific epitopes, and yet allow an immune response capable of eliminating cells infected with most or all strains of HIV (see WO 91/02805).
  • the gene delivery vehicle preferably directs the expression of at least one immunogenic portion of a feline immunodeficiency virus antigen.
  • Suitable antigens in this regard include pl5gag, p24gag, plOgag, pl3pol, p62pol, pl5pol and p36pol.
  • gene delivery vehicles are provided which direct the expression of gp68env, gp27env and rev.
  • "rev” is understood to refer to the antigen corresponding to the rev open reading frame (see Phillips et al., First International Conference of Feline Immunodeficiency Virus researchers, University of California, Davis, CA, September 4-7, 1991).
  • the gene delivery vehicle preferably directs the expression of at least one immunogenic portion of an FeLV antigen selected from pl5gag, pl2gag, p27gag, plOgag, pl4pol, p80pol, p46pol, gp70env and pl5env.
  • gene delivery vehicles are provided which direct the expression of gp85env.
  • the gene delivery vehicle preferably directs the expression of HBeAg, HBcAg, HBsAgs, ORF 5, ORF 6, the HBV pol antigen, or any combination of these antigens (e.g., HBeAg and HBcAg).
  • the HBsAgs are selected from the group consisting of S, pre-Sl, and pre-S2.
  • gene delivery vehicles are provided which direct the expression of an immunogenic portion of antigen X, or co- express this antigen with an immunomodulatory factor.
  • the gene delivery vehicle directs the expression of the hepatitis C core antigen, antigen El, antigen E2/NS1, antigen NS2, antigen NS3, antigen NS4, antigen NS5, or combinations thereof.
  • gene delivery vehicles are provided which direct the co-expression of at least one immunogenic portion of a hepatitis B antigen and at least one immunogenic portion of a hepatitis C antigen.
  • gene delivery vehicles are provided which direct the expression of an immunogenic portion of a polyprotein antigen, or co-express this antigen with an immunomodulatory factor.
  • altered cellular component refers to proteins and other cellular constituents which are either associated with rendering a cell tumorigenic, or are associated with tumorigenic cells in general, but are not required or essential for rendering the cell tumorigenic.
  • the cellular components may be essential to normal cell growth and regulation, and include for example, proteins which regulate intracellular protein degradation, transcriptional regulation, cell-cycle control, and cell-cell interaction. After alteration, the cellular components no longer perform their regulatory functions, and hence the cell may experience uncontrolled growth.
  • the cellular component may be altered by a point mutation, by a deletion, or by a chromosomal translocation.
  • the altered cellular components include, ras , p53 , Rb , altered protein encoded by the Wilms' tumor gene, ubiquitin , mucin , DCC, APC, MCC, neu, an altered receptor, or polypeptides resulting from chromosomal translocations such as bcr/abl.
  • non-tumorigenic altered cellular components are provided, including for example, ⁇ ras ⁇ , ⁇ ras ⁇ , and ⁇ ras 61.
  • gene delivery vehicles which direct the expression of several altered cellular components, including, for example, a gene delivery vehicle which directs the expression of both ras and p53 , or a gene delivery vehicle which directs the expression or ras*, mucin*, and DCC.
  • gene delivery vehicles which direct the expression of at least one immunogenic non-tumorigenic form of a normal cell component associated with the tumorigenic state (i.e., a tumor associated antigen or TAA).
  • a normal cell component associated with the tumorigenic state i.e., a tumor associated antigen or TAA.
  • Suitable components in this regard include antigens such as MART 1, MAGE 1 and 3, and tyrosine hydroxylase.
  • the gene delivery vehicles can be used to specifically immunosuppress certain responses by the immune system.
  • T cell activation requires the interaction of the antigen specific T-cell receptor with antigen presented on the surface of antigen presenting cells (APC) and secondary signals delivered by the interaction of costimulatory molecules on the surface of T-cells (CD4, CD8, LFA-1, CD2, CD3, CD28, CTLA4, etc.) and APC (MHC ⁇ , MHC I, ICAM-1, ICAM-2, LFA-3, B7.1, B7.2, B7.3, etc.).
  • APC antigen presenting cells
  • a gene delivery vehicle encoding an allergen such as casein, glutin, bee venom or albumin, an autoantigen such as myelin basic protein or pancreatic ⁇ cells or a foreign antigen present on donor graft tissue such as MHC encoded alloantigens and antisense and/or ribozyme sequences for down-regulation of B7.1, B7.2, and B7.3 is administered at multiple sites to induce a specific immunosuppression to a particular antigen.
  • an allergen such as casein, glutin, bee venom or albumin
  • an autoantigen such as myelin basic protein or pancreatic ⁇ cells
  • a foreign antigen present on donor graft tissue such as MHC encoded alloantigens and antisense and/or ribozyme sequences for down-regulation of B7.1, B7.2, and B7.3 is administered at multiple sites to induce a specific immunosuppression to a particular antigen.
  • gene delivery vehicles encoding an allergen, an autoantigen or a foreign antigen present on donor graft tissue may be co-injected with a second gene delivery vehicle encoding soluble CTLA4, CD28, or B7.1-.3 in order to induce specific immunosuppression to a particular antigen.
  • gene delivery vehicles including for example viral vectors, nucleic acid vectors with or without transfection enhancers such as local anesthetics, liposomes or polycation condensing agents, and bacteria.
  • Suitable viral vectors include recombinant retroviruses or a recombinant virus selected from the group consisting of poliovirus, rhinovirus, pox virus (e.g., the canary pox virus or the vaccinia virus), influenza virus, adenovirus, parvovirus (e.g., the adeno-associated virus, B19 or MVN), herpes virus, SV40, HIV, measles, alpha viruses such as Sindbis virus or corona virus.
  • pox virus e.g., the canary pox virus or the vaccinia virus
  • influenza virus e.g., the canary pox virus or the vaccinia virus
  • adenovirus e.g., the adeno-associated virus, B19 or MVN
  • herpes virus e.g., the adeno-associated virus, B19 or MVN
  • herpes virus SV40
  • HIV measles
  • alpha viruses such as Sindbis virus
  • the gene delivery vehicle may be administered at multiple sites to a patient directly, for example by direct injection, or alternatively, through the use of target cells transduced ex vivo.
  • the present invention also provides pharmaceutical compositions (including, for example, various adjuvants) suitable for administering the gene delivery vehicles.
  • Figure 1A is a graph depicting the results of a CTL assay performed on animals injected with a representative gene delivery vehicle at multiple sites.
  • Figure IB is a graph depicting the results of a CTL assay comparable to Figure 1 A performed on animals injected with the same representative gene delivery vehicle at a single site.
  • Figure 2A is a graph depicting the results of a CTL assay performed on animals injected with a representative gene delivery vehicle at multiple sites.
  • Figure 2B is a graph depicting the results of a CTL assay comparable to Figure 2A performed on animals injected with the same representative gene delivery vehicle at a single site.
  • Immunogenic portion refers to a portion of the respective antigen which is capable, under the appropriate conditions, of causing an immune response (i.e., cell-mediated or humoral).
  • "Portions” may be of variable size, but are preferably at least 8 amino acids long, and may include the entire protein molecule. Representative assays which may be utilized to determine immunogenicity (e.g., cell-mediated immune response), are described in more detail below.
  • Cell mediated immune responses may be mediated through Major Histocompatability Complex ("MHC") class I presentation, MHC class ⁇ presentation, or both.
  • MHC Major Histocompatability Complex
  • Gene delivery vehicle refers to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest.
  • the gene delivery vehicle will generally include promoter elements and may include a signal that directs polyadenylation.
  • the gene delivery vehicle includes a sequence which, when transcribed, is operably linked to the sequence(s) or gene(s) of interest and acts as a translation initiation sequence.
  • the gene delivery vehicle may also include a selectable marker such as Neo, SV2 Neo, TK, hygromycin, phleomycin, histidinol, or DHFR, as well as one or more restriction sites and a translation termination sequence.
  • the gene delivery vehicle is a retrovirus
  • a packaging signal and long terminal repeats (LTRs) appropriate to the retrovirus used will be included(if these are not already present).
  • the gene delivery vehicle is a Sindbis virus
  • the vehicle RNA will include a 5' sequence which is capable of initiating transcription, as well as sequences which, when expressed, code for biologically active Sindbis non-structural proteins (i.e., NS1, NS2, NS3, and NS4).
  • the Sindbis gene delivery vehicle should include a Sindbis RNA polymerase recognition sequence, and a viral junction region, which may, in certain embodiments, be modified in order to either prevent or inhibit viral transcription of the subgenomic fragment.
  • the gene delivery vehicle may also include nucleic acid molecule(s) which are of a size sufficient to allow production of viable virus, as well as one or more restriction sites.
  • nucleic acid molecule(s) which are of a size sufficient to allow production of viable virus, as well as one or more restriction sites.
  • ELVS eucaryotic layered vector system
  • the Sindbis vector genome is transfected as a DNA molecule to give a eucaryotic layered vector system (ELVS)
  • ELVS eucaryotic layered vector system
  • it should additionally include a 5' RNA polymerase II promoter which is capable of initiating the synthesis of viral RNA from cDNA, and a 3' sequence which controls transcription termination and splice recognition.
  • Immunomodulatory factor refers to factors which, when manufactured by one or more of the cells involved in an immune response, or, which when added exogenously to the cells, causes the immune response to be different in quality or potency from that which would have occurred in the absence of the factor.
  • the quality or potency of a response may be measured by a variety of assays known to one of skill in the art including, for example, in vitro assays which measure cellular proliferation (e.g., -1H thymidine uptake), and in vitro cytotoxic assays (e.g., which measure 51 - release) (see, Warner et al., AIDS Res. and Human Retroviruses 7:645-655, 1991).
  • Immunomodulatory factors may be active both in vivo and ex vivo.
  • Representative examples of such factors include cytokines, such as interleukins 2, 4, 6, 12 and 15 (among others), alpha interferons, beta interferons, gamma interferons, GM-CSF, G-CSF, and tumor necrosis factors (TNFs).
  • Other immunomodulatory factors include, for example, CD3, ICAM-1, ICAM-2, LFA-1, LFA-3, MHC class I molecules, MHC class II molecules, B7.1-.3, b2-microglobulin, chaperones, or analogs thereof.
  • immunomodulatory factors can be produced in a number of known ways (Ellis and Gerety, J. Med Virol. 57:54-58, 1990), including chemical synthesis (Bergot et al., Applied Biosystems Peptide Synthesizer User Bulletin No. 16, 1986, Applied Biosystems, Foster City, California) and DNA expression in recombinant systems, such as the insect-derived baculovirus system (Doerfler, Current Topics in Immunology 757:51-68, 1986), mammalian-derived systems (such as CHO cells) (Berman et al., J. Virol.
  • the present invention is directed toward the administration of gene delivery vehicles at multiple sites in order to stimulate an immune response, preferably a cell-mediated immune response.
  • an immune response preferably a cell-mediated immune response.
  • the ability to recognize and defend against foreign pathogens is central to the function of the immune system. This system, through immune recognition, is capable of distinguishing "self from "non-self (foreign) and attacking non-self, which is essential to ensure that defensive mechanisms are directed towards invading entities rather than against host tissues.
  • TCR clonotypic T cell receptors
  • MHC self major histocompatibility complex
  • each T cell has specificity for one peptide antigen displayed on one, self MHC molecule.
  • a second protective mechanism is that T and B cells of the immune system demonstrate long-term memory after encountering antigen, which is the principle of vaccination. These memory lymphocytes respond more rapidly and effectively to a second antigen encounter than the primary exposure.
  • the phrase "multiple sites” is equivalent to two or more sites.
  • at least two of the sites are associated with or involve alternate lymphoid tissues (e.g., different lymph nodes, spleen, tonsils, bone marrow, thymus, liver).
  • alternate lymphoid tissues e.g., different lymph nodes, spleen, tonsils, bone marrow, thymus, liver.
  • the antigen expressed via the gene delivery vehicles generally traffics centrally to the nearest lymph node, following the pathway for lymphatic or blood circulation.
  • antigen injected intramuscular to the deltoid muscle (or any upper arm site) will normally traffic to the axillary lymph nodes, while injection to the thigh or buttocks traffics to the inguinal lymph nodes.
  • intraperitoneal injection should traffic to the mesenteric lymph nodes and the spleen.
  • intravenous injection of gene delivery vehicles should traffic primarily to the spleen, and next to several lymph nodes.
  • Intradermal and subcutaneous injection proceeds similar to intramuscular injections, in that antigen travels centrally to the nearest lymph node. Administration orally or sublingually generally traffics to the mesenteric lymph nodes, while oral, nasal, rectal, and vaginal administration of antigen generally travels to the nearest central lymph node. Given the routing of antigen as described herein, one can readily predict which lymph nodes will be involved.
  • the precise lymph node involved may be confirmed through a lymph node biopsy, preferably in association with a diagnostic method (e.g., fluorescent beads, PCR).
  • a diagnostic method e.g., fluorescent beads, PCR.
  • the gene delivery vehicles are administered immediately following one another up to 12 hours apart, with an interval of less than 1 hour being particularly preferred. Where several different gene delivery vehicles and/or routes of administration are utilized, it will be evident that the timing between administrations need not be uniform, but rather may be staggered or even random within the overall time of administration.
  • Gene delivery vehicles which direct the expression of one or more heterologous nucleotide sequences are provided.
  • Gene delivery vehicles refers to recombinant vehicles, such as viral vectors (Jolly, Cancer Gen. Therapy 7:51-64, 1994), nucleic acid vectors, naked DNA, cosmids, bacteria, and certain eukaryotic cells (including producer cells;), that are capable of eliciting an immune response within an animal.
  • Representative examples of such gene delivery vehicles include poliovinis (Evans et al., Nature 559:385-388, 1989; and Sabin, J. Biol.
  • viral carriers may be homologous, non-pathogenic(defective), replication competent virus (e.g., Overbaugh et al., Science 259:906-910,1988), and nevertheless induce cellular immune responses, including CTL.
  • an immune response may also be generated by administration of a bacterium which expresses the immunogenic portion(s) on its cell surface.
  • a bacterium which expresses the immunogenic portion(s) on its cell surface.
  • Representative examples include BCG (Stover, Nature 557:456-458, 1991), salmonella (Newton et al., Science 244:10-12, 1989) and E. coli. (Cline et al., Pharmac. Ther. 29:69, 1985, and Friedmann et al., Science 244:1215, 1989).
  • Naked DNA or nucleic acid molecules are also suitable for use as gene delivery vehicles within the present invention (WO 90/11092).
  • gene delivery vehicles may be either DNA or RNA and, in certain embodiments, are linked to killed adenovirus (Curiel et al., Hum. Gene. Ther. 5:147-154, 1992).
  • Other suitable vehicles include DNA- ligand (Wu et al., J. Biol. Chem. 264:16985-16987, 1989), lipid-DNA combinations (Feigner et al., Proc. Natl. Acad Sci. USA 84:1413 7417, 1989), liposomes (Wang et al., Proc. Natl. Acad Sci. 54:7851-7855, 1987) and microprojectiles (Williams et al., Proc. Natl. Acad Sci. 55:2726-2730,1991).
  • This approach takes advantage of the fact that latex beads, when incubated with cells in culture, are efficiently transported and concentrated in the perinuclear region. The beads will then be transported into cells when injected into muscle. DNA coated latex beads will be efficiently transported into cells after endocytosis is initiated by the latex beads, and thus increase the gene transfer and expression efficiency of this method.
  • This method may be improved further by treatment of the beads to increase their hydrophobicity and thereby facilitate the disruption of the endosome and release of the DNA into the cytoplasm.
  • ex vivo procedures may be used in which cells are removed from an animal, modified, and placed into the same or another animal.
  • Cells that can be modified include, but are not limited to, fibroblasts, endothelial cells, hepatocytes, epithelial cells, lymphocytes, keratinocytes, thyroid follicular cells and bone marrow cells. It will be evident that one can utilize any of the gene delivery vehicles noted above for introduction into tissue cells in an ex vivo context. Protocols for physical and chemical methods of uptake include calcium phosphate precipitation, direct microinjection of DNA into intact target cells, and electroporation.
  • MHC-matched animals Although not always, see, e.g., Yamamoto et al., "Efficacy of Experimental FIV Vaccines," 1st International Conference of FIV researchers, University of California at Davis, September 1991).
  • a variety of animal cells may be utilized within the context of the present invention, including for example, human, macaque, dog, rat, and mouse cells.
  • Cells may be removed from a variety of locations including, for example, from a selected tumor.
  • the above-described methods may additionally comprise the steps of depleting fibroblasts or other non-contaminating tumor cells subsequent to removing tumor cells from a warm-blooded animal, and/or the step of inactivating the cells, for example, by irradiation.
  • a gene delivery vehicle may be inserted into non-tumorigenic cells, including for example, cells from the skin (dermal fibroblasts), or from the blood (e.g., peripheral blood leukocytes).
  • T cell subset or stem cells may also be specifically removed from the blood (see, for example, PCT WO 91/16116, entitled “Immunoselection Device and Method”).
  • Vector constructs may then be used to modify the removed cells utilizing any of the above-described techniques, followed by return of the cells to the warm-blooded animal.
  • the gene delivery vehicle is a retrovirus.
  • Retroviruses are RNA viruses with a single positive strand genome which, in general, are nonlytic. Upon infection, the retrovirus reverse transcribes its RNA into DNA, forming a provirus which is inserted into the host cell genome.
  • Preparation of retroviral constructs for use in the present invention is described in greater detail in an application entitled "Recombinant Retroviruses", herein incorporated by reference.
  • the retroviral genome can be divided conceptually into two parts.
  • the "trans-acting" portion consists of the region coding for viral structural proteins, including the group specific antigen (gag) gene for synthesis of the core coat proteins; the pol gene for the synthesis of the reverse transcriptase and integrase enzymes; and the envelope (env) gene for the synthesis of envelope glycoproteins.
  • the "cis-acting" portion consists of regions of the genome that is finally packaged into the viral particle. These regions include the packaging signal, long terminal repeats (LTR) with promoters and polyadenylation sites, and two start sites for DNA replication.
  • LTR long terminal repeats
  • the internal or "trans-acting" part of the cloned provirus is replaced by the gene of interest to create a "vector construct".
  • the vector construct When the vector construct is placed into a cell where viral packaging proteins are present (see WO 92/05266), the transcribed RNA will be packaged as a viral particle which , in turn, will bud off from the cell. These particles are used to transduce tissue cells, allowing the vector construct to integrate into the cell genome. Although the vector construct expresses its gene product, the virus carrying it is replication defective because the trans-acting portion of the viral genome is absent.
  • Various assays may be utilized in order to detect the presence of any replication competent infectious retrovirus.
  • One preferred assay is the extended S ⁇ " assay.
  • Numerous retroviral gene delivery vehicles may be utilized within the context of the present invention, including for example those disclosed within EP 415,731; WO 90/07936; WO 91/0285, WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No. 5,219,740; WO 93/11230; WO 93/10218; Vile and Hart, Cancer Res. 55:3860-3864, 1993; Vile and Hart, Cancer Res. 55:962-967, 1993; Ram et al., Cancer Res. 55:83-88, 1993; Takamiya et al., J. Neurosci. Res. 55:493-503, 1992; Baba et al., J.
  • retroviral vectors include murine leukemia amphotropic or xenotropic, or VsVg pseudotype vectors (see, WO 92/14829, incorporated herein by reference).
  • Retroviral gene delivery vehicles of the present invention may be readily constructed from a wide variety of retroviruses including, for example, B-, C-, and D-type retroviruses, as well as spumaviruses and lentiviruses (see, RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985). Briefly, viruses are often classified according to their morphology as seen under electron microscopy. Type "B” retroviruses appear to have an eccentric core, while type “C” retroviruses have a central core. Type “D” retroviruses have a morphology intermediate between type B and type C retroviruses.
  • retroviruses include a variety of xenotropic retroviruses (e.g., NZB-X1, NZB-X2, and NZB9.1 (see, O'Neill et al., J Vir. 55:100-106, 1985)) and polytropic retroviruses (e.g., MCF and MCF-MLV (see, Kelly et al., J. Vir. 45(l):291-298, 1983)).
  • xenotropic retroviruses e.g., NZB-X1, NZB-X2, and NZB9.1
  • polytropic retroviruses e.g., MCF and MCF-MLV (see, Kelly et al., J. Vir. 45(l):291-298, 1983)
  • retroviruses may be readily obtained from depositories or collections such as the American Type Culture Collection ("ATCC"; Rockville, Maryland), or isolated from known sources using commonly available techniques.
  • ATCC American Type Culture Collection
  • retroviruses for the preparation or construction of retroviral gene delivery vehicles of the present invention include retroviruses such as Avian Leukosis Virus, Bovine Leukemia Virus, Murine Leukemia Virus, Mink-Cell Focus- Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis Virus, and Rous Sarcoma Virus.
  • retroviruses such as Avian Leukosis Virus, Bovine Leukemia Virus, Murine Leukemia Virus, Mink-Cell Focus- Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis Virus, and Rous Sarcoma Virus.
  • Particularly preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe, J. Virol. 19:19-25, 1976), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC No.
  • Rous Sarcoma Viruses include Bratislava, Bryan high titer (e.g., ATCC Nos. VR-334, VR-657, VR-726, VR-659, and VR-728), Bryan standard, Carr-Zilber, Engelbreth-Holm, Harris, Prague (e.g., ATCC Nos. VR-772 and 45033), and Schmidt-Ruppin (e.g. ATCC Nos. VR-724, VR-725, VR-354).
  • retroviral gene delivery vehicles Any of the above retroviruses may be readily utilized in order to assemble or construct retroviral gene delivery vehicles given the disclosure provided herein, and standard recombinant techniques (e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Kunkle, PNAS 52:488, 1985).
  • portions of the retroviral gene delivery vehicles may be derived from different retroviruses.
  • retrovector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.
  • retrovector constructs comprising a 5' LTR, a tRNA binding site, a packaging signal, one or more heterologous sequences, an origin of second strand DNA synthesis and a 3' LTR, wherein the vector construct lacks gag/pol or env coding sequences.
  • LTRs Long Terminal Repeats
  • U5 Long Terminal Repeats
  • U3 Long Terminal Repeats
  • These elements contain a variety of signals which are responsible for the biological activity of a retrovirus, including for example, promoter and enhancer elements which are located within U3.
  • LTRs may be readily identified in the provirus due to their precise duplication at either end of the genome.
  • a 5' LTR should be understood to include a 5' promoter element and sufficient LTR sequence to allow reverse transcription and integration of the DNA form of the vector.
  • the 3' LTR should be understood to include a polyadenylation signal, and sufficient LTR sequence to allow reverse transcription and integration of the DNA form of the vector.
  • the tRNA binding site and origin of second strand DNA synthesis are also important for a retrovirus to be biologically active, and may be readily identified by one of skill in the art.
  • retroviral tRNA binds to a tRNA binding site by Watson-Crick base pairing, and is carried with the retrovirus genome into a viral particle.
  • the tRNA is then utilized as a primer for DNA synthesis by reverse transcriptase.
  • the tRNA binding site may be readily identified based upon its location just downstream from the 5* LTR.
  • the origin of second strand DNA synthesis is, as its name implies, important for the second strand DNA synthesis of a retrovirus. This region, which is also referred to as the poly-purine tract, is located just upstream of the 3' LTR.
  • certain preferred retrovector constructs which are provided herein also comprise a packaging signal, as well as one or more heterologous sequences, each of which is discussed in more detail below.
  • retrovector constructs which lack both gag/pol and env coding sequences.
  • the phrase "lacks gag/pol or env coding sequences" should be understood to mean that the retrovector does not contain at least 20, preferably at least 15, more preferably at least 10, and most preferably at least 8 consecutive nucleotides which are found in gag/pol or env genes, and in particular, within gag/pol or env expression cassettes that are used to construct packaging cell lines for the retrovector construct.
  • retrovector constructs which lack gag/pol or env sequences may be accomplished by preparing vector constructs which lack an extended packaging signal.
  • extended packaging signal refers to a sequence of nucleotides beyond the minimum core sequence which is required for packaging, that allows increased viral titer due to enhanced packaging.
  • the minimum core packaging signal is encoded by the sequence beginning from the end of the 5' LTR up through the Pst I site.
  • the extended packaging signal of MoMLV includes the sequence beyond nucleotide 567 up through the start of the gag/pol gene (nucleotide 621), and beyond nucleotide 1560.
  • retrovector constructs which lack extended packaging signal may be constructed from the MoMLV by deleting or truncating the packaging signal prior to nucleotide 567.
  • retrovector constructs are provided wherein the packaging signal that extends into, or overlaps with, retroviral gag/pol sequence is deleted or truncated.
  • the packaging signal is deleted or truncated prior to the start of the gag/pol gene.
  • the packaging signal is terminated at nucleotide 570, 575, 580, 585, 590, 595, 600, 610, 615, or 617.
  • retrovector constructs which include a packaging signal that extends beyond the start of the gag/pol gene.
  • packaging cell lines for the production of recombinant viral particles wherein the 5' terminal end of the gag/pol gene in a gag/pol expression cassette has been modified to contain codons which are degenerate ⁇ or gag.
  • retrovector constructs are provided comprising a 5' LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis and a 3' LTR, wherein the retrovector construct does not contain a retroviral nucleic acid sequence upstream of the 5' LTR.
  • the phrase "does not contain a retroviral nucleic acid sequence upstream of the 5' LTR" should be understood to mean that the retrovector does not contain at least 20, preferably at least 15, more preferably at least 10, and most preferably at least 8 consecutive nucleotides which are found in a retrovirus, and more specifically, in a retrovirus which is homologous to the retrovector construct.
  • the retrovector constructs do not contain a env coding sequence upstream of the 5' LTR.
  • retrovector constructs comprising a 5' LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis and a 3' LTR, wherein the retrovector construct does not contain a retroviral packaging signal sequence downstream of the 3* LTR.
  • packaging signal sequence should be understood to mean a sequence sufficient to allow packaging of the RNA genome.
  • the gene delivery vehicle is a Sindbis viral vector. Briefly, the Sindbis virus is the prototype member of the alphavirus genus of the togavirus family.
  • the unsegmented genomic RNA (49S RNA) of Sindbis virus is approximately 11,703 nucleotides in length, contains a 5' cap and a 3' polyadenylated tail, and displays positive polarity.
  • Infectious enveloped Sindbis virus is produced by assembly of the viral nucleocapsid proteins onto the viral genomic RNA in the cytoplasm and budding through the cell membrane embedded with viral encoded glycoproteins. Entry of virus into cells is by endocytosis through clatharin coated pits, fusion of the viral membrane with the endosome, release of the nucleocapsid, and uncoating of the viral genome.
  • the genomic 49S RNA serves as template for synthesis of the complementary negative strand.
  • This negative strand in turn serves as template for genomic RNA and an internally initiated 26S subgenomic RNA.
  • the Sindbis viral nonstructural proteins are translated from the genomic RNA while structural proteins are translated from the subgenomic 26S RNA. All viral genes are expressed as a polyprotein and processed into individual proteins by post translational proteolytic cleavage.
  • the packaging sequence resides within the nonstructural coding region, therefore only the genomic 49S RNA is packaged into virions.
  • Sindbis vector constructs comprising a 5' sequence which is capable of initiating transcription of a Sindbis virus, a nucleotide sequence encoding Sindbis non-structural proteins, a viral junction region which has been inactivated such that viral transcription of the subgenomic fragment is prevented, and a Sindbis RNA polymerase recognition sequence.
  • the viral junction region has been modified such that viral transcription of the subg ⁇ ao ic fragment is reduced.
  • Sindbis vector constructs comprising a 5' sequence which is capable of initiating transcription of a Sindbis virus, a nucleotide sequence encoding Sindbis non-structural proteins, a first viral junction region which has been inactivated such that viral transcription of the subgenomic fragment is prevented, a second viral junction region which has been modified such that viral transcription of the subgenomic fragment is reduced, and a Sindbis RNA polymerase recognition sequence.
  • Sindbis cDNA vector constructs comprising the above-described vector constructs, in addition to a 5' promoter which is capable of initiating the synthesis of viral RNA from cDNA, and a 3' sequence which controls transcription termination.
  • the vector constructs described above contain no Sindbis structural proteins in the vector constructs the selected heterologous sequence may be located downstream from the viral junction region; in the vector constructs described above having a second viral junction, the selected heterologous sequence may be located downstream from the second viral junction region, where the heterologous sequence is located downstream, the vector construct may comprise a polylinker located between the viral junction region and said heterologous sequence, and preferably the polylinker does not contain a wild-type Sindbis virus restriction endonuclease recognition sequence.
  • Sindbis gene delivery vehicles are utilized, the modified cells are fully viable and healthy, and no other viral antigens (which may well be immunodominant) are expressed.
  • Such an approach may be extended to the expression of a peptide having multiple epitopes, one or more of the epitopes being derived from different proteins.
  • this aspect of the invention allows efficient stimulation of cytotoxic T lymphocytes (CTL) directed against antigenic epitopes, and peptide fragments of antigens encoded by sub-fragments of genes, through intracellular synthesis and association of these peptide fragments with MHC class I molecules.
  • CTL cytotoxic T lymphocytes
  • Sindbis vector constructs as well as numerous similar vector constructs, may be readily prepared essentially as described in U.S. Serial No. 08/198,450, which is incorporated herein by reference in its entirety.
  • the efficiency of packaging and hence, viral titer is to some degree dependent upon the size of the sequence to be packaged.
  • additional non-coding sequences may be added to the gene delivery vehicle.
  • gene delivery vehicles may be constructed to include a promoter such as SV40 (see, Kriegler et al., Cell 55:483, 1984), cytomegalovirus (“CMV”) (see, Boshart et al., Cell 47:521-530, 1991), or an internal ribosomal binding site ("IRBS").
  • a promoter such as SV40 (see, Kriegler et al., Cell 55:483, 1984), cytomegalovirus (“CMV”) (see, Boshart et al., Cell 47:521-530, 1991), or an internal ribosomal binding site ("IRBS”).
  • CMV cytomegalovirus
  • IRBS internal ribosomal binding site
  • the gene delivery vehicles provided herein direct the expression of a substance capable of preventing, inhibiting, stabilizing or reversing infectious, cancerous, auto-immune or immune diseases.
  • diseases include viral infections such as HTV, HBV and HPV, melanomas, autoimmune diseases such as diabetes, graft vs. host disease, multiple sclerosis, rheumatoid arthritis uveitis, Alzheimer's disease and heart disease.
  • the present invention is directed, in part, toward methods for stimulating a specific immune response, either humoral or cell-mediated, to an antigen or pathogenic antigen.
  • the present invention provides a method for stimulating a specific immune response by using gene delivery vehicles that direct the expression of an antigen or modified form thereof in susceptible target cells capable of initiating such an immune response to the antigen.
  • Expression of the nucleic acid encoded protein may be transient or stable with time.
  • the gene delivery vehicle is preferably designed to express a modified form of the antigen which will stimulate an immune response and which has reduced pathogenicity relative to the native antigen. This immune response is achieved when cells present antigens in the correct manner, i.e., in the context of the MHC class I and/or ⁇ molecules, along with various accessory molecules.
  • Heterologous sequences which may be carried by the gene delivery vehicle include, for example, sequences which encode antigens which stimulate an immune response, and immunomodulatory factors which assist or inhibit an immune response.
  • the gene delivery vehicle may contain two or more heterologous sequences.
  • the antigen generated from the recombinant retroviral genome may be of a form which will elicit either or both an HLA class I- or class Il-restricted immune response.
  • the antigen is preferably selected from gp 160, gp 120, and gp 41, which may be modified to reduce their pathogenicity.
  • the selected antigen is modified to reduce the possibility of syncytia, to avoid expression of epitopes leading to a disease enhancing immune response, to remove immunodominant, but strain-specific epitopes or to present several strain- specific epitopes, and allow a response capable of eliminating cells infected with most or all strains of HTV.
  • the strain-specific epitopes can be further selected to promote the stimulation of an immune response within an animal which is cross-reactive against other strains of HTV.
  • Antigens from other HTV genes or combinations of genes, such as gag, pol, rev, vif, nef, prot, gag/pol, gag/prot, etc., may also provide protection in particular cases.
  • HTV is only one example. This approach will be effective against many virally linked diseases or cancers where a characteristic antigen (which does not need to be a membrane protein) is expressed, such as in HPV and cervical carcinoma, HTLV-I- induced leukemias, prostate-specific antigen (PSA) and prostate cancer, mutated p53 and colon carcinoma and various cellular genes such as MAGE 1, MAGE 3, MART 1, tyrosinase and melanoma.
  • a characteristic antigen which does not need to be a membrane protein
  • the hepatitis B virus presents several different antigens, including among others, three HB “S” antigens (HBsAgs), an HBc antigen (HBcAg), an HBe antigen (HBeAg), and an HBx antigen (HBxAg) (see WO 93/15207 and Blum et al., "The Molecular Biology of Hepatitis B Virus," UG 5(5):154-158, 1989).
  • HBeAg results from proteolytic cleavage of P22 precore intermediate and is secreted from the cell.
  • HBeAg is found in serum as a 17 kD protein.
  • the HBcAg is a protein of 183 amino acids
  • the HBxAg is a protein of 145 to 154 amino acids, depending on subtype.
  • HBsAg synthesized in animal cells is glycosylated, assembled and secreted into the cell supernatant (Tiollais et al., Nature 577:489-495, 1985).
  • Three different env proteins are encoded by the S region of the HBV genome, which contains three translation start codons (Heerman et al., J. Virol 52:396-402, 1984; Tiollais et al., Nature 577:489-495, 1985).
  • the large, middle, and major env proteins initiate translation at the first, second and third ATG and the synthesis proceeds to the end of the ORF.
  • the preS], preS2 and the S gene segments of this ORF are located between the first and second ATG, the second and third ATG, and the third ATG and the end of the ORF, respectively.
  • the three segments encode 119, 55 or 226 amino acids, respectively.
  • the preS2 product binds pHSA (Machida et al., Gastroenterology 56:910-918, 1984; Michel et al., Proc. Natl. Acad Sci. USA 57:7708-7712, 1985; Persing et al., Proc. Natl. Acad Sci. USA 52:3440-3444, 1985).
  • hepatocytes express a receptor for HSA it has been suggested that pHSA may act as an intermediate receptor, binding to middle S protein and to hepatocyte, resulting virus attachment (Michel et al., Proc. Natl. Acad Sci. USA 57:7708-7712, 1985).
  • the major and large env proteins are either non-glycosylated (p24, p39) or are glycosylated at a site within the S region (gp27, gp42).
  • the middle env protein is glycosylated at a site within the pre-S2 region (gp33) and may also be glycosylated in the S region (gp36).
  • various immunogenic portions of the above-described antigens may be combined in order to present an immune response when administered by the gene delivery vehicles described herein.
  • particular combinations of antigens may be preferred for administration in particular geographic regions.
  • epitopes that are found in all human hepatitis B virus S antigen samples are defined as determinant "a”.
  • mutually exclusive subtype determinants however have also been identified by two-dimensional double immunodifiusion (Ouchterlony, Progr. Allergy 5:1, 1958).
  • a gene delivery vehicle for administration which is appropriate to the particular hepatitis B virus subtype which is prevalent in the geographical region of administration.
  • Subtypes of a particular region may be determined by two-dimensional double immunodifiusion or, preferably, by sequencing the S open reading frame of HBV virus isolated from individuals within that region.
  • HBV pol also presented by HBV are pol ("HBV pol"), ORF 5, and ORF 6 antigens.
  • HBV pol the polymerase open reading frame of HBV encodes reverse transcriptase activity found in virions and core-like particles in infected liver.
  • the polymerase protein consists of at least two domains: the amino terminal domain encodes the protein that primes reverse transcription, and the carboxyl terminal domain which encodes reverse transcriptase and RNase H activity.
  • Immunogenic portions of HBV pol may be determined utilizing methods known in the art, and administered using the gene delivery vehicle in order to generate an immune response within a warm-blooded animal.
  • HBV antigens such as ORF 5 and ORF 6, (Miller et al., Hepatology 9:322-327, 1989), may be expressed utilizing gene delivery vehicles as described herein.
  • ATCC No. 45020 contains the total genomic DNA of hepatitis B (extracted from purified Dane particles) (see, Figure 3 of Blum et al., 77G 5(5): 154-158, 1989) in the Bam HI site of pBR322 (Moriarty et al., Proc. Natl. Acad Sci. USA 75:2606-2610, 1981).
  • a gene delivery vehicle which directs the expression of a non-tumorigenic, altered cellular component, such as a ras (ras ) gene (see WO 93/10814).
  • a ras ras
  • the ras gene is an attractive target because it is causally linked to the neoplastic phenotype, and indeed may be necessary for the induction and maintenance of tumorigenesis in a wide variety of distinct cancers, such as pancreatic carcinoma, colon carcinoma and lung adenocarcinoma.
  • ras genes are found in pre-neoplastic tumors, and therefore immune intervention therapy may be applied prior to detection of a malignant tumor.
  • Normal ras genes are non-tumorigenic and ubiquitous in all mammals. They are highly conserved in evolution and appear to play an important role in maintenance of the cell cycle and normal growth properties.
  • the normal ras protein is a G-protein which binds GTP and has GTPase activity, and is involved in transmitting signals from the external milieu to the inside of the cell, thereby allowing a cell to respond to its environment.
  • Ras genes on the other hand alter the normal growth regulation of neoplastic cells by uncoupling cellular behavior from the environment, thus leading to the uncontrolled proliferation of neoplastic cells.
  • ras gene Mutation of the ras gene is believed to be an early event in carcinogenesis (Kumar et al., "Activation of ras Oncogenes Preceding the Onset of Neoplasia," Science 245:1101-1104, 1990), which, if treated early, may prevent tumorigenesis.
  • Ras genes occur in a wide variety of cancers, including for example, pancreatic, colon, and lung adenocarcinomas (see Table 1 below). TABLE 1
  • Table 3 summarizes known in vivo mutations (codons 12, 13 and 61) which activate human ras, as well as potential mutations which have in vitro transforming activity.
  • Potential mutations with in vitro transforming activity were produced by the systematic substitution of amino acids for the normal codon (e.g., other amino acids were substituted for the normal glycine at position 12).
  • Such mutations while not presently known to occur in humans or animals, may serve as the basis for an anti-cancer immunotherapeutic if they are eventually found to arise in vivo.
  • novel proteins encoded by these sequence(s) may be used as a marker of tumorigenic cells, and an immune response directed against these novel coding regions may be utilized to destroy tumorigenic cells containing the altered sequences (ras ).
  • a gene delivery vehicle which directs the expression of an altered p53 (p53 ) gene.
  • p53 is a nuclear phosphoprotein which was originally discovered in extracts of transformed cells, and thus was initially classified as an oncogene (Linzer and Levine, Cell 17:43-52, 1979; Lane and Crawford, Nature 275:261-263, 1979). It was later discovered that the original p53 cDNA clones were mutant forms of p53 (Hinds et al., J. Virol. 65:739-746, 1989). It now appears that p53 is a tumor suppressor gene, which negatively regulates the cell cycle, and that mutation of this gene may lead to tumor formation.
  • Certain alterations of the p53 gene may be due to certain specific toxins.
  • Bressac et al. (Nature 550:429-431, 1991) describes specific G to T mutations in codon 249, in patients affected with hepatocellular carcinoma.
  • One suggested causative agent of this mutation is aflatoxin Bi, a liver carcinogen which is known to be a food contaminant in Africa.
  • a gene delivery vehicle which directs the expression of an altered Rb (Rb ) gene.
  • Rb Rb
  • retinoblastoma is a childhood eye cancer assodated with the loss of a gene locus designated Rb, which is located in chromosome band 13ql4.
  • a gene from this region has been cloned which produces a nuclear phosphoprotein of about HOkd (Friend et al., Nature 323:643, 1986; Lee et al., Science 235:1394, 1987; and Fung et al., Science 256:1657, 1987).
  • Rb is believed to be a negative regulator of cellular proliferation, and has a role in transcriptional control and cell-cycle regulation. Rb binds to at least seven proteins found in the nucleus, and in particular, appears to be involved with a cellular transcription factor which has been designated both E2F (Bagchi et al., Cell 62:659-669, 1990) and DRTF (Shivji and La Thangue, MoL Cell. Biol. 77:1686-1695, 1991). Rb is believed to restrict cellular growth by sequestering a variety of nuclear proteins involved in cellular proliferation.
  • deletions within the Rb gene have been detected which evidence that the Rb gene may be responsible for tumorigenicity. These deletions include, for example, a deletion in exon 21 in a prostate cancer and bladder cancer cell line (Bookstein et al., Science 247:712-715, 1990; Horowitz et al., Science 243:931, 1989), a deletion of exon 16 in a small-cell carcinoma of the lung (Shew et al., Cell Growth andDiff. 7:17, 1990), and a deletion between exons 21 and 27 (Shew et al., Proc. Natl. Acad Sci. USA 87:6, 1990).
  • a gene delivery vehicle which directs the expression of an altered gene which causes Wilms' tumor.
  • Wilms' tumor is typically found in children younger than 16 years of age. One child in 10,000 will develop this tumor, which comprises approximately 5% of childhood cancers. The tumor usually presents itself as a large abdominal mass which is surrounded by a fibrous pseudocapsule. Approximately 7% of the tumors are multifocal in one kidney, and 5.4% are involved with both kidneys.
  • the Wilms' tumor gene has been localized to chromosome l lpl3, and a cDNA clone (wtl) has been isolated that is characteristic of a tumor suppressor gene (Call et al., Cell 60:509, 1990; Gessler et al., Nature 343:144, 1990; Rose et al., Cell 60:495, 1990; and Haber et al., Cell 67: 1257, 1990).
  • the wtl gene encodes a protein which contains four zinc fingers and a glutamine and proline rich amino terminus. Such structures are believed to be associated with transcriptional and regulatory functions.
  • Mutations of the Wilms 1 tumor gene include the insertion of lysine, threonine, and serine between the third and forth zinc fingers.
  • a wtl protein which contains such insertions does not bind to the EGR-1 site.
  • a second alternative mutation results in the insertion of about 17 amino acids in the region immediately NH2-terminal to the zinc finger domain (Madden et al., Science 255:1550-1553, 1991; Call et al., Cell 60:509, 1990; Gessler et al., Nature 343:144, 1990; Rose et al., Cell 60:495, 1990; Haber et al., Cell 67:1257, 1990; and Buckler et al., Mol. Cell. Biol. 77:1707, 1991).
  • novel protein(s) containing novel coding sequences may be used as a marker of tumorigenic cells, and an immune response directed against these novel coding region(s) may be utilized to destroy tumorigenic cells containing the altered sequence(s) or gene(s), which cause Wilms' tumor.
  • a gene delivery vehicle which directs the expression of an altered mucin.
  • Mucins are large molecular weight glycoproteins which contain approximately 50% carbohydrate.
  • Polymorphic epithelial mucin (PEM) is a tumor-associated mucin (Girling et al., Int. J. Cancer 45:1072-1076, 1989) which is found in the serum of cancer patients.
  • the full- length cDNA sequence has been identified (Gendler et al., J. Biol. Chem. 265(25): 15286- 15293, 1990; Lan et al., J. Biol. Chem.
  • a gene delivery vehicle which directs the expression of an altered DCC (deleted in colorectal carcinomas) gene.
  • DCC deficiency C-cell-associated tumor suppressor gene
  • NCAM neural cell-adhesion molecule
  • contactin contactin
  • the DCC gene is expressed in normal colonic mucosa, but its expression is reduced or absent in the majority of colorectal carcinomas (Solomon, Nature 545:412-414,
  • a gene delivery vehicle which directs the expression of MCC or APC.
  • MCC mutated in colorectal cancer
  • APC have been identified as tumor suppressor genes (Kinzler et al., Science 257:1366-1370, 1991) which undergo mutation in familial adenomatous polyposis (FAP).
  • FAP familial adenomatous polyposis
  • Alterations as described above result in the production of protein(s) containing novel coding sequence(s).
  • the novel protein(s) encoded by these sequence(s) may be used as a marker of tumorigenic cells, and an immune response directed against these novel coding region(s) may be utilized to destroy tumorigenic cells containing the altered sequence(s) or gene(s) which cause DCC, APC, or MCC.
  • ubiquitin is a cellular protein which is involved in cell-cycle control and DNA replication. Other functions of ubiquitin include intracellular protein degradation, heat-shock response, transcriptional regulation, cell-cycle control, and cell-cell interaction. Ubiquitin is believed to be a marker molecule that targets proteins for a variety of metabolic fates, and a cDNA sequence which encodes this protein has been identified (Lund et al., "Nucleotide sequence analysis of a cDNA encoding human ubiquitin reveals that ubiquitin is synthesized as a precursor," /. Biol. Chem. 263:4926-4931, 1985).
  • a mutant ubiquitin has recently been identified in a human colon carcinoma cell line (Mafune et al., Arch-Surg. 126:462-466, 1991).
  • This tumor cell contains a novel fusion protein consisting of a hybrid ubiquitin-ribosomal protein S27a.
  • the fusion junction of this protein results in a novel nonself protein sequence which may be immunogenic, and therefore used to eliminate tumor cells carrying this fusion protein.
  • a gene delivery vehicle which directs the expression of altered bcr/abl. Briefly, in tumor cells from almost all patients with chronic myelogenous leukemia, the Philadelphia chromosome, a fusion of chromosomes 9 and 22, directs the synthesis of the fused P210D cr/fa bl protein.
  • This hybrid gene encodes a 210kD phosphoprotein with disregulated protein-kinase activity which leads to the chronic myelogenous leukemia (Daley et al., Science 247:824-829, 1990; Shtivelman et al., Nature 315:550-554, 1985; Ben-Neriah et al., Science 233:212-214, 1986; and Shtivelman et al., Cell 47:277-284, 1986).
  • the fusion junction of these two chromosomes results in a novel nonself protein sequence which may be immunogenic, and thus used to eliminate tumor cells carrying this fusion protein.
  • gene delivery vehicles which direct the expression of a substance such as MAGE 1, MAGE
  • MART-1 tyrosine hydroxylase or a protein that has been considered “normal,” but is associated with, or an immune response (especially a cellular immune response) against the protein is associated with, the presence of tumors in a patient.
  • At least one immunogenic portion of a substance may be incorporated into a gene delivery vehicle.
  • the immunogenic portion(s) which are incorporated into the gene delivery vehicle may be of varying length, although it is generally preferred that the portions be at least 8 amino acids long, and may include an entire protein. Immunogenicity of a particular sequence is often difficult to predict.
  • peptide epitopes may be predicted utilizing class I peptide binding motifs, such as the motif described by Falk et al. for HLA A2.1 (Nature 557:290, 1991). From this analysis, peptides may be synthesized and used as targets in an in vitro cytotoxic assay.
  • ELISA which detects the presence of antibodies against the newly introduced vector-encoded proteins
  • assays which test for T helper cells such as gamma-interferon assays, IL-2 production assays, and proliferation assays.
  • Immunogenic portions may also be selected by other methods.
  • the HLA A2.1/K D transgenic mouse and the HLA-B27 transgenic mouse have been shown to be useful as models for human T-cell recognition of viral antigens.
  • the murine T-cell receptor repertoire recognizes the same antigenic determinants recognized by human T-cells.
  • the CTL response generated in the HLA A2.1/K 0 transgenic mouse is directed toward virtually the same epitope as those recognized by human CTLs of the HLA A2.1 haplotype (Vitiello et al., J. Exp.
  • immunogenic portions for incorporation into vector constructs include HBeAg, HBcAg, and HBsAgs.
  • Additional immunogenic portions of the hepatitis B virus may be obtained by truncating the coding sequence at various locations including, for example, the following sites: Bst UI, Ssp l, Ppu Ml, and Msp l (Valenzuela et al., Nature 250:815-19, 1979; Valenzuela et al., Animal Virus Genetics: ICN/UCLA Symp. Mol. Cell Biol., 1980, B. N. Fields and R. Jaenisch (eds.), pp. 57-70, New York: Academic).
  • the methods described herein may also be applied to the treatment of hepatitis C infections.
  • RNA of HCV has recently been determined to have a sequence of 9379 nucleotides (Choo et al., Proc. Natl. Acad Sci. USA 55:2451-2455, 1991; Choo et al., Brit. Med Bull. 46(2):423-441, 1990; Okamoto et al., J. Gen. Vir. 72:2697-2704, 1991; see also Genbank Accession No. M67463, Intelligenetics (Mountain View, California). This sequence expresses a polyprotein precursor of 3011 amino acids, which has significant homology to proteins of the flavivirus family.
  • the polyprotein precursor is cleaved to yield several different viral proteins, including C (nucleocapsid protein) El, E2/NS1, and non-structural proteins NS2, NS3, NS4, and NS5 (Houghton et al., Hepatology 74:381-388, 1991).
  • C nucleocapsid protein
  • E2/NS1 non-structural proteins
  • NS2, NS3, NS4, and NS5 Houghton et al., Hepatology 74:381-388, 1991.
  • Preferred immunogenic portion(s) of hepatitis C may be found in the C and NS3-NS4 regions since these regions are the most conserved among various types of hepatitis C virus (Houghton et al., Hepatology 74:381-388, 1991).
  • Particularly preferred immunogenic portions may be determined by a variety of methods.
  • identification of immunogenic portions of the polyprotein may be predicted based upon amino acid sequence.
  • various computer programs which are known to those of ordinary skill in the art may be utilized to predict class I binding peptides.
  • peptide epitopes which can bind HLA A2.1 molecules may be predicted utilizing the HLA A2.1 motif described by Falk et al. (Nature 557:290, 1991). From this analysis, peptides are synthesized and used as targets in an in vitro cytotoxic assay.
  • methods for destroying hepatitis B carcinoma cells comprising the step of administering to a warm ⁇ blooded animal a gene delivery vehicle which directs the expression of an immunogenic portion of antigen X, such that an immune response is generated.
  • Sequences which encode the HBxAg may readily be obtained by one of skill in the art given the disclosure provided herein.
  • a 642 bp Neo I-Taq I is recovered from ATCC 45020, and inserted into gene delivery vehicles as described above for other hepatitis B antigens.
  • the X antigen is a known transactivator which may function in a manner similar to other potential oncogenes (e.g., El A).
  • El A potential oncogenes
  • Various methods may be utilized to render the X antigen non- tumorigenic including, for example, by truncation, point mutation, addition of premature stop codons, or phosphorylation site alteration.
  • the sequence or gene of interest which encodes the X antigen is truncated. Truncation may produce a variety of fragments, although it is generally preferable to retain greater than or equal to 50% of the encoding gene sequence.
  • any truncation leave intact some of the immunogenic sequences of the gene product.
  • multiple translational termination codons may be introduced into the gene. Insertion of termination codons prematurely terminates protein expression, thus preventing expression of the transforming portion of the protein.
  • the X gene or modified versions thereof may be tested for tumorigenicity in a variety of ways. Representative assays include tumor formation in nude mice, colony formation in soft agar, and preparation of transgenic animals, such as transgenic mice.
  • gene delivery vehicles may be prepared which direct the co-expression of several of the above-described immunogenic portions (as well as immunomodulatory factors, if desired).
  • gene delivery vehicles may be prepared which direct the co-expression of both an immunogenic portion of the hepatitis B antigen, as well as an immunogenic portion of the hepatitis C polyprotein.
  • Such vehicles may be administered as described above and below, in order to prevent or treat acute and chronic hepatitis infections of either type B or C.
  • gene delivery vehicles may be prepared which direct the co-expression of both an immunogenic portion of the hepatitis B X antigen, as well as an immunogenic portion of the hepatitis C polyprotein.
  • Such vehicles may similarly be administered in order to treat hepatocellular carcinoma which is associated with either hepatitis B or C.
  • a vector may also be utilized as a prophylactic treatment for the disease.
  • a gene delivery vehicle may express (either separately or as one construct) all or immunogenic portions of HBcAg, HBeAg, HBsAgs, HBxAg, as well as immunogenic portions of HCV antigens as discussed below.
  • the gene delivery vehicle may also co-express an immunomodulatory factor, such as alpha interferon (Finter et al., Drugs 42(5):749-765, 1991; U.S. Patent No. 4,892,743; U.S. Patent No.
  • ICAM-1 Altman et al., Nature 555:512-514, 1989
  • ICAM-2 LFA-1
  • LFA-3 MHC class I molecules
  • MHC class ⁇ molecules MHC class ⁇ molecules
  • b2-microglobulin, chaperones CD3, B7.1-.3
  • MHC-linked transporter proteins or analogs thereof Powis et al., Nature 554:528-531, 1991.
  • sequences which encode the above-described antigens, proteins or factors may be readily obtained from a variety of sources, including for example depositories such as the American Type Culture Collection (ATCC, Rockville, Maryland), or from commercial sources such as British Bio-Technology Limited (Cowley, Oxford England).
  • Representative examples include BBG 12 (containing the GM-CSF gene coding for the mature protein of 127 amino adds), BBG 6 (which contains sequences encoding gamma interferon), ATCC No. 39656 (which contains sequences encoding TNF), ATCC No. 20663 (which contains sequences encoding alpha interferon), ATCC Nos. 31902, 31902, and 39517 (which contain sequences encoding beta interferon), ATCC No.
  • Sequences which encode the above-described feline leukemia virus and feline immunodeficiency virus antigens may be prepared as described within the references cited above, or obtained from a variety of sources.
  • sequences which encode the envelope protein of FeLV may be readily obtained from the American Type Culture Collection ("ATCC”; Rockville, Maryland) (see, for example, ATCC Nos. 39528, 39529, and 39530).
  • ATCC American Type Culture Collection
  • AIDS Repository Disvision of AIDS, National Institute of Allergy and Infectious Disease, Bethesda, Maryland; see NIH Publication No.
  • N2-1536 holds a deposit of a plasmid clone which contains a sequence encoding a full-length, replication competent FeLV (e.g., clone p61E-FeLV, Catalog No. 109), as well as a deposit of a plasmid clone which contains a sequence encoding a feline immunodeficiency virus (e.g., clone pFTV-14-Petaluma, Catalog No. 851).
  • a plasmid clone which contains a sequence encoding a full-length, replication competent FeLV e.g., clone p61E-FeLV, Catalog No. 109
  • a plasmid clone which contains a sequence encoding a feline immunodeficiency virus e.g., clone pFTV-14-Petaluma, Catalog No. 851).
  • Sequences which encode the above-described altered cellular components may be obtained from a variety of sources.
  • plasmids which contain sequences that encode altered cellular products may be obtained from a depository such as the American Type Culture Collection (ATCC, Rockville, Maryland), or from commercial sources such as Advanced Biotechnologies (Columbia, Maryland).
  • ATCC American Type Culture Collection
  • ATCC No. 41000 containing a G to T mutation in the 12th codon of ras
  • ATCC No. 41049 containing a G to A mutation in the 12th codon.
  • plasmids which encode normal cellular components may also be obtained from depositories such as the ATCC (see, for example, ATCC No. 41001 which contains a sequence which encodes the normal ras protein, ATCC No. 57103 which encodes abl; and ATCC Nos. 59120 or 59121 which encode the bcr locus) and mutated to form the altered cellular component.
  • ATCC ATCC No. 41001 which contains a sequence which encodes the normal ras protein
  • ATCC No. 57103 which encodes abl
  • ATCC Nos. 59120 or 59121 which encode the bcr locus
  • cDNA sequences which encode the above heterologous sequences may be obtained from cells which express or contain the sequences. Briefly, within one embodiment mRNA from a cell which expresses the gene of interest is reverse transcribed with reverse transcriptase using oligo dT or random primers. The single stranded cDNA may then be amplified by PCR (see, U.S. Patent Nos. 4,683,202, 4,683,195, and 4,800,159. See also, PCR Technology: Principles and Applications for DNA Amplification, Erlich (ed.), Stockton Press, 1989) utilizing oligonucleotide primers complementary to sequences on either side of desired sequences.
  • a double stranded DNA is denatured by heating in the presence of heat stable Taq polymerase, sequence spedfic DNA primers, ATP, CTP, GTP and TTP. Double-stranded DNA is produced when synthesis is complete. This cycle may be repeated many times, resulting in a factorial amplification of the desired DNA.
  • Sequences which encode the above-described proteins may also be synthesized, for example, on an Applied Biosystems Inc. DNA synthesizer (e.g., APB DNA synthesizer model 392 (Foster City, California). Routes of Administration
  • Suitable methods include, for example, various physical methods such as direct DNA injection (Acsadi et al., Nature 552:815-818, 1991), and microprojectile bombardment (Williams et al., PNAS 55:2726-2730, 1991).
  • the gene delivery vehicle can be injected into the interstitial space of tissues including muscle, brain, liver, skin, spleen or blood (see, WO 90/11092). Administration may also be accomplished by intravenous injection or direct catheter infusion into the cavities of the body (see, WO 93/00051), discussed in more detail below.
  • administration of the gene delivery vehicles at multiple sites be via at least two injections.
  • suitable modes of administration include intramuscular, intradermal and subcutaneous injections, with at least one of the injections preferably being intramuscular.
  • two or more of the injections are intramuscular.
  • administration via injections is preferred, it will be evident that the gene delivery vehicles may be administered through multiple topical or separate ocular administrations.
  • additional routes are suitable for use within the present invention when combined with one or more of the routes briefly noted above, including intraperitoneal, intracranial, oral, rectal, nasal, vaginal and sublingual administration.
  • Transdermal or topical application of a gene delivery vehicle may be used as a route of administration because the skin is the most expansive and readily accessible organ of the human body.
  • Transdermal delivery systems are capable of delivering a drug through intact skin so that it reaches the systemic circulation in sufficient quantity to be therapeutically beneficial.
  • TDS provides a variety of advantages, including elimination of gastrointestinal abso ⁇ tion problems and hepatic first pass effect, reduction of dosage and dose intervals, and improved patient compliance.
  • TDS The major components of TDS are a controlled release device composed of polymers, the drug to be administered, excipients, and enhancers, and a fastening system to fix the device to the skin.
  • polymers include gelatin, gum arabic, paraffin waxes, and cellulose acetate phthalate (Sogibayasi et al., J. Controlled Release 29:177-185, 1994).
  • These polymers can be dermatologically formulated into aqueous, powder or oil phases. The various combinations can produce lotions, pastes, ointments, creams, and gels, with the aid ofemulsifiers.
  • Topical administration of gene delivery vehicles as described herein may be accomplished by encapsulation in liposomes.
  • Hyaluronic acid has been used as a bioadhesive ligand for the formation of liposomes to enhance adherence and retention to the extracellular matrix in cases of burns and wound healing (Yerushalmi et al., Arch. Biochem. and Biophys. 575:267-273, 1994).
  • Methods of liposome preparation can be tailored to control size and morphology.
  • Drug molecules can be either inco ⁇ orated in the aqueous space or intercalated into the lipid bilayer of liposomes, depending upon the physiochemical characteristics of the drug.
  • ionophoresis may be used to cause increased penetration of ionized substances into or through the skin by the application of an electrical field.
  • This method has the advantage of being able to deliver the drug in a pulsatile manner (Singh et al., Dermatology 757:235-238, 1993).
  • Ocular administration of a gene delivery vehicle may also be utilized.
  • the avascular components of the eye allow for topical therapy to be most effective for a number of disease states such as glaucoma, inflammation, and he ⁇ es keratitis (Wearley, Crit. Rev. in Therap. Drug Car. Sys. 5:331-394, 1991).
  • Formulations of a gene delivery vehicle in inert ingredients such as buffers, chelating agents, antioxidants, and preservatives can be inco ⁇ orated into ophthalmic dosage forms intended for multiple dose use. Formulations also may consist of aqueous suspensions, ointments, gels, inserts, bioadhesives, microparticles, and nanoparticles.
  • the nasal cavity also offers an alternative route of administration for a gene delivery vehicle.
  • the human nasal cavities have a total surface area of approximately 150 cm 2 and are covered by a highly vascular mucosal layer.
  • a respiratory epithelium comprised of columnar cells, goblet cells, and ciliary cuboidal cells line most of the nasal cavity (Chien et al., Crit. Rev. in Therap. Drug Car. Sys. 4:67-194, 1987).
  • the subepithelia contains a dense vascular network and the venous blood from the nose passes directly into the systemic circulation, avoiding first-pass metabolism in the liver. Thus, delivery to the upper region of the nasal cavity may result in slower clearance and increased bioavailability.
  • the absence of cilia in this area is an important factor in the increased effectiveness of nasal sprays as compared to drops.
  • the addition of viscosity-building agents such as methycellulose can change the pattern of deposition and clearance of intranasal applications. Additionally, bioadhesives can be used as a means to prolong residence time in the nasal cavity.
  • Various formulations comprising sprays, drops, and powders, with or without the addition of abso ⁇ tive enhancers have been investigated (see Wearley
  • Oral administration of a gene delivery vehicle includes sublingual, buccal, and gastrointestinal delivery.
  • Sublingual and buccal (cheek) delivery allow for rapid systemic abso ⁇ tion of compounds which bypass hepatic first-pass metabolism and degradation in the stomach and intestines.
  • Buccal delivery devices can be designed unidirectional for oral mucosal abso ⁇ tion only. Additionally these devices can prevent diffusion-limiting- mucus buildup to allow for enhanced abso ⁇ tion. Delivery through the gastrointestinal track allows for precise targeting for drug release.
  • genes can be specifically delivered to areas in the stomach, duodenum, jejunum, ileum, cecum, colon, or rectum.
  • Oral formulations include tablets, capsules, aqueous suspensions, and gels.
  • bioadhesive polymers may contain bioadhesive polymers, hydrodynamically balanced systems, gastroinflatable delivery devices, intragastric retention shapes, enteric coatings, excipients, or intestinal so ⁇ tion promoters (Ritschel, Meth. Find. Exp. Clin. Pharmacol. 75:313-336, 1991).
  • the human rectum has a surface area of between 200 to 400 cm 2 and is abundant in blood and lymphatic vessels. This offers an alternative route of administration of a gene delivery vehicle. Depending on the actual site of administration, it may be possible to bypass first-pass metabolism by the liver. Targeting of the systemic circulation can be achieved by delivering the vehicle to an area behind the internal rectal sphincter. This allows abso ⁇ tion directly into the inferior vena cava thereby bypassing the portal circulation and avoiding metabolism in the liver. The liver can be targeted by delivering the vehicle to the region of the ampulla recti which allows abso ⁇ tion into the portal system (Ritschel, supra). Pulmonary administration of gene delivery vehicles can be accomplished through aerosolization.
  • Aerosol therapy is very common in obstructive bronchial diseases but can be used for topical treatment of the respiratory tract as well as the treatment of systemic diseases (Kohler, Lung Suppl. :677-684, 1990).
  • the surface area of the lung is approximately 75 m 2 and requires only one puff of an aerosol to cover this entire area within seconds.
  • Abso ⁇ tion occurs quickly because the walls of the alveoli in the deep lung are extremely thin. Abso ⁇ tion and clearance depends on a number of factors including particle size and solubility (Wearley, supra).
  • Gene delivery vehicles can also be administered into the urinary bladder via catheterization (Morris et al., J. ofUrol. 152:506-509, 1994). This intravesical therapy can be used for the treatment of bladder cancer.
  • the rationale for intravesical therapy is that high concentrations of drug may contact tumor-bearing mucosa for prolonged periods. The likelihood of tumor implantation after resection maybe reduced by destroying viable cancer cells, as well as providing a cytotoxic effect on residual carcinoma. There is also minimal toxicity owing to limited systemic abso ⁇ tion.
  • the vaginal mucosa consists of stratified squamous epithelium.
  • Gene delivery vehicles can be administered through the vaginal orifice onto the mucosa.
  • Formulations include ointments, creams, and suppositories.
  • gene delivery vehicles of the present invention may also be manipulated by a variety of methods known in the art, in order to render the expressed substance more immunogenic. Representative examples of such methods include: adding sequences that correspond to T helper epitopes; promoting cellular uptake by adding sequences encoding hydrophobic residues; or any combination of these (see generally, Hart, op. cit., Milich et al., Proc. Natl. Acad Sci. USA 55:1610-1614, 1988; Willis, Nature 540:323-324, 1989; Griffiths et al., J. Virol. 65:450-456, 1991).
  • compositions comprising one of the above-described gene delivery vehicles, such as a recombinant retrovirus or recombinant virus selected from the group consisting of poliovirus, rhinovirus, pox virus, canary pox virus, vaccinia virus, influenza virus, adenovirus, parvovirus, adeno-associated virus, he ⁇ es virus, SV40, HIV, measles, corona and Sindbis virus in combination with a pharmaceutically acceptable carrier or diluent.
  • the composition may be prepared either as a liquid solution, or as a solid form (e.g., lyophilized) which is suspended in a solution prior to administration.
  • the composition may be prepared with suitable carriers or diluents for surface administration, injection, oral, or rectal administration.
  • the recombinant virus is utilized at a concentration ranging from 0.25% to 25%, and preferably about 5% to 20% before formulation.
  • the gene delivery vehicle will constitute about 1 ug of material per dose, with about 10 times this amount material (10 ug) as copurified contaminants.
  • the composition is prepared in 0.1-1.0 ml of aqueous solution formulated as described below.
  • Pharmaceutically acceptable carriers or diluents are nontoxic to recipients at the dosages and concentrations employed.
  • Representative examples of carriers or diluents for injectable solutions include water, isotonic saline solutions which are preferably buffered at a physiological pH (such as phosphate-buffered saline or Tris-buffered saline), mannitol, dextrose, glycerol, and ethanol, as well as polypeptides or proteins such as human serum albumin.
  • a preferred composition comprises a recombinant virus in a 10 mg/ml mannitol, 1 mg ml HSA, 20 mM Tris, pH 7.2 and 150 mM NaCl.
  • the gene delivery vehicle since the gene delivery vehicle represents approximately 1 ug of material, it may be less than 1% of high molecular weight material, and less than 1/100,000 of the total material (including water).
  • This composition is stable at -70°C for at least six months.
  • the composition may be injected intravenously (i.v.) or subcutaneously (s.c), although it is generally preferable to inject it intramuscularly (i.m.).
  • the individual doses normally used are l ⁇ to 10 ⁇ c.f.u. (colony forming units of neomycin resistance titered on HT1080 cells). These are generally administered at one- to four-week intervals for three or four doses initially. Subsequent booster shots may be given as one or two doses after 6-12 months, and thereafter annually.
  • Oral formulations may also be employed with carriers or diluents such as cellulose, lactose, mannitol, poly(DL-lactide-co-glycolate) spheres, and/or carbohydrates such as starch.
  • the composition may take the form of, for example, a tablet, gel capsule, pill, solution, or suspension, and additionally may be formulated for sustained release.
  • preparation of a suppository may be accomplished with traditional carriers such as polyalkalene glucose, or a triglyceride.
  • compositions of the present invention may also include factors which stimulate cell division, and hence, uptake and inco ⁇ oration of a recombinant retroviral vector.
  • factors which stimulate cell division, and hence, uptake and inco ⁇ oration of a recombinant retroviral vector include melanocyte stimulating hormone (MSH), for melanomas or epidermal growth factor (EGF) for breast or other epithelial carcinomas.
  • MSH melanocyte stimulating hormone
  • EGF epidermal growth factor
  • the gene delivery vehicle may direct the expression of an immunomodulatory factor in addition to at least one substance. If the gene delivery vehicle, however, does not express an immunomodulatory factor which is a cytokine, this cytokine may be included in the above-described compositions, or may be administered separately (concurrently or subsequently) with the above-described compositions.
  • the immunomodulatory factor is preferably administered according to standard protocols and dosages as prescribed in The Physician's Desk Reference.
  • alpha interferon may be administered at a dosage of 1-5 million units/day for 2-4 months, and IL-2 at a dosage of 10,000-100,000 units/kg of body weight, 1-3 times/day, for 2-12 weeks.
  • Gamma interferon may be administered at dosages of 150,000-1,500,000 units 2-3 times/week for 2-12 weeks.
  • EXAMPLE 1 Mice were injected three times at weekly intervals at multiple sites or at a single site with a representative gene delivery vehicle, N2 IIIB env (lO ⁇ CFU/ml). Specifically, ten mice were injected at two intramuscular sites (right and left hind flank muscles) and ten mice were injected at one intramuscular site (right hind flank muscle). After seven days, a CTL assay (Warner et al., AIDS Res. and Hum. Retroviruses 7(8):645- 655, 1991) was performed on each animal.
  • Figure 1A depicts the results of the CTL assay for animals injected at multiple sites;
  • Figure IB representing CTL assay results for animals injected at a single site.
  • Each bar within Figures 1 A and IB represents the percent (%) net target cell lysis associated with an individual mouse. As illustrated in Figure 1A, nine out of ten mice exhibited target cell lysis of greater than 60% (average of 76%) at an effecto ⁇ target ratio of 100: 1. By comparison, as illustrated in Figure IB, only three out of ten mice achieved 60% target cell lysis at a effecto ⁇ target ratio of 100: 1 (average of 39%).
  • EXAMPLE 2 Within this experiment, five mice were injected two times at a weekly interval at multiple sites. Specifically, two intramuscular sites (left- and right-hand limbs) were injected with N2 IIIB env that was diluted 1:2 (0.5X concentration). A separate group of five mice were injected two times at a weekly interval at one site (right hind limb) with N2 ⁇ TB env at a concentration of IX. The two groups of mice received identical vector particle doses.

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Abstract

Méthodes de stimulation d'une réponse immunitaire humorale ou cellulaire chez un animal à sang chaud par administration en plusieurs sites d'un ou plusieurs véhicules distributeurs de gènes. Chacun de ces véhicules dirige l'expression d'au moins une substance dans des cellules hôtes modifiées avec le véhicule de manière à provoquer une réponse immunitaire. Dans les variantes préférées, la substance exprimée émet une réponse immunitaire induite par la cellule et de préférence une réponse immunitaire par HLA restreinte à la catégorie I.
PCT/US1995/016471 1994-12-30 1995-12-15 Administration directe en plusieurs sites de vehicules distributeurs de genes WO1996020731A1 (fr)

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WO1999019501A1 (fr) * 1997-10-14 1999-04-22 Institute For Vaccine Development Vecteur d'origine retrovirale d'adn non integrant a forte expression de proteines et antigenes immunogenes secretes
WO2000000218A1 (fr) * 1998-06-26 2000-01-06 Aventis Pasteur Immunisation a ciblage mucosal
US7238673B2 (en) 1989-03-31 2007-07-03 The Regents Of The University Of Michigan Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor

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WO1990011092A1 (fr) * 1989-03-21 1990-10-04 Vical, Inc. Expression de sequences de polynucleotides exogenes chez un vertebre
WO1991002805A2 (fr) * 1989-08-18 1991-03-07 Viagene, Inc. Retrovirus de recombinaison apportant des constructions de vecteur a des cellules cibles
WO1993000051A1 (fr) * 1991-06-28 1993-01-07 The Regents Of The University Of Michigan Traitement de pathologies par instillation sitospecifique de cellules ou par transformation sitospecifique de cellules et trousses prevues a cet effet

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WO1990011092A1 (fr) * 1989-03-21 1990-10-04 Vical, Inc. Expression de sequences de polynucleotides exogenes chez un vertebre
WO1991002805A2 (fr) * 1989-08-18 1991-03-07 Viagene, Inc. Retrovirus de recombinaison apportant des constructions de vecteur a des cellules cibles
WO1993000051A1 (fr) * 1991-06-28 1993-01-07 The Regents Of The University Of Michigan Traitement de pathologies par instillation sitospecifique de cellules ou par transformation sitospecifique de cellules et trousses prevues a cet effet

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IRWIN, M.J. ET AL., KEYSTONE SYMPOSIA ON MOLECULAR & CELLULAR BIOLOGY, JOURNAL OF CELLULAR BIOCHEMISTRY SUPPL. 17E, 1993, MARCH 29- APRIL 25, 1993, pages 79, XP002003658 *
JOLLY, D.J. ET AL., KEYSTONE SYMPOSIA ON MOLECULAR & CELLULAR BIOLOGY, JOURNAL OF CELLULAR BIOCHEMISTRY SUPPL. 18A. 1994, JANUARY 4-23, 1994, pages 220, XP002003657 *
WARNER, J.F. ET AL., TENTH INTERNATIONAL CONFERENCE ON AIDS AND THE INTERNATIONAL CONFERENCE ON STD, VOL. 2. THE GLOBAL CHALLENGE OF AIDS: TOGETHER FOR THE FUTURE. MEETING, YOKOHAMA.JAPAN, AUGUST 7-12, 1994, pages 116, XP002003659 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7238673B2 (en) 1989-03-31 2007-07-03 The Regents Of The University Of Michigan Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor
WO1999019501A1 (fr) * 1997-10-14 1999-04-22 Institute For Vaccine Development Vecteur d'origine retrovirale d'adn non integrant a forte expression de proteines et antigenes immunogenes secretes
WO2000000218A1 (fr) * 1998-06-26 2000-01-06 Aventis Pasteur Immunisation a ciblage mucosal

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