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WO1989005349A1 - Procede servant a combattre des infections virales - Google Patents

Procede servant a combattre des infections virales Download PDF

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Publication number
WO1989005349A1
WO1989005349A1 PCT/AU1988/000474 AU8800474W WO8905349A1 WO 1989005349 A1 WO1989005349 A1 WO 1989005349A1 AU 8800474 W AU8800474 W AU 8800474W WO 8905349 A1 WO8905349 A1 WO 8905349A1
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WO
WIPO (PCT)
Prior art keywords
cells
nucleotide sequence
transformed
hiv
bone marrow
Prior art date
Application number
PCT/AU1988/000474
Other languages
English (en)
Inventor
Andrew John Hapel
Ming-Chiu Fung
Jeffrey Robert Hahn
Original Assignee
The Australian National University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Australian National University filed Critical The Australian National University
Publication of WO1989005349A1 publication Critical patent/WO1989005349A1/fr

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Classifications

    • 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
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/18—Erythrocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • 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
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—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
    • C12N2740/16211—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

  • This invention relates to a method of combating viral infections, and in particular it relates to a method of combating infections with retroviruses.
  • Retroviruses are RNA viruses that can make a DNA copy of their genome using a virus-encoded enzyme called reverse transcriptase.
  • the DNA copy (ies) can become randomly integrated into the genome of eukaryotic cells. These integrated viral genomes may remain silent, may produce infectious virus, or may be oncogenic. Rarely, infection with a retrovirus is lytic for the infected cell. Large numbers of retroviruses have been isolated from mice and many have no obvious pathologic effect. However because retroviruses contain one genes, which may be derived from cellular analogues that control cell division, they have been extensively studied in an attempt to understand oncogenesis.
  • Retroviruses that affect man include the human T-lymphotropic viruses HTLVI and HLTVII, which can cause leukaemias, and the human immunodeficiency virus (HIV or HTLVIII) which causes the acquired immunodeficiency
  • the AIDS virus is somewhat unique in that it causes a lytic infection of lymphocytes, thus inducing a severe and chronic suppression of the immune response of the host, ultimately resulting in a complete collapse of the body's defences against infection and the
  • AIDS sufferers normally die as the result of uncontrolled secondary infections due to viruses, fungi, protozoa and bacteria, or as a result of uncontrolled cancers or the like.
  • the total number of ret ovirus- infected individuals in the western world may be 10
  • Retroviruses are divided into groups based on their ability to interfere with infection of their host cell by other retroviruses. When a cell is infected with a virus in a particular interference group, no other virus in that group can subsequently infect the cell, that is the cell
  • the AIDS virus, HIV includes major proteins in its outer envelope which are glycoproteins known as gpl20 and gp41.
  • the virus initially produces a large envelope protein, gpl60, before splitting it into the two smaller proteins.
  • HIV infects cells primarily because the glycoprotein gpl20 which is positioned on the envelope of the virus and on the membrane of cells infected with HIV, binds to a cell surface determinant called CD4, which is found predominantly on helper type thymus-derived lymphocytes (3,4).
  • CD4 + thymus-derived lymphocytes are the primary target of HIV, and infection and subsequent loss of these cells results in immunosuppression and the catastrophic adventitius sequelae that are characteristic of HIV infection.
  • AIDS a major defect in AIDS is loss of immune function, particularly T-cell mediated immune function as a result of lymphocyte destruction by the AIDS virus.
  • One means for reconstituting immune function would be by transplantation of histocompatible blood cells or bone marrow stem cells, however in the HIV infected patient these transplanted cells would of course themselves be infected and destroyed. It is one object of the present invention to provide a method whereby such cells can be transplanted without resultant destruction by the virus.
  • the present invention provides a method for combating viral infection in a human or animal patient, which comprises administering to the patient histocompatible blood cells or bone marrow stem cells which have been transformed with a nucleotide sequence coding for a protein or polypeptide which, on expression by the cells, will reduce the rate at which superinfecting virus particles can enter the cells.
  • the present invention provides a therapeutic or prophylactic composition for combating viral infection in a human or animal patient, which comprises histocompatible blood cells or bone marrow stem cells which have been transformed with a nucleotide sequence coding for a protein or polypeptide which, on expression by the cells, will reduce the rate at which superinfecting virus particles can enter the cells.
  • the present invention has particular application in combating retroviral infection, most particularly HIV infection, and in one particularly preferred embodiment of the present invention the histocompatible blood cells or bone marrow stem cells are transformed with a nucleotide sequence or gene coding for all or an immunologically-active portion of a HIV envelope protein.
  • the cells are transformed with a nucleotide sequence or gene coding for all or an immunologically-active portion of the HIV envelope proteins gpl60, gpl20 or gp41, or any portion of the gpl60 gene encoding an "interfering" polypeptide.
  • selected blood cells expressing HIV envelope proteins may be useful as potent immunogens inducing strong cell mediated immunity to HIV.
  • retroviral vectors have become well known (5a, 5b, 6) and available. These vectors can enter cells and express the genes or nucleotide sequences they carry without producing infectious virus.
  • Such vectors include, for example, non-replicating viral genomes which do not contain the gene for reverse transcriptase and so cannot reproduce, or modified viral genomes which do not contain genes for structural components of the virus envelope and so cannot make infectious virus.
  • retroviral vectors are used to construct artificial HIV-like viral genomes.
  • constructs do not have the pathogenicity of HIV since they cannot replicate in the transformed cells, however they do contain the gene or similar sequence for gpl ⁇ O, gpl20 or gp41 under control of either a modified retroviral promoter or some other promoter.
  • the transformed cells may be used in accordance with the present invention for combating appropriate viral infections.
  • they may be used therapeutically in the treatment of infected patients or they may be used prophylactically in order to prevent or minimise infection by the virus.
  • bone marrow stem cells are transformed with a retroviral vector which carries the gene coding for the envelope glycoprotein gpl60 or gpl20 from HIV.
  • This gene enables the transformed bone marrow cells to express g ⁇ l60 or gpl20 which will bind to the cell surface determinant CD4 of helper type thymus-derived lymphocytes. The surface receptors of these cells will thus become saturated with gpl20, and not be available for interaction with super-infecting HIV particles.
  • the methods used in this aspect of the invention include construction of a viral vector containing the gene for gpl60 downstream of a suitable promoter, and the use of this vector to transfect bone marrow stem cells and lymphocytes in vitro. The cells expressing gpl60 or gpl20 and gp41 can then be exposed to HIV, and examined for production of infectious virus and/or reverse transcriptase (an indicator of retroviral infection) .
  • the present invention particularly relates to combating retroviral infections, and in addition to its use in relation to HIV or HLTVIII described in detail, the invention may be used in therapeutic or prophylactic treatment of leukaemias resulting from HTLVI or HLTVII infections in humans. In the treatment of such leukaemias, known chemotherapy and/or radiotherapy procedures are used to destroy the leukaemic cells, and the patient may then be treated by administration of appropriately transformed cells in accordance with the present invention.
  • the present invention is further illustrated in one embodiment by the following Example which relates to the construction of cells transformed with the HIV gpl60 gene.
  • the retroviral shuttle vector, fpGV-1, (5a, 5b) was used to express the HIV-1 envelope glycoprotein gpl60.
  • fpGV-1 was derived from the HT-1 strain of Moloney
  • SMV Sarcoma Virus
  • HIV-1 envelope glycoprotein gpl60 gene was excised from the commercially available plasmid pBHIO (Biotech Research
  • Figure 3 The strategy of 0 cloning of the gpl60 gene into the retroviral shuttle vector fpGV-1 is shown in Figure 3.
  • the pBHIO plasmid DNA was digested with restriction endonuclease Xhol and the sticky ends of the DNA were blunt-ended using T4 DNA polymerase. The DNA was then digested with restriction *5 endonuclease Sail and separated on a 1% low melting point agarose gel. A 3.Ikb DNA fragment was isolated from the gel using hot phenol extraction method.
  • Vector fpGV-1 plasmid DNA was digested with restriction endonuclease
  • DNA was ligated with the 3.Ikb Xhol (blunt-ended)-£ ⁇ 11 DNA fragment isolated from pBHIO and transformed into E.coli
  • HIV-F1 contains the HIV-1 envelope glycoprotein gpl60 gene which is orientated in the same transcription direction as the SV40 early promoter (shown in Figure 3).
  • Peripheral blood mononuclear cells (10 6 /ml) were stimulated with concanavalin A (10 ⁇ g/ml) for 4 days.
  • Activated T cells were washed, subcultured at 3x10-**/ml in tissue culture medium containing optimum growth concentrations of interleukin-2 (IL-2).
  • the activated cells were stored in liquid N 2 .
  • cells were thawed and treated with OKT8 for 30 min on ice followed by rabbit complement for 1 hr at 37°C.
  • Treated cells were washed and cultured in round well trays in IL-2 at 2xl0 4 /well in 200 ⁇ l.
  • Growth of the activated T cells was inactivated by periodic addition of IL-2 and by stimulation with 5 ⁇ g/ml of phytohaemagglutinin and a 0.05% suspension of sheep red blood cells.
  • Activated T cells were monitored by FACs analysis for T cell surface markers. Populations generated were 90-95% CD4 + , 0% CD8 + , with remaining cells being CD16 + NK cells.
  • COS-1 cells were seeded at 5 10 ⁇ per 60mm diameter petri dish, grown overnight in Dulbecco's modified Eagle's medium (DMEM) (Flow Labs, Sydney, Australia), supplemented with 10% fetal calf serum (FCS), then transfected with HIV-F1 (20 ⁇ g DNA per 10 6 cells) by calcium phosphate precipitation and glycerol shock (15% glycerol for three minutes) . The cells were then washed in DMEM containing 10% FCS, rested in 5ml of the same medium and -incubated at 37°C in 5% C0 2 .
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • CD4 + lymphocytes produced as described above were transfected by allowing them to settle with the CaP0 4 precipitate, decanting off supernatant, then applying glycerol shock as above. Cells were resuspended in growth medium and plated in 96 well round bottom plates at 2xl0 4 cells/well.
  • transfection of large numbers of bone marrow cells or lymphocytes can be most effectively achieved using the electroporation technique.
  • cells suspended in buffer containing positive retroviral vector are exposed to an electric pulse. This causes temporary pore formation in the cell membrane allowing the vector to enter the cell.
  • Transplant cells transfected in this way and containing, for example, the gene or part of the gene for gpl60, could be injected directly back into the patient without prolonged culture.

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Abstract

Le procédé décrit, qui sert à combattre une infection virale chez un être humain ou un animal, consiste à administrer au patient des cellules sanguines histocompatibles ou des cellules souches de moelle osseuse qui ont été transformées avec une séquence de nucléotides codant pour une protéine ou un polypeptide qui, par expression par les cellules, réduit le rythme auquel les particules virales surinfectieuses peuvent pénétrer dans les cellules. La séquence de nucléotides peut être constituée par une séquence ou un gène codant pour la totalité ou pour une partie immunologiquement active d'une protéine d'enveloppe d'HIV, telle que les protéines gp160, gp120 ou gp41.
PCT/AU1988/000474 1987-12-09 1988-12-09 Procede servant a combattre des infections virales WO1989005349A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
AUPI582387 1987-12-09
AUPI5823 1987-12-09
AUPI7629 1988-04-07
AUPI762988 1988-04-07

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WO1989005349A1 true WO1989005349A1 (fr) 1989-06-15

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Cited By (64)

* Cited by examiner, † Cited by third party
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EP0386882A1 (fr) * 1989-02-06 1990-09-12 Dana Farber Cancer Institute Provirus d'HIV défectif pour l'empaquetage, lignées cellulaires et leur utilisation
FR2655855A1 (fr) * 1989-12-20 1991-06-21 Pasteur Institut Protection genetique d'animaux contre l'infection par le virus leucemogene bovin, et provirus et virus recombinant derive de ce virus.
EP0584348A1 (fr) * 1992-03-11 1994-03-02 Auragen, Inc. Vaccin genetique pour virus d'immunodefience
WO1995006120A1 (fr) * 1993-08-25 1995-03-02 Rhone-Poulenc Rorer S.A. Cellules recombinantes de la lignee monocyte-macrophage pour la therapie genique
WO1996006177A2 (fr) * 1994-08-22 1996-02-29 Connaught Laboratories Limited Particules semblables a des retrovirus rendues non-infectieuses par une pluralite de mutations
US5691177A (en) * 1988-03-21 1997-11-25 Guber; Harry E. Recombinant retroviruses expressing a protein that converts a pro-drug into a cytotoxic agent
US5693522A (en) * 1991-11-29 1997-12-02 Chiron Viagene, Inc. Anti-cancer immunotherapeutics
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