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WO1999030742A1 - Emploi therapeutique de vecteurs lentiviraux - Google Patents

Emploi therapeutique de vecteurs lentiviraux Download PDF

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Publication number
WO1999030742A1
WO1999030742A1 PCT/US1998/025720 US9825720W WO9930742A1 WO 1999030742 A1 WO1999030742 A1 WO 1999030742A1 US 9825720 W US9825720 W US 9825720W WO 9930742 A1 WO9930742 A1 WO 9930742A1
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WIPO (PCT)
Prior art keywords
vector
cells
hiv
gene
transgene
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PCT/US1998/025720
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English (en)
Inventor
Luigi Naldini
Jin-Ping Song
Original Assignee
Luigi Naldini
Song Jin Ping
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 Luigi Naldini, Song Jin Ping filed Critical Luigi Naldini
Priority to AU18035/99A priority Critical patent/AU749059B2/en
Priority to CA002314683A priority patent/CA2314683A1/fr
Priority to KR1020007006423A priority patent/KR20010033062A/ko
Priority to JP2000538720A priority patent/JP2002508338A/ja
Priority to EP98962895A priority patent/EP1037669A4/fr
Publication of WO1999030742A1 publication Critical patent/WO1999030742A1/fr

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    • 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
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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

Definitions

  • the instant invention relates to the use of lentiviral vectors per se for a therapeutic benefit.
  • the vector need not contain a transgene with antiviral activity.
  • Figure 1 depicts four graphs of Gag p24 antigen expression in human SupTl lymphocytes transduced with lentiviral vector at different multiplicity of infection (M.O.I.; rectangles, triangles, ellipses) or in control non-transduced cells (lozenges) after infection with different amounts of HIV.
  • Figure 2 depicts Gag p24 antigen expression and cell survival after HIV infection of human primary CD4 + lymphocytes transduced with either a lentiviral vector (triangles) or a murine leukemia virus based vector
  • the instant invention provides use of a lentiviral vector.
  • the vector can be one which carries a foreign gene with an anti-viral activity, however, that is not a prerequisite in the practice of the instant invention.
  • the lentiviral genome and the proviral DNA have the three genes found in retroviruses: gag, pol and env, which are flanked by two long terminal repeat (LTR) sequences.
  • the gag gene encodes the internal structural (matrix, capsid and nucleocapsid) proteins; the pol gene encodes the RNA-directed DNA polymerase (reverse transcriptase) , a protease and an integrase; and the env gene encodes viral envelope glycoproteins .
  • the 5' and 3' LTR's serve to promote transcription and polyadenylation of the virion RNA's.
  • the LTR contains all other cis-acting sequences necessary for viral replication.
  • Lentiviruses have additional genes including vif, vpr, tat, rev, vpu, nef and vpx (in HIV-1, HIV-2 and/or SIV) .
  • Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the Psi site) . If the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the cis defect prevents encapsidation of genomic RNA. However, the resulting mutant remains capable of directing the synthesis of all virion proteins.
  • the vectors of interest are those which have an intact
  • a vector of interest contains a packaging signal sequence comprising the leader sequence downstream of the LTR and until the beginning of the gag gene.
  • the vector may also contain an additional portion of the gag gene to enhance packaging.
  • the vector of interest also includes a part of the env gene containing the Rev Response Element (RRE) , and it may or may not include a splice acceptor site downstream of the RRE.
  • the vectors of interest may contain one or more transgenes, or foreign nucleic acid, and preferably a transgene with anti-viral activity. However, a vector of interest need not contain a heterologous gene.
  • heterologous or foreign nucleic acid sequence is linked operably to a regulatory nucleic acid sequence.
  • heterologous nucleic acid sequence refers to a sequence that originates from a foreign species, or, if from the same species, it may be substantially modified from the original form. Alternatively, an unchanged nucleic acid sequence that is not expressed normally in a cell is a heterologous nucleic acid sequence.
  • operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • the heterologous sequence is linked to a promoter, resulting in a chimeric gene.
  • the heterologous nucleic acid sequence is preferably under control of either the viral LTR promoter-enhancer signals or of an internal promoter, and retained signals within the retroviral LTR can still bring about efficient expression of the transgene.
  • the foreign gene can be any transcribable nucleic acid of interest.
  • the foreign gene encodes a polypeptide.
  • the polypeptide has some therapeutic benefit.
  • the polypeptide may supplement deficient or nonexistent expression of an endogenous protein in a host cell.
  • the polypeptide can confer new properties on the host cell, such as a chimeric signalling receptor, see U.S. Pat. No. 5,359,046.
  • the artisan can determine the appropriateness of a foreign gene practicing techniques taught herein and known in the art. For example, the artisan would know whether a foreign gene is of a suitable size for encapsidation and whether the foreign gene product is expressed properly.
  • modulate it may be desirable to modulate the expression of a gene regulating molecule in a cell by the introduction of a molecule by the method of the invention.
  • modulate envisions the suppression of expression of a gene when it is over-expressed or augmentation of expression when it is under-expressed.
  • nucleic acid sequences that interfere with the expression of a gene at the translational level can be used.
  • the approach can utilize, for example, antisense nucleic acid, ribozymes or triplex agents to block transcription or translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or triplex agent, or by cleaving same with a ribozyrae.
  • the target of those molecules is the lentiviral RNA.
  • the RNA may be a sequence of the virus not present in the vector or that has been mutated in the vector.
  • Antisense nucleic acids are DNA or RNA molecules which are complementary to at least a portion of a specific mRNA molecule (Weintraub, Sci. Am. (1990) 262:40). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA forming a double-stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA since the cell will not translate a mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides or more are preferred since such are synthesized easily and are less likely to cause problems than larger molecules when introduced into the target cell. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (Marcus-Sakura, Anal. Biochem. (1988) 172:289).
  • the antisense nucleic acid can be used to block expression of a viral protein or a dominantly active gene product, such as amyloid precursor protein that accumulates in Alzheimer's disease. Such methods are also useful for the treatment of Huntington's disease, hereditary Parkinsonism and other diseases. Antisense nucleic acids are also useful for the inhibition of expression of proteins associated with toxicity.
  • oligonucleotide to stall transcription can be by the mechanism known as the triplex strategy since the oligomer winds around double-helical DNA, forming a three-strand helix. Therefore, the triplex compounds can be designed to recognize a unique site on a chosen gene (Maher et al., Antisense Res and Dev. (1991) 1(3):227; Helene, Anticancer Drug Dis. (1991) 6(6):569).
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode those RNA's, it is possible to engineer molecules that recognize and cleave specific nucleotide sequences in an RNA molecule (Cech, J. Amer. Med Assn. (1988) 260:3030). A major advantage of that approach is only mRNA's with particular sequences are inactivated.
  • nucleic acid encoding a biological response modifier.
  • immunopotentiating agents including nucleic acids encoding a number of the cytokines classified as “interleukins", for example, interleukins 1 through 12.
  • interferons include gamma interferon (7-IFN) , tumor necrosis factor (TNF) and granulocyte-macrophage colony stimulating factor (GM-CSF) .
  • TNF tumor necrosis factor
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • Nucleic acids encoding growth factors, toxic peptide ⁇ , ligands, receptors or other physiologically important proteins also can be introduced into cells.
  • the transgene also can be an inducible toxic molecule.
  • the method of the invention may also be useful for neuronal, glial, fibroblast or mesenchy al cell transplantation, or "grafting", which involves transplantation of cells infected with the recombinant lentivirus of the invention ex vivo, or infection in vivo into the central nervous system or into the ventricular cavities or subdurally onto the surface of a host brain.
  • grafting Such methods for grafting will be known to those skilled in the art and are described in Neural Grafting in the Mammalian CNS, Bjorklund & Stenevi, eds. (1985) .
  • gene transfer could introduce a normal gene into the affected tissues for replacement therapy, as well as to create animal models for the disease using antisense mutations. For example, it may be desirable to insert a
  • Factor VIII or IX encoding nucleic acid into a lentivirus for infection of a muscle, spleen or liver cell.
  • the promoter sequence may be homologous' or heterologous to the desired gene sequence.
  • a wide range of promoters may be utilized, including a viral or a mammalian promoter, and an inducible promoter.
  • Cell or tissue specific promoters can be utilized to target expression of gene sequences in specific cell populations. Suitable mammalian and viral promoters for the instant invention are available in the art.
  • the nucleic acid construct referred to as the transfer vector having the packaging signal and the heterologous cloning site, also contains a selectable marker gene.
  • Marker genes are utilized to assay for the presence of the vector, and thus, to confirm infection and integration. The presence of a marker gene ensures the selection and growth of only those host cells which express the inserts.
  • Typical selection genes encode proteins that confer resistance to antibiotics and other toxic substances, e.g., histidinol, puromycin, hygromycin, neomycin, methotrexate etc. and cell surface markers.
  • nucleic acid sequence refers to any nucleic acid molecule, preferably DNA, as discussed in detail herein.
  • the nucleic acid molecule may be derived from a variety of sources, including DNA, cDNA, synthetic
  • nucleic acid sequences may comprise genomic DNA which may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with promoter regions, poly A sequences or other associated sequences.
  • Genomic DNA may be extracted and purified from suitable cells by means well known in the art.
  • messenger RNA mRNA
  • mRNA messenger RNA
  • the recombinant lentivirus produced by the method of the invention is a derivative of human immunodeficiency virus (HIV) .
  • HIV human immunodeficiency virus
  • the vectors of interests are produced using known methods.
  • the vectors of interest can be introduced into cells either as the nucleic acid or encapsidated as a virus particle.
  • An artisan is familiar with methods for encapsidating a lentiviral vector of interest.
  • the vectors are introduced into target cells using methods known by those of skill in the art.
  • the vectors can be introduced into human cell lines by calcium phosphate transfection, lipofection or electroporation, generally together with a dominant selectable marker, such as neo, DHFR, Gin synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones.
  • a dominant selectable marker such as neo, DHFR, Gin synthetase or ADA
  • the selectable marker gene can be the transgene.
  • a likely means for transforming host cells with a vector of interest is by infecting cells with virus particles carrying a vector of interest.
  • the vector of interest would be encapsidated using known packaging systems, such as that taught in U.S. Pat. No. 5,686,279 and in Naldini et al. Science (1996) 272:263-267. Briefly, using either a stable packaging cell line or by transient transfection, the vector of interest is introduced into a cell which packages the vector of interest into viral particles.
  • the virus particles are obtained from the culture medium, treated as known in the art to provide a virus preparation.
  • the target cell then is exposed to the virus preparation. That can be via in vivo administration means, wherein the virus preparation is administered to a host, for example, in a parenteral form. Alternatively, cells from the host can be retrieved and maintained in culture where those cells are exposed to the virus preparation. Once transformed, stably or not, the cells then can be returned to the host.
  • the vector carry a transgene.
  • transgene is one which itself has a therapeutic effect.
  • the vectors of interest should have a place in current therapy of diseases associated with lentivirus.
  • Plasmids of the invention employs standard ligation and restriction techniques which are well understood in the art (see Maniatis et al., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1982). Isolated plasmids, DNA sequences or synthesized oligonucleotides are cleaved, tailored and religated in the form desired.
  • Site-specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are understood in the art, and the particulars of which are specified by the manufacturer of the commercially available restriction enzymes, see, e.g. New England Biolabs, Product Catalog.
  • suitable restriction enzyme or enzymes
  • about 1 ⁇ g of plasmid or DNA sequences is cleaved by one unit of enzyme in about 20 ⁇ l of buffer solution.
  • an excess of restriction enzyme is used to ensure complete digestion of the DNA substrate. Incubation times of about one hour to two hours at about 37 °C are workable, although variations can be tolerated.
  • cleaved fragments After each incubation, protein is removed by extraction with phenol/chloroform, which may be followed by ether extraction, and the nucleic acid recovered from aqueous fractions by precipitation with ethanol. If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general description of size separations is found in Methods of Enzymology 65:499-560 (1980).
  • Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTP's) using incubation times of about 15 to 25 minutes at 20°C in 50 mM Tris
  • Ligations can be performed in 15-50 ⁇ l volumes under the following standard conditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 33 mg/ml BSA, 10 mM-50 mM NaCl and either 40 ⁇ M ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0°C (for "sticky end” ligation) or 1 M ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14°C
  • Intermolecular "sticky end” ligations are usually performed at 33-100 ⁇ g/ l total DNA concentrations (5-100 mM total end concentration) .
  • Intermolecular blunt end ligations (usually employing a 10-30 fold molar excess of linkers) are performed at l ⁇ M total ends concentration.
  • Lentiviral vectors (Naldini et al., supra and Proc. Natl. Acad. Sci. (1996) 93:11382-11388) have been used to infect human cells growth-arrested in vitro and to transduce neurons after direct injection into the brain of adult rats.
  • the vector was efficient at transferring marker genes in vivo into the neurons and long term expression in the absence of detectable pathology was achieved.
  • the viral titer is determined by infection of, for example, 10 6 NIH 3T3 cells or 10 5 HeLa cells with an appropriate amount of viral supernatant, in the presence of 8 ⁇ g/ml polybrene (Sigma Chemical Co. , St. Louis, MO) . Forty-eight hours later, the transduction efficiency is assayed.
  • polybrene Sigma Chemical Co. , St. Louis, MO
  • the mechanism of the resistance was mapped to a post-integration step and shown to be dependent on an intact HIV LTR in the vector.
  • transcription from the vector LTR was enhanced greatly, resulting in increased expression of the transgene.
  • the vector RNA competes effectively with the viral RNA's both for binding the transactivators and for packaging by the budding viral particles, resulting in inhibition of viral replication and mobilization and spreading of the vector.
  • Viral particles collected from the infected transduced cells were less infectious than virus collected from infected non-transduced cells, and transferred efficiently the transgene into naive cells.
  • the instant vector will find use alone, either containing a transgene or not, and preferably the transgene has an antiviral activity; or in combination with another vector carrying a transgene with antiviral activity, wherein the instant vector does or does not contain a transgene.
  • the viral particles can be further purified from the viral supernatants as known in the art.
  • the viral particles or vector nucleic acid can be administered to a host with a disorder associated with or caused by a lentivirus using known techniques.
  • vector means both a bare recombinant lentiviral vector and recombinant lentiviral particle. Simply dissolving a vector in Hanks' balanced saline solution or phosphate buffered saline is sufficient to provide a solution useful for injection. There are no known restrictions on the carriers or other components that can be coadministered with the vector (although compositions that degrade the virion or polynucleotides thereof should be avoided in the normal manner with vectors) .
  • compositions can be prepared as injectable formulations to be delivered intramuscularly, including implantable pumps (known by those of skill in the art and described, for example, in U.S. Pat. No. 5,474,552). Numerous formulations for injection are known and can be used in the practice of the instant invention.
  • the vectors can be used with any pharmaceutically acceptable carrier for ease of administration and handling.
  • aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose.
  • Solutions of the vector as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.
  • a dispersion of viral particles also can be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, the preparations contain a preservative to prevent the growth of microorganisms.
  • the sterile aqueous media employed are obtainable by standard techniques well-known to those skilled in the art.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that administration by a syringe is possible.
  • the formulation must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) , suitable mixtures thereof and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the vector in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze drying which yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.
  • the therapeutic compounds of this invention may be administered to a host alone or in combination with pharmaceutically acceptable carriers.
  • pharmaceutically acceptable carriers As noted above, the relative proportions of active ingredient and carrier are determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmaceutical practice.
  • the dosage of the instant therapeutic agents which will be most suitable for prophylaxis or treatment will vary with the form of administration, the particular recombinant vector chosen and the physiological characteristics of the particular patient under treatment. Generally, small dosages will be used initially and, if necessary, will be increased by small increments until the optimum effect under the circumstances is reached. Exemplary dosages are within the range of 10 8 up to approximately 5 x 10 15 particles in a total volume of 3-10 ml.
  • Plasmid pHR'-CMV-Neo was derived by substituting the BamHI-XhoI fragment of pHR'-CMV-LacZ containing the E.coli LacZ gene with a BamHI-XhoI fragment containing the neomycin phosphotransferase gene of E.coli (Beck et al., Gene (1982) 19:327-336).
  • pHR2 is a lentiviral transfer vector in which 124 base pairs (bp) of nef sequences upstream of the 3' LTR in pHR have been replaced with a polylinker both to reduce HIV-1 sequences and to facilitate transgene cloning.
  • pHR2 was derived from pHR'-CMV-LacZ by replacing the 4.6 kilobase (kb) Clal-StuI fragment with an 828 bp Clal-StuI fragment generated by PCR using pHR'-CMV-LacZ as the template and with the o 1 igonuc 1 eot ide primer, 5'-
  • TTATAATGTCAAGGCCTCTC-3 ' (SEQ ID NO: ) in a three part ligation with a 4.4 kb Stul-Ncol fragment and a 4.5 kb Ncol-Clal fragment from pHR'-CMV-LacZ.
  • Plasmid pHR2-PGK-GFP was derived by cloning the XhoI-BamHI fragment of pRT43.3PGKF3 (WO 97/07225) containing the human PGK promoter (GenBank Accession number #M11958 nucleotides 2-516) and the Ba HI-NotI fragment of plasmid of pEGFPl (Clontech) containing a codon usage-optimized and improved version of the Green Fluorescent Protein (GFP) of A. victoria and a Notl-SacII linker, into the Xhol and SacII sites of pHR2.
  • GFP Green Fluorescent Protein
  • Human SupTl T-lymphoblastoid cells were obtained by ATCC.
  • Human CD4 + primary blood lymphocytes (PBL) were separated from buffy coats from donations, stimulated with 2.5 ⁇ g/ml phytohemagglutinin or Dynal beads coated with OKT3 and CD28 antibodies for 2 days, then washed and cultured with 100 U/ml of interleukin 2 (Chiron) in AIM-V medium (Gibco) .
  • the SupTl cells or PBL were transduced either with lentivector or a urine leukemia virus (MLV) vector carrying the same transgene overnight in the presence of 2 ⁇ g/ml polybrene, then washed and selected for transgene expression after 48 hrs.
  • MMV urine leukemia virus
  • Cells transduced with vectors carrying the neomycin resistance gene were selected in medium containing 1 mg/ml G418, then cultured in normal medium for virus challenge.
  • Cells transduced with vectors carrying the green fluorescent protein (GFP) as transgene were selected by cell sorting.
  • GFP green fluorescent protein
  • HIV-1 virions were produced either by 293T cells transfected with the proviral infectious molecular clone R8, or by SupTl cells chronically infected with R8 virus.
  • R8 is a ly phocytotropic HIV-1 hybrid of the HXB2-D and NL43 isolates that expresses all HIV reading frames (Gallay et al. , Cell (1995) 83:569-576).
  • the virus stock was titered on HeLa P4 cells and had an infectivity of 1,000 to 3,000 infectious units/ng p24.
  • the cells were washed twice after overnight incubation with the virus in the presence of 2 ⁇ g/ml polybrene, and further cultured for up to 3 weeks. Every 3-4 days, the conditioned medium was collected and HIV replication was determined by accumulation of HIV-1 Gag p24 in the medium by a commercially available ELISA kit (DuPont) .
  • CD4 + PBL's were transduced with either lentivector (pHR2-PGK-GFP) or the MLV retrovector carrying the same GFP transgene driven by the human PGK promoter, and sorted for transgene expression.
  • the selected populations then were challenged with HIV virus as described above. Both the non-transduced cells (indicated in the figure by diamonds) and sorted cells transduced by the MLV retrovector (indicated by squares) yielded similar levels of p24 antigen in the culture medium.
  • the cells transduced by the lentiviral yielded sharply reduced p24 even after inoculation with high doses of HIV (100 ng p24 equivalent of virus) .
  • transgene expression was augmented significantly after infection with the HIV virus.

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Abstract

L'invention porte sur un vecteur lentiviral inhibant la propagation d'un lentivirus dans une cellule.
PCT/US1998/025720 1997-12-12 1998-12-11 Emploi therapeutique de vecteurs lentiviraux WO1999030742A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
AU18035/99A AU749059B2 (en) 1997-12-12 1998-12-11 Therapeutic use of lentiviral vectors
CA002314683A CA2314683A1 (fr) 1997-12-12 1998-12-11 Emploi therapeutique de vecteurs lentiviraux
KR1020007006423A KR20010033062A (ko) 1997-12-12 1998-12-11 렌티바이러스성 벡터의 치료학적 용도
JP2000538720A JP2002508338A (ja) 1997-12-12 1998-12-11 レンチウィルスベクターの治療使用
EP98962895A EP1037669A4 (fr) 1997-12-12 1998-12-11 Emploi therapeutique de vecteurs lentiviraux

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US6957997P 1997-12-12 1997-12-12
US60/069,579 1997-12-12

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WO1999030742A1 true WO1999030742A1 (fr) 1999-06-24

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Cited By (10)

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US6656706B2 (en) 1999-12-23 2003-12-02 The United States Of America As Represented By The Department Of Health And Human Services Molecular clones with mutated HIV gag/pol, SIV gag and SIV env genes
US6712612B1 (en) 2000-12-12 2004-03-30 Genecure Llc Safe and stable retroviral helper cell line and related compositions and methods
EP1741782A2 (fr) 2000-05-10 2007-01-10 Sanofi Pasteur Limited Polypeptides immunogéniques codés par des minigènes mage et leurs utilisations
EP1964573A2 (fr) 1999-10-22 2008-09-03 Aventis Pasteur Limited Procédé d'induction et/ou amélioration d'une réponse immune vers des antigènes de tumeurs
EP2181704A2 (fr) 2002-12-30 2010-05-05 Angiotech International Ag Liberation de medicaments a partir d'une compostion polymere a gelification rapide
US8501464B2 (en) 2003-04-24 2013-08-06 Ospedale San Raffaele S.R.L. Lentiviral vectors carrying synthetic bi-directional promoters and uses thereof
US9593340B2 (en) 2007-10-15 2017-03-14 Admedus Vaccines Pty Ltd. Expression system for modulating an immune response
WO2017096432A1 (fr) 2015-12-09 2017-06-15 Admedus Vaccines Pty Ltd Composition immunomodulatrice pour le traitement
US9795658B2 (en) 2010-04-20 2017-10-24 Admedus Vaccines Pty Ltd Expression system for modulating an immune response
US11285191B2 (en) 2016-07-26 2022-03-29 The Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Immunostimulatory compositions and uses therefor

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5650309A (en) * 1995-05-16 1997-07-22 The Regents Of The University Of California Viral vectors

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996028565A1 (fr) * 1995-03-16 1996-09-19 Hisamitsu Pharmaceutical Co., Inc. Nouveau recombinant genique
US6013516A (en) * 1995-10-06 2000-01-11 The Salk Institute For Biological Studies Vector and method of use for nucleic acid delivery to non-dividing cells
EP0871757B1 (fr) * 1995-11-28 2003-03-12 The Johns Hopkins University School Of Medicine Replication conditionnelle de vecteurs viraux et leur utilisation
WO1997020052A1 (fr) * 1995-11-28 1997-06-05 Clinical Technologies, Inc. Vih recombine, cellules d'encapsidation modifiees et methode de traitement du syndrome d'immunodeficience acquise
EP0882134B8 (fr) * 1996-02-21 2009-09-02 Genetic Immunity kft. Procedes et compositions pour l'immunisation genetique protectrice et therapeutique
AU723313B2 (en) * 1996-03-05 2000-08-24 Regents Of The University Of California, The Recombinant live feline immunodeficiency virus and proviral DNA vaccines
DE69737597D1 (de) * 1996-07-22 2007-05-24 Us Gov Health & Human Serv Vektoren zur inhibierung von viralem und tumorwachstum
JP2001513643A (ja) * 1997-03-06 2001-09-04 ウーベルラ、クラウス レンチウイルスをベースとするベクター及びベクター系
GB9704977D0 (en) * 1997-03-11 1997-04-30 Univ Glasgow FIV vaccine
EP1003894A2 (fr) * 1997-07-18 2000-05-31 Chiron Corporation Vecteurs lentiviraux

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5650309A (en) * 1995-05-16 1997-07-22 The Regents Of The University Of California Viral vectors

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BERKHOUT B, SILVERMAN R H, JEANG K-T: "TAT TRANS-ACTIVATES THE HUMAN IMMUNODEFICIENCY VIRUS THROUGH A NASCENT RNA TARGET", CELL, CELL PRESS, US, vol. 59, 20 October 1989 (1989-10-20), US, pages 273 - 282, XP002916548, ISSN: 0092-8674, DOI: 10.1016/0092-8674(89)90289-4 *
LISZIEWICHZ J, ET AL.: "INHIBITION OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 REPLICATION BY REGULATED EXPRESSION OF A POLYMERIC TAT ACTIVATION RESPONSE RNA DECOY AS A STRATEGY FOR GENE THERAPY IN AIDS", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 90, 1 September 1993 (1993-09-01), US, pages 8000 - 8004, XP002916547, ISSN: 0027-8424, DOI: 10.1073/pnas.90.17.8000 *
See also references of EP1037669A4 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1964573A2 (fr) 1999-10-22 2008-09-03 Aventis Pasteur Limited Procédé d'induction et/ou amélioration d'une réponse immune vers des antigènes de tumeurs
US6656706B2 (en) 1999-12-23 2003-12-02 The United States Of America As Represented By The Department Of Health And Human Services Molecular clones with mutated HIV gag/pol, SIV gag and SIV env genes
US8076100B2 (en) 1999-12-23 2011-12-13 The United States Of America As Represented By The Department Of Health And Human Services Molecular clones with mutated HIV gag/pol, SIV gag and SIV env genes
EP1741782A2 (fr) 2000-05-10 2007-01-10 Sanofi Pasteur Limited Polypeptides immunogéniques codés par des minigènes mage et leurs utilisations
US6712612B1 (en) 2000-12-12 2004-03-30 Genecure Llc Safe and stable retroviral helper cell line and related compositions and methods
EP2181704A2 (fr) 2002-12-30 2010-05-05 Angiotech International Ag Liberation de medicaments a partir d'une compostion polymere a gelification rapide
US8501464B2 (en) 2003-04-24 2013-08-06 Ospedale San Raffaele S.R.L. Lentiviral vectors carrying synthetic bi-directional promoters and uses thereof
US9593340B2 (en) 2007-10-15 2017-03-14 Admedus Vaccines Pty Ltd. Expression system for modulating an immune response
US9795658B2 (en) 2010-04-20 2017-10-24 Admedus Vaccines Pty Ltd Expression system for modulating an immune response
WO2017096432A1 (fr) 2015-12-09 2017-06-15 Admedus Vaccines Pty Ltd Composition immunomodulatrice pour le traitement
US11285191B2 (en) 2016-07-26 2022-03-29 The Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Immunostimulatory compositions and uses therefor

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JP2002508338A (ja) 2002-03-19
KR20010033062A (ko) 2001-04-25
AU1803599A (en) 1999-07-05
EP1037669A4 (fr) 2003-04-02
EP1037669A1 (fr) 2000-09-27
AU749059B2 (en) 2002-06-20

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