WO1997048277A1 - Vecteurs retroviraux a structure modulaire et leurs utilisations - Google Patents

Vecteurs retroviraux a structure modulaire et leurs utilisations Download PDF

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WO1997048277A1
WO1997048277A1 PCT/US1997/008805 US9708805W WO9748277A1 WO 1997048277 A1 WO1997048277 A1 WO 1997048277A1 US 9708805 W US9708805 W US 9708805W WO 9748277 A1 WO9748277 A1 WO 9748277A1
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ltr
transactivator
promoter
retroviral
construct according
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PCT/US1997/008805
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WO1997048277A9 (fr
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Fred H. Gage
Steven T. Suhr
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The Salk Institute For Biological Studies
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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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the present invention relates to novel retroviral constructs, methods for the preparation thereof, as well as various uses therefor.
  • retroviruses for gene therapy are maintaining transgene expression after cellular infection.
  • mitotically active cells in culture such as skin fibroblasts, hepatocytes, or astroglia
  • retroviruses readily infect and express the integrated transgene.
  • expression of the retroviral transcriptional cassette is blocked.
  • retroviral genomes are introduced into embryonic cells, either by normal viral infection or by injection of proviral DNA into the pronucleus, the virus is able to integrate into the host genome, but transgenes introduced in this way are not expressed.
  • Cells with regulated transgenes have historically been produced by the simultaneous integration of several plasmids into the host cell by transfection. This results in the formation of very few stably transduced cells having all of the required components for regulation intact. Moreover, since variable numbers of plasmids integrate into the host cell genome, individual cells may have very different inductive properties.
  • viruses that have potentially much greater range of application to gene therapy, production of transgenic animals, and gene transfer to developing embryos than previously available retroviral vectors.
  • retroviral vectors having modified long terminal repeats (LTRs) which enable high level and ligand-modulatable expression of a desired gene product, even after prolonged periods of cellular quiescence.
  • LTRs long terminal repeats
  • Invention vectors overcome proviral transcriptional inactivation which occurs in cultured primary cells that are growth arrested due to environmental constraints such as contact inhibition and/or nutrient starvation.
  • Invention vectors represent a class of retroviral vectors in which LTR-promoted proviral expression in infected cells may be maintained or increased, even in situations generally considered to be non-permissive for retroviral vectors.
  • gene transfer vectors with the capacity for prolonged transgene expression for either in vivo or ex vivo gene therapy as gene transfer vectors with the capacity for modulated transgene expression for either in vivo or ex vivo gene therapy application
  • gene transfer vectors for efficient production of transgenic animals as vectors for efficient gene transfer to developing embryos; as vectors with inducible high titers; and the like.
  • Figure 1 provides a schematic representation of retroviral vectors according to the invention (designated
  • LTR refers to retroviral iong-terminal repeats
  • 4E refers to a modified
  • LTR containing ecdysone response elements EcREs
  • ⁇ refers to retroviral packaging signals
  • Neo r and l, L-His r
  • CMV cytomegalovirus and SV40 promoters, respectively
  • IRS internal ribosomal entry site
  • RXR refers to the human RXR alpha open reading .frame (ORF)
  • Transgene refers to any gene of interest.
  • FIG. 2 presents a summary of the inductive properties of native and hybrid EcR, RXR ⁇ , and Usp constructs in the presence and absence of an ecdysteroid (e.g., 1 ⁇ M muristerone A (MurA) ) .
  • Figure 2A presents a schematic of the receptors used in transient transfection experiments.
  • RXR ⁇ refers to the human retinoid X receptor alpha
  • EcR refers to the Drosophila ecdysone receptor
  • Usp refers to the Drosophila ultraspiracle receptor
  • V refers to the fused 80 amino acid T domain of HSV VP16.
  • Figure 2B presents the luciferase activity induced in the presence or absence of MurA, employing native proteins involved in ecdysteroid response (i.e., EcR, RXR, Usp) , either alone or in combination.
  • native proteins involved in ecdysteroid response i.e., EcR, RXR, Usp
  • Figure 2C presents the luciferase activity induced in the presence or absence of MurA, employing either native or hybrid receptor-complex protein combinations, wherein hybrid receptors comprise N-terminal fusions containing the HSV VP16 T domain (referred to herein as CVRXR, CVUsp, CVEcR, CofVEcR, C2VE, EcRV and CVEnV; see Figure 2A) .
  • Figure 2D presents the luciferase activity induced in the presence or absence of MurA, employing EcR hybrids containing the VP16 T domain, in combination with native hRXR ⁇ .
  • Figure 3 illustrates the ecdysteroid-induced transactivation of native and mutant LTRs containing ecdysteroid response elements.
  • Figure 3A presents a schematic representation of the native MLV LTR (designated in the Figure as 3'LTR).
  • the LTR is composed of the U3 region (containing Nhel and Xbal sites) , the core enhancers, basal transcriptional activation signals, and the R and U5 regions. Native R and U5 regions were left unchanged in these constructs. Transcription is initiated at the U5-R border (depicted with an arrow) .
  • Enhancerless constructs were produced by simultaneous digestion of both the Xbal and Nhel sites, followed by religation with or without tandem 24-bp EcREs (depicted by an E) . Constructs with intact enhancers were digested at either the Xbal or Nhel site, and tandem EcREs inserted as shown in the figure.
  • Figure 3B presents the luciferase activity induced in the presence or absence of 1 ⁇ M MurA, employing mutant and native LTR constructs transiently transfected into CV-1 cells.
  • Figure 3C presents a comparison of the luciferase activity induced by MurA employing the enhancer-containing construct, 4X, in two different cell types, CV-1 and PA317.
  • constructs comprising: a retroviral psi ( ⁇ ) packaging signal, a 5' long terminal repeat (LTR) and a 3' LTR, wherein one or both of said LTRs comprises an exogenous regulatory element responsive to a transactivator, a transactivator under the expression control of a promoter, a heterologous gene encoding a protein of interest, wherein said heterologous gene is maintained under the control of said regulatory element responsive to said transactivator, and optionally, a selectable marker.
  • LTR 5' long terminal repeat
  • RNA derived from such constructs can be packaged in infectious virions employing standard techniques, e.g., by passaging such constructs through packaging cell lines, as described, for example, by Miller in Human Gene Therapy 1:5-14 (1990).
  • retroviral psi ( ⁇ ) packaging signals useful in the practice of the present invention can be obtained from any of a variety of sources, such as, for example, MLV, HIV, SIV, RSV, MMTV, Foamy virus, and the like.
  • Exemplary psi ( ⁇ ) packaging signals contemplated for use herein include ⁇ -proline, ⁇ -glutamine, ⁇ -plus, and the like.
  • Retroviral long terminal repeats contemplated for use in the practice of the present invention can be obtained from a wide variety of sources.
  • 5' LTRs and 3' LTRs employed for the preparation of invention constructs can independently be obtained from MLV, HIV, SIV, RSV, MMTV, Foamy virus, and the like.
  • 5' LTRs and/or 3' LTRs employed for the preparation of invention constructs comprise an exogenous regulatory element responsive to the transactivator present in the construct.
  • One such exogenous regulatory element is an operator which is responsive to a ligand-mediated receptor which confers responsiveness to antibiotics.
  • Exemplary operators contemplated for use in this aspect of the invention include the tetracycline-analog regulated operator, the TET operator, the Lac operator, and the like.
  • Additional operators contemplated for use herein include response elements which are bound by fusion variants of a ligand-mediated member of the steroid/thyroid superfamily of receptors, such as the progesterone/GAL4 fusion transactivator (which binds the GAL4 response element) , and the like.
  • presently preferred sites for insertion of operators into native MLV LTRs are Nhel and Xbal.
  • 5' LTRs and/or 3' LTRs employed for the preparation of invention constructs comprise at least one hormone response element.
  • the 5' LTRs and/or the 3' LTRs comprise a plurality of hormone response elements.
  • Hormone response elements contemplated for use in the practice of the present invention can readily be inserted into any convenient restriction site in either the 5' or 3' LTR.
  • presently preferred sites for insertion of hormone response elements into native MLV LTRs are Nhel and Xbal.
  • Hormone response elements contemplated for use herein typically comprise at least two half-sites (in either direct repeat or inverted repeat orientation to one another) , separated by a spacer of 0-5 nucleotides.
  • half-site refers to a contiguous 6 nucleotide sequence that is bound by a particular member of the steroid/thyroid superfamily of receptors. Each half- site is typically separated by a spacer of 0 up to about 5 nucleotides. Typically, two half-sites with a corresponding spacer make up a hormone response element.
  • Hormone response elements can be incorporated in multiple copies into various transcription regulatory regions.
  • Preferred hormone response elements employed in the practice of the present invention comprise a first half-site and a second half-site, separated by a spacer of 0-5 nucleotides; wherein each half-site has the sequence:
  • each M is independently selected from A or C; with the proviso that at least 4 nucleotides of each -RGBNNM- group of nucleotides are identical with the nucleotides at comparable positions of the sequence -AGGTCA-.
  • Exemplary half-sites having the -RGBNNM- motif for use in preparing response elements useful in the practice of the present invention include, for example, half-sites selected from -AGGGCA-, -AGTTCA-, -AGGTAA-, -AGGTCA-, -GGTTCA-, -GGGTTA-, -GGGTGA-, -AGGTGA-, -GGGTCA-, and the like.
  • a particularly preferred first half-site is -AGTGCA-.
  • Ecdysone response elements contemplated for use herein are short cis-acting sequences (i.e., having about
  • ecdysone 12-20 bp
  • a suitable ligand such as ecdysone or muristerone A
  • ecdysone response element has been previously described, see, e.g., Yao et al., Cell., 71:63-
  • 5' LTRs and/or 3' LTRs employed for the preparation of invention constructs can be modified by the deletion therefrom of enhancer sequences. Such constructs are particularly useful for expression of heterologous genes where very low background levels are desired in the absence of induction.
  • Transactivators contemplated for use in the practice of the present invention can be selected from: ligand-mediated members of the steroid/thyroid superfamily of receptors, or fusion variants thereof containing at least the ligand binding domain of a member of the superfamily, ligand-mediated receptors which confer responsiveness to antibiotics, constitutive transactivators, and the like.
  • Ligand-mediated members of the steroid/thyroid superfamily of receptors contemplated for use in the practice of the present invention include hormone binding proteins that operate as ligand-dependent transcription factors, including identified members of the steroid/thyroid superfamily of receptors for which specific ligands have not yet been identified (referred to hereinafter as "orphan receptors") .
  • Exemplary members of the steroid/thyroid superfamily of receptors include steroid receptors such as glucocorticoid receptor (GR) , mineralocorticoid receptor (MR) , estrogen receptor (ER) , progesterone receptor (PR) , androgen receptor (AR) , vitamin D 3 receptor (VDR) , and the like; plus retinoid receptors, such as the various isoforms of retinoic acid receptor (e.g., RAR ⁇ , RAR / 3, or RAR ⁇ ) , the various isoforms of retinoid X receptor (e.g., RXR ⁇ , RXR/3, or RXR ⁇ ) , and the like (see, e.g., U.S.
  • GR glucocorticoid receptor
  • MR mineralocorticoid receptor
  • ER estrogen receptor
  • PR progesterone receptor
  • AR vitamin D 3 receptor
  • retinoid receptors such as the various isoforms of
  • TR thyroid receptor
  • TR ⁇ TR ⁇
  • insect derived receptors such as the ecdysone receptor, and the like
  • other gene products which, by their structure and properties, are considered to be members of the superfamily, as defined hereinabove, including the various isoforms thereof.
  • DNA-binding domains of all members of the steroid/thyroid superfamily of receptors are related, consisting of 66-68 amino acid residues, and possessing about 20 invariant amino acid residues, including nine cysteines.
  • a member of the superfamily can be characterized as a protein which contains these 20 invariant amino acid residues.
  • the highly conserved amino acids of the DNA-binding domain of members of the superfamily are as follows:
  • X designates non-conserved amino acids within the DNA-binding domain; an asterisk denotes the amino acid residues which are almost universally conserved, but for which variations have been found in some identified hormone receptors; and the residues enclosed in parenthesis are optional residues (thus, the DNA-binding domain is a minimum of 66 amino acids in length, but can contain several additional residues) .
  • a presently preferred receptor contemplated for use herein is the insect-derived ecdysone receptor (as well as fusion variants thereof) , since this receptor (as well as ligand and response elements therefor) is not endogenous to mammalian cells contemplated for treatment in accordance with the present invention.
  • Fusion variants of ligand-mediated members of the steroid/thyroid superfamily of receptors contemplated herein include fusion proteins comprising at least the ligand binding domain of a member of the superfamily and a DNA binding domain capable of binding a response element not endogenous to the LTR.
  • fusion proteins are progesterone/GAL4 chimera.
  • transactivators contemplated for use herein include, e.g., homeobox proteins, zinc finger proteins, hormone receptors, helix-turn-helix proteins, helix-loop- helix proteins, basic-Zip proteins (bZip) , / 0-ribbon factors, and the like. See, for example, Harrison, S., "A Structural Taxonomy of DNA-binding Domains," Nature. 353:715-719.
  • Homeobox DNA-binding proteins suitable for use herein include, for example, HOX, STF-1 (Leonard et al., 1993, Mol. Endo.. 7:1275-1283), Antp, Mat ⁇ -2, INV, and the like. See, also, Scott et al.
  • Constitutive transactivators contemplated for use in the practice of the present invention include ecdysone receptors containing multiple Vpl6 activation domains, Vpl6-GAL4 fusions, tetracycline transactivator variants, TTA variants, and the like.
  • Transactivators contemplated for use in the practice of the present invention may function in the presence or absence of a heterologous partner.
  • Examples of transactivators that typically function in the absence of a heterologous partner are tetracycline-controlled transactivators, steroidogenic factor-1 (SF-1) , nerve growth factor-IB (NGF-1B) , and the like.
  • many members of the steroid/thyroid superfamily of receptors typically function in the presence of heterologous partners therefor, i.e., as multimers.
  • at least one member of such multimeric species is a member of the steroid/thyroid superfamily.
  • Such multimeric species commonly comprise a member of the steroid/thyroid superfamily, associated with a silent partner therefor.
  • Exemplary silent partners include RXR, Usp, and the like.
  • transactivators and/or multimeric partners therefor can be further modified by the introduction of activation domains thereto.
  • Activation domains contemplated for use herein are typically derived from transcription factors and comprise a contiguous sequence of amino acids that functions to activate gene expression when associated with a suitable DNA-binding domain and a suitable ligand binding domain.
  • the activation domain can be positioned at any convenient site within the transactivator, i.e., at the carboxy terminus, the amino terminus or between the ligand binding domain and the DNA binding domain of the transactivator.
  • Suitable activation domains can be obtained from a variety of sources, e.g., from the N-terminal region of a member of the steroid/thyroid superfamily of receptors, from a transcription factor activation domain, such as, for example, VP16 or GAL4 activation domains, and the like.
  • the presently most preferred activation domain contemplated for use in the practice of the present invention is obtained from the C-terminal region of the VP16 protein.
  • Promoters contemplated for control of expression of transactivators employed in the practice of the present invention include inducible, constitutive and/or tissue specific promoters.
  • Inducible promoters contemplated for use in the practice of the present invention comprise transcription regulatory regions that do not function to transcribe mRNA unless inducing conditions are present.
  • suitable inducible promoters include DNA sequences corresponding to: the E. coli lac operator responsive to IPTG (see Nakamura et al., Cell. 18:1109-1117, 1979); the metallothionein promoter metal-regulatory-elements responsive to heavy-metal (e.g. zinc) induction (see Evans et. al, U.S. Patent No. 4,870,009), the phage T71ac promoter responsive to IPTG (see Studier et al., Meth. Enzvmol.. 185: 60-89, 1990; and U.S. #4,952,496), the heat- shock promoter; the TK minimal promoter; the CMV minimal promoter; a synthetic promoter; and the like.
  • Exemplary constitutive promoters contemplated for use in the practice of the present invention include the CMV promoter, the SV40 promoter, the DHFR promoter, and the like.
  • tissue specific promoters contemplated for use in the practice of the present invention include the GH promoter, the NSE promoter, the GFAP promoter, neurotransmitter promoters (e.g., tyrosine hydroxylase, TH, choline acetyltransferase, ChAT, and the like) , promoters for neurotropic factors (e.g. , a nerve growth factor promoter, NT-3, BDNF promoters, and the like), and so on.
  • neurotransmitter promoters e.g., tyrosine hydroxylase, TH, choline acetyltransferase, ChAT, and the like
  • promoters for neurotropic factors e.g. , a nerve growth factor promoter, NT-3, BDNF promoters, and the like
  • Heterologous genes contemplated for use in the practice of the present invention include wild type genes and/or a therapeutic genes.
  • Exemplary wild type genes are genes which encode products: the substantial absence of which leads to the occurrence of a non-normal state in a subject; or a substantial excess of which leads to the occurrence of a non-normal state in a subject.
  • Wild type genes are those that are native to cells of a particular type. Such genes may be undesirably overexpressed, or may not be expressed in biologically significant levels.
  • a synthetic or natural gene coding for human insulin would be exogenous genetic material to a yeast cell (since yeast cells do not naturally contain insulin genes)
  • a human insulin gene inserted into a human skin fibroblast cell would be a wild type gene with respect to that cell since human skin fibroblasts contain genetic material encoding human insulin, although human skin fibroblasts do not express human insulin in biologically significant levels.
  • Therapeutic genes contemplated for use in the practice of the present invention include those which encode products: which are toxic to the cells in which they are expressed; or which impart a beneficial property to a subject.
  • therapeutic gene refers to a gene which imparts a beneficial function to the host in which such gene is expressed.
  • Therapeutic genes are those that are not naturally found in host cells. For example, a synthetic or natural gene coding for wild type human insulin would be therapeutic when inserted into a skin fibroblast cell so as to be expressed in a human host, where the human host is not otherwise capable of expressing functionally active human insulin in biologically significant levels.
  • therapeutic genes are expressed at a level that provides a therapeutically effective amount of the corresponding therapeutic protein.
  • Heterologous genes useful in the practice of the present invention include genes that encode biologically active proteins of interest, such as, e.g., secretory proteins that can be released from said cell; enzymes that can metabolize a toxic substance to produce a non-toxic substance, or that metabolize an inactive substance to produce a useful substance; regulatory proteins; cell surface receptors; and the like.
  • Useful genes include genes that encode blood clotting factors such as human factors VIII and IX; genes that encode hormones such as insulin, parathyroid hormone, luteinizing hormone releasing factor (LHRH) , alpha and beta seminal inhibins, and human growth hormone; genes that encode proteins such as enzymes, the absence of which leads to the occurrence of an abnormal state; genes encoding cytokines or lymphokines such as interferons, granulocytic macrophage colony stimulating factor (GM-CSF) , colony stimulating factor-1 (CSF-1) , tumor necrosis factor (TNF) , and erythropoietin (EPO) ; genes encoding inhibitor substances such as alpha.,-antitrypsin; genes encoding substances that function as drugs, e.g., genes encoding the diphtheria and cholera toxins; and the like.
  • cytokines or lymphokines such as interferons, granulocytic macrophage colony stimulating factor
  • nucleic acid sequence information for a desired protein can be located in one of many public access databases, e.g., GENBANK, EMBL, Swiss-Prot, and PIR, or in many biology related journal publications. Thus, those of skill in the art have access to nucleic acid sequence information for virtually all known genes. Those of skill in the art can either obtain the corresponding nucleic acid molecule directly from a public depository or the institution that published the sequence.
  • the skilled artisan can employ routine methods, e.g., polymerase chain reaction (PCR) amplification, to isolate the desired nucleic acid molecule from the appropriate nucleic acid library.
  • PCR polymerase chain reaction
  • antibiotic resistance genes genes which enable cells to process metabolic intermediaries, and the like.
  • exemplary antibiotic resistance genes include genes which impart tetracycline resistance, genes which impart ampicillin resistance, neomycin resistance, hygromycin resistance, puroroycin resistance, and the like.
  • genes which enable cells to process metabolic intermediaries include genes which enable cells to incorporate L-histidinol, genes encoding thymidine kinase, genes encoding xanthine-guanine phosphoribosyl 97/48277 PC17US97/08805
  • Additional components which can optionally be incorporated into invention constructs include genes encoding those proteins required for retroviral packaging, e.g., the pol gene, the gag gene and the env gene.
  • MARVs for "modular assembly retroviral vectors”
  • LTR promoters that respond to specific constitutive or ligand-dependent transcription factors encoded by nucleic acids which have been introduced into the recombinant retroviral vectors.
  • MARV vectors Three general elements combine to form MARV vectors: 1) native or mutated LTRs containing regulatory elements responsive to a transactivator, wherein said regulatory elements are inserted into various locations within the promoter, 2) transactivator(s) , optionally modified to provide a user defined level of expression in the absence of ligand and higher-level expression in the presence of ligand, and 3) retroviral packaging signal, wherein these elements are arranged with endogenous promoters and (optionally) selectable marker genes to result in MARV viruses with other user-definable characteristics.
  • the first generation of MARV vectors were designed to respond to insect hormones (known as ecdysteroids) to stimulate transcription from the viral LTR.
  • ecdysteroids insect hormones
  • An example of a receptor complex for ecdysteroids is composed of the Drosophila ecdysone receptor (EcR) and the human retinoid X receptor (RXR) or the Drosophila ultraspiracle receptor.
  • EcR Drosophila ecdysone receptor
  • RXR human retinoid X receptor
  • a hybrid EcR CofVEcR; see Figure 2A
  • This receptor combination results in 20-100-fold transcription induction in the presence of the ecdysteroid muristerone A.
  • the two-plasmid system is typically provided with antibiotic resistance markers, which enable the selection and characterization of infected cells in vitro.
  • a retroviral vector system which, when introduced into the genome of a suitable host along with a co-functioning partner therefor, enables ligand-mediated expression of an exogenous protein.
  • One vector of the invention vector system comprises: a retroviral psi ( ⁇ ) packaging signal, a 5' long terminal repeat (LTR) and a 3' LTR, wherein one or both of said LTRs optionally comprises an exogenous regulatory element responsive to a transactivator, a transactivator under the expression control of a promoter, and optionally, a selectable marker, and
  • the other vector of the invention vector system i.e., the co-functioning partner for the above-described vector
  • MARSHA comprises: a retroviral psi ( ⁇ ) packaging signal, a 5' long terminal repeat (LTR) and a 3' LTR, wherein one or both of said LTRs comprises an exogenous regulatory element responsive to a transactivator, a heterologous gene encoding a protein of interest, wherein said heterologous gene is maintained under the expression control of said regulatory element responsive to said transactivator, and optionally, a selectable marker.
  • the above-described vectors can be introduced into a suitable host at the same time, or sequentially, employing techniques which are well known in the art.
  • the vector containing the heterologous gene could be introduced into the host first, followed by the vector encoding the transactivator.
  • the vector encoding the transactivator could be introduced into the host first, followed by the vector containing the heterologous gene.
  • a MARV vector can be designed to encode both subunits of a dimeric receptor and an antibiotic resistance gene (see Figure 1) .
  • a "covector” (referred to herein as MARSHA) is designed to encode the heterologous gene and a second antibiotic resistance gene (see Figure 1) .
  • the MARSHA vector carrying the heterologous gene also has LTRs modified to promote high- level expression only in the presence of the MARV encoded transactivator and exogenous ligand (see Figure 1) .
  • Co- infected primary mammalian cells can then be selected using both antibiotics, resulting in a cell population that is dependent on ligand for high-level expression of the heterologous gene.
  • heterologous gene(s) By introducing all of the necessary regulatory machinery, plus heterologous genes, selectable markers, and receptors, on the MARV retroviruses, highly efficient insertion of heterologous gene(s) into targeted cells can be achieved.
  • This characteristic of the invention viral system results in individual cells with similar properties resulting in general cell populations with reproducibly characteristic properties of heterologous gene induction. The rapidity and reproducibility of this system makes it useful for ex vivo gene therapy (i.e., for gene transfer to primary cells) , particularly in cases where speed is critical to disease treatment.
  • the above-described viral constructs address several important problems confronted in the use of retroviruses in application of therapeutic gene transfer strategies to a variety of human diseases.
  • the retroviral vectors of the invention are capable of prolonged gene expression under conditions where conventional integrated retroviruses are no longer transcriptionally active.
  • the present invention allows the level of gene expression to be regulated through the application of exogenous factor(s) .
  • exogenous factor(s) In many applications of gene transfer to ameliorate a disease phenotype, only intermittent expression of the therapeutic heterologous gene may be necessary or even desirable.
  • the level of heterologous gene expression may be modified.
  • individual ligand-responsive LTRs have different characteristics of expression which may be included depending on the specific application. For example, the 4X LTR (see Figure 3A) has a high level of basal expression, which can be increased even further through the application of muristerone A (MurA) .
  • the 4E LTR has low basal expression, which can be substantially increased through treatment with hormone.
  • the basal and induced expression levels of transgene expression may be "customized".
  • a method of making a modular assembly retroviral vector comprising individually introducing each of the following components into a separate restriction site(s) of DNA engineered to have multiple restriction sites: a retroviral psi ( ⁇ ) packaging signal, a 5' long terminal repeat (LTR) and a 3' LTR, wherein one or both of said LTRs comprises an exogenous regulatory element responsive to a transactivator, a transactivator under the expression control of a promoter, a heterologous gene encoding a protein of interest, wherein said heterologous gene is maintained under the control of a regulatory element responsive to said transactivator, and optionally, a selectable marker.
  • a variety of additional components can optionally be incorporated into the above-described modular assembly retroviral vectors, such as, for example, genes encoding those proteins required for retroviral packaging, e.g., the pol gene, the gag gene and the env gene.
  • invention constructs can be prepared in a very straightforward manner employing a wide variety of readily available starting materials.
  • modular assembly retroviral vectors prepared as described above, e.g., MARVs comprising DNA engineered to have multiple restriction sites therein, wherein each of the following components is inserted into a separate restriction site(s) thereof: a retroviral psi ( ⁇ ) packaging signal, a 5' long terminal repeat (LTR) , and a 3' LTR.
  • MARVs MARVs comprising DNA engineered to have multiple restriction sites therein, wherein each of the following components is inserted into a separate restriction site(s) thereof: a retroviral psi ( ⁇ ) packaging signal, a 5' long terminal repeat (LTR) , and a 3' LTR.
  • a modular assembly retroviral vector comprising the following components: a retroviral psi ( ⁇ ) packaging signal, a 5' long terminal repeat (LTR) , and a 3' LTR, wherein each component of said vector is bounded by a relatively rare restriction site(s) , such that each of said components can be independently removed and/or inserted into separate restriction site(s) on said vector.
  • the modular assembly retroviral vectors of the invention can not only be prepared in a very straightforward manner employing a wide variety of readily available starting materials, in addition, the design of such vectors renders them amenable to rapid interchange of each of the retroviral components with replacement components from preexisting vectors.
  • transgenic animals capable of prolonged and regulated expression of heterologous gene(s)
  • said method comprising introducing retroviral construct(s) of the invention, as described hereinabove, into early-stage embryos or stem cells.
  • transgenic animal refers to an animal that contains one or more inheritable expression constructs containing one or more heterologous gene(s) under the transcription control of an operator or hormone response element as described herein.
  • an invention transgenic animal also contains one or more inheritable expression constructs containing a member of the steroid/thyroid superfamily of receptors that functions as a silent partner for the active receptor subunit (e.g., RXR) .
  • introducing embraces a variety of techniques for contacting target cells with retroviral construct(s) of the invention, such as, for example, by direct injection of such constructs into host cells, by co-injection and/or co-infection of host cells with retroviral construct(s) of the invention, in combination with separate retroviral construct(s) which encode those proteins required for retroviral packaging, by injection and/or infection of host cells with retroviral construct(s) of the invention which themselves contain sequence which encode those proteins required for retroviral packaging, by injection and/or infection with infectious virions prepared from retroviral construct(s) of the invention, and the like.
  • transgenic animals using a particular nucleic acid construct are well-known in the art.
  • the first line will express, for example, RXR and a modified EcR (e.g., VpEcR) .
  • Tissue specificity is conferred by the selection of tissue-specific promoters (e.g., T-cell specific) that will then direct the expression of the receptors.
  • a second line contains a regulatory element controlling the expression of an heterologous gene.
  • an invention transgenic animal contains one or more expression constructs containing nucleic acid encoding an ecdysone receptor, exogenous RXR, and an heterologous gene under the transcription control of an ecdysone response element. It has been found that in transgenic mice containing an ecdysone response element and expressing ecdysone receptor and RXR, muristerone treatment can activate gene expression. Thus, with tissue specific expression of ecdysone receptor and RXR and timely hormone treatment, inducible gene expression can be achieved with spatial, dosage, and temporal specificity.
  • Ligands contemplated for use herein are compounds which, inside a cell, bind to the transactivator, thereby creating a ligand/receptor complex, which in turn can bind to an appropriate regulatory element.
  • Preferred ligands contemplated for use in the practice of the present invention are characterized as not normally being present in the cells of the subject, meaning that the ligand is exogenous to the subject. Ecdysteroids, for example, are not naturally present in mammalian systems. Thus, in accordance with the invention method, unless and until an ecdysteroid is administered to the subject, substantially no expression of the desired heterologous gene occurs.
  • ligand e.g., ecdysteroid
  • Ligand can be administered in a variety of ways, as are well-known in the art. For example, such ligands can be administered topically, orally, intravenously, intraperitoneally, intravascularly, and the like.
  • ecdysone and "ecdysteroid” as interchangeably used herein, are employed herein in the generic sense (in accordance with common usage in the art) , referring to a family of ligands with the appropriate binding and transactivation activity (see, for example, Cherbas et al. , in Biosynthesis, metabolism and mode of action of invertebrate hormones (ed. J. Hoffmann and M. Porchet) , p. 305-322; Springer-Verlag, Berlin).
  • An ecdysone therefore, is a compound which acts to modulate gene transcription for a gene maintained under the control of an ecdysone response element.
  • 20-Hydroxy-ecdysone (also known as /?-ecdysone) is the major naturally occurring ecdysone. Unsubstituted ecdysone (also known as o-ecdysone) is converted in peripheral tissues to /J-ecdysone. Analogs of the naturally occurring ecdysones are also contemplated within the scope of the present invention. Examples of such analogs, commonly referred to as ecdysteroids, include ponasterone A, 26-iodoponasterone A, muristerone A, inokosterone, 26-mesylinokosterone, and the like.
  • ex vivo methods for the treatment of a subject in need of gene therapy comprising introducing the retroviral construct of the invention (or both partners of the retroviral vector system of the invention) , as described hereinabove, into cells obtained from said subject, reintroducing the modified cells prepared as described above into said subject, and optionally administering, to said host, ligand for the transactivator.
  • Heterologous genes contemplated for use in accordance with the present invention include genes encoded by dopaminergic neurons (useful, for example, for the treatment of Parkinson's disease) , cholinergic neurons (useful, for example, for the treatment of Alzheimer's disease) , hippocampal pyramidal neurons (also useful for the treatment of Alzheimer's disease) , norepinephrine neurons (useful, for example, for the treatment of epilepsy), spinal neurons (useful, for example, for the treatment of spinal injury) , glutamatergic neurons (useful, for example, for the treatment of schizophrenia) , cortical neurons (useful, for example, for the treatment of stroke and brain injury), motor and sensory neurons (useful, for example, for the treatment of amyotrophic lateral sclerosis) , and the like.
  • MARV was constructed in a modular fashion with individual elements of the recombinant retrovirus inserted sequentially.
  • MARV started as a polylinker composed of 5' Notl-MluI-NruI-EcoRI-AscI-Pmll-BstBI-BamHI-Hindlll-Hpal- Clal-Nsil-Kpnl 3' sites.
  • the retroviral ⁇ gin (see Adam and Miller in J. Virol. (52 . :3802 (1988) and Barklis et al., in Cell £7:391 (1986)) was inserted Smal-EcoRI into the NruI-EcoRI sites of the MARV polylinker.
  • the internal CMV promoter was inserted BamHI-Clal into the BamHI-Clal sites of the evolving MARV vector. All LTRs destined for insertion into the 5' location were produced by low- cycle/high-fidelity PCR production and end primers with compatible Notl-Mlul sites for insertion into the Notl-Mlul sites of MARV. 3' LTRs were generated with Clal-Nsil compatible ends for insertion into these sites. The R region of the 3' LTR was included in the downstream primer sequence. Transgenes, receptor cDNAs, or selectable marker genes were inserted into the remaining polylinker from the EcoRI site through the BamHI site, or the remaining polylinker after the internal promoters (Hindlll-Clal) . By proceeding in this fashion, and taking advantage of the modular design to swap in new modifications, a variety of MARV and MARSHA vectors were constructed, including those schematized in Figure 1.
  • the resulting vectors were characterized by restriction digests and mapping, amplified by large-scale plasmid preparation, and prepared for transfection into packaging cells by standard methods.
  • Transient retroviral production in 293 and 293T cells has been previously described (see Pear et al., in Proc Natl Acad Sci USA £0:8392 (1993)). Forty-eight hours after transient retroviral production, the conditioned media was removed, filtered through 0.45 mm filters, and frozen at -70°C until use.
  • 10-cm dishes of primary-cultured Fisher rat abdominal fibroblasts (Rosenberg et al., Science 242:1575 (1988)) at approximately 50% density were infected with 1/10 volume of virus-containing media and 8 ⁇ g polybrene/ml for 48 hours. Infected cells were then selected under the appropriate antibiotic until non-infected cells were cleared from the population (approximately 10 days) . Resistant colonies were trypsinized, pooled and passaged. The resulting population was then infected with the second virus, and reselected again in media containing both antibiotics. The infection and selection process for the second round of viral infection proceeded as described for the first round. The final population of doubly resistant primary fibroblasts were then pooled and passaged to 6 or 24-well Costar plates for assay in vitro.
  • the short (24 hour) course was performed in 24- well Costar dishes with cells at approximately 80% confluency.
  • Five ⁇ l of murA (final concentration of 1 ⁇ M) or vehicle (20% EtOH) was added to the 1 ml of media in each well.
  • Eight hours after first murA administration the cells were all washed in PBS and the media was replaced.
  • /3-galactosidase histochemical reaction was performed on 1.5% glutaraldehyde-fixed cells essentially as described by Shimohama et al. in Brain Res Mol Brain Res 5_:271 (1989) in a 37"C environment for 2 hours.
  • the 4-day time course was performed in an identical fashion, except that the cell population was plated at only 20% confluency at the start of the experiments (and grew to near-total confluency by the end of the 4 day period) and neither MurA nor media was changed or replaced.
  • MMBG fibroblasts were plated in triplicate at high density in 24-well plates. Within 48-72 hours of plating, the cells reached 100% confluency within the wells. At this point, 1 ⁇ M murA or vehicle was added to one group of plates, while others were left completely untreated. The wells were then placed in a 37°C-10% C0 2 environment for the next 25 days without any media changes. On day 23, the plates that had received neither vehicle nor murA on day 1 were removed and half of the wells were given vehicle and the other half were given 1 ⁇ M murA for 40 hours.
  • MMGH fibroblasts were produced by infection of a MARV-infected fibroblast line with the MARSHA-GH virus. Cells were selected under both antibiotics as described previously. After preliminary examination of murA-induced hGH production in the bulk population, the MMGH population was plated at high density in 6-well Costar plates. Forty- eight hours after the initial plating and when the cells were essentially completely growth inhibited by contact, the medium of all plates was replaced with DME containing 2% FBS. Under these conditions, primary rat fibroblasts stop dividing even if they are not confluent, and settle out into a distended morphology with prominent nuclei characteristic of severely growth-arrested fibroblasts.
  • the native MLV LTR was subcloned into a modified version of the cloning vector pBSK for manipulation and mutation of internal sequences. Tandem EcREs were inserted as described with reference to the constructs described in Figure 2. Mutated LTRs were inserted into a derivative of the pBLluc vector replacing all original TK promoter sequences with the modified LTRs. 100 ng of CofVEcR, 50 ng of CTRL and 100 ng of LTR-reporter and pCHHO internal control were cotransfected, treated and quantified as described with reference to the data presented in Figure 2.
  • EcR, RXR ⁇ , and Usp open reading frames were subcloned without internal polyadenylation signals into the cloning vector pBSK (Stratagene, La Jolla, CA) for further manipulation of the cDNA sequences.
  • Unique Sfil sites were inserted by PCR mutagenesis into the cDNA sequences of RXR ⁇ and Usp overlapping the ATG initiation condon.
  • a similar modification was inserted into the Ncol site approximately 210 bp into the EcR ORF.
  • EcR C-terminal modification of EcR was performed in an identical fashion except that the inserted Sfil compatible site overlapped the TAG termination condon, recreating it after the site insertion.
  • a plasmid encoding the HSV VP16 r domain was used as a template for PCR amplification of ⁇ domain sequences with compatible and in- frame ends relative to the inserted Sfil sites in the receptor proteins. All modified receptor proteins were inserted into the vector LNCX (A.D. Miller, GenBank Ace.No. M28247) for expression in transient transfection assays.
  • the E41uc promoter is 4-tandem EcREs inserted into the BamHI site of plasmid TK-luc which, briefly, is an 180 bp TK minimal promoter with flanking polyadenylation signals to both polyadenylate the 3' end of the nascent luciferase mRNA, and to prevent read-through at the 5' end upstream of the inserted EcREs.
  • the sequences of the EcREs were as described (see Thomas et al., in Nature 362:471 (1993)) with compatible BamHI/B ⁇ lll. or Xbal/Nhel overhangs for insertion into either the pBLluc vector or LTR constructs described in Figure 3.
  • Transient transfections were performed by calcium-phosphate coprecipitation employing standard methods (see Sambrook et al., in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, New York, New York (1989)). All tissue culture experiments were performed using DMEM 10% FBS in a 10% C0 2 incubator unless otherwise specified. All transfections were performed in triplicate in 24-well Costar plates using CV-1 cells at an approximately 5X10 4 plating density. Ecdysone receptor plasmid constructs were delivered at 100 ng/well, dimer partner constructs at 50 ng/well, and reporter constructs and internal controls (pCHHO) at 100 ng/well for a total of 350 ng DNA/well. Immediately following transfection, l ⁇ m murA (Sigma, St.
  • Phase contrast photography was conducted on fields of 0-gal stained MARV/MARSHA- ⁇ gal coinfected fibroblasts treated with MurA at time 0 and continued for 8 hours with wells stained at 0, 2, 4, 6, 8 and 24 hour intervals.
  • 6 hours the first hint of /3-gal activity is observed in only one or two cells/field.
  • 8 hours of treatment many cells are clearly responding to added hormone.
  • Fibroblasts cultured in conditions of contact inhibition with no media changes or other nutrient replenishment for 25 days continue to be strongly positive 97/48277 PC17US97/08805
  • Fibroblasts cultured in conditions of contact inhibition with no media changes or other nutrient replenishment for 25 days and then stimulated with 1 uM MurA for the last 40 hours of culture reveals that quiescent, nutrient starved fibroblasts continue to respond strongly to hormone, indicating that the machinery of induction is present at sufficient levels to result in high-level stimulation of transgene expression. Again, most (>80%) of the cells indicate high-level /3-gal activity.
  • invention constructs as a gene transfer vector for prolonged transgene expression for either in vivo or ex vivo gene therapy applications
  • TH tyrosine hydroxylase
  • invention constructs allows one to overcome the loss of transgene expression by providing stimulation of the retroviral LTR promoter through either ligand-activated transactivating complexes or through constitutive transactivating receptor variants. In this way, transgene expression may be maintained for longer periods of time, even indefinitely if desired.
  • Example 6 Use of invention constructs as a gene transfer vector for modulated transgene expression for either in vivo or ex vivo gene therapy application
  • L-dopa Treatment of Parkinson's disease with the chemical precursor of dopamine, L-dopa, has proven effective in ameliorating many of the deficits of Parkinsonism. With time, however, patients become refractory to L-dopa therapy, with the deleterious effects of chronic treatment outweighing even the serious symptoms of the disease itself. Eventually, patients are left with few therapeutic options. While the transplantation of TH expressing cells may be effective when constantly producing low-levels of L-dopa, a potentially far more beneficial approach would be to allow the physician some degree of control over L-dopa production in the patient. This would allow sufficient control to ensure that the transgenic factor is expressed at appropriate therapeutic levels.
  • the transgene may be allowed to become quiescent and transcriptionally inactive until needed again. Because the transcriptional induction of the invention retroviral constructs is dependent on an exogenous ligand, expression of an integrated therapeutic transgene can be placed under the control of the physician and patient.
  • Example 7 Use of invention constructs as a gene transfer vector for efficient production of transgenic animals
  • Transgenic animals are generally produced by either pronuclear injection of DNA or by transfection of embryonic stem (ES) cells followed by selection and 97/48277 PC17US97/08805
  • the retroviral constructs of the present invention are capable of overcoming the transcriptional block to result in germ-line transgenic animals with full expression from the integrated transgene.
  • the level of transcription may still be regulated by controlling the supply of ligand to the transgenic animal.
  • the increased efficiency of producing transgenic animals by retroviral infection should open up the way to producing mutant animals of a variety of species previously impractical for genetic modification because of the potential cost of producing a large number of non-positive animals by classical methods.
  • invention constructs of the present invention can effectively overcome the block of viral expression in embryonic cells
  • invention constructs are a potent tool in the delivery of transgenes to somatic tissues of a developing embryo. With many diseases, considerable damage is done during embryonic development so that therapies applied after birth are essentially ineffective to ameliorate the disease phenotype.
  • Retroviral constructs of the present invention can infect cells of the embryo and can provide therapeutic factors to the developing fetus either constitutively, or under the regulation of exogenously produced ligand.
  • retroviruses as gene transfer agents
  • titers of retroviruses from existing producer cell lines are only on the order of 1X10 4 or 1X10 5 .
  • expression of the retrovirus may be induced by greater than ten-fold, resulting in correspondingly higher titers of infectious virus.

Abstract

L'invention concerne des vecteurs rétroviraux nouveaux ayant de longues répétitions terminales qui assurent l'expression de produits géniques spécifiques à un niveau élevé et avec modulation de ligand, même après des périodes prolongées de quiescence cellulaire. Les vecteurs ainsi conçus surmontent l'inactivation de transcription provirale intervenant dans les cellules primaires cultivées dont la croissance est interrompue sous l'effet des contraintes du milieu (par exemple, inhibition de contact et/ou suppression des nutriments). Ces vecteurs définissent une classe de vecteurs rétroviraux dont l'expression provirale induite par de longues répétitions terminales dans les cellules infectées peut être maintenue ou accrue, même sous des conditions généralement considérées étant non permissives pour les vecteurs rétroviraux.
PCT/US1997/008805 1996-06-20 1997-05-22 Vecteurs retroviraux a structure modulaire et leurs utilisations WO1997048277A1 (fr)

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GB2331522A (en) * 1996-10-17 1999-05-26 Oxford Biomedica Ltd Lentiviral vectors
WO1998017816A1 (fr) * 1996-10-17 1998-04-30 Oxford Biomedica (Uk) Limited Vecteurs lentiviraux
US6235522B1 (en) 1996-10-17 2001-05-22 Oxford Biomedica (Uk) Limited Lentiviral vectors
GB2331522B (en) * 1996-10-17 2001-05-23 Oxford Biomedica Ltd Lentiviral vectors
US7056699B2 (en) 1996-10-29 2006-06-06 Oxford Biomedia (Uk) Limited Lentiviral LTR-deleted vector
US6924123B2 (en) 1996-10-29 2005-08-02 Oxford Biomedica (Uk) Limited Lentiviral LTR-deleted vector
US6555107B2 (en) 1997-09-24 2003-04-29 The Regents Of The University Of California Lentiviral nucleic acids and uses thereof
WO1999058155A1 (fr) * 1998-05-14 1999-11-18 The Salk Institute For Biological Studies Formulations utiles dans la modulation de l'expression des genes exogenes des systemes mammaliens, et produits correspondants
US7045315B2 (en) 1998-05-14 2006-05-16 The Salk Institute For Biological Studies Methods for modulating expression of exogenous genes in mammalian systems
EP1165842A4 (fr) * 1999-03-16 2004-07-07 Dana Farber Cancer Inst Inc Systeme vectoriel lentiviral pour le criblage de grande quantite
EP1165842A1 (fr) * 1999-03-16 2002-01-02 Dana-Farber Cancer Institute, Inc. Systeme vectoriel lentiviral pour le criblage de grande quantite
WO2000075351A1 (fr) * 1999-06-09 2000-12-14 Syngenix Limited Vecteurs a encapsidation deficiente bases sur le siv
EP1083230A1 (fr) * 1999-09-10 2001-03-14 Academisch Medisch Centrum Amsterdam Replicon viral et virus dépendant d'agents inducteurs
AU780984B2 (en) * 1999-09-10 2005-04-28 Academisch Medisch Centrum Bij De Universiteit Van Amsterdam Viral replicons and viruses dependent on inducing agents
JP2003509059A (ja) * 1999-09-10 2003-03-11 アカデミス ジーケンハイス ベイ デ ウニフェルシテイト ファン アムステルダム 誘導物質に依存するウイルスレプリコンおよびウイルス
WO2001020013A3 (fr) * 1999-09-10 2001-09-27 Amc Amsterdam Replicons viraux et virus dependants d'agents inducteurs
WO2001020013A2 (fr) * 1999-09-10 2001-03-22 Academisch Medisch Centrum Bij De Universiteit Van Amsterdam Replicons viraux et virus dependants d'agents inducteurs
US7109029B2 (en) 2001-02-23 2006-09-19 Cell Genesys, Inc. Vector constructs

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