WO1994021806A1 - Systeme d'administration commande par des facteurs associes au vih et a la cellule - Google Patents

Systeme d'administration commande par des facteurs associes au vih et a la cellule Download PDF

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WO1994021806A1
WO1994021806A1 PCT/GB1994/000568 GB9400568W WO9421806A1 WO 1994021806 A1 WO1994021806 A1 WO 1994021806A1 GB 9400568 W GB9400568 W GB 9400568W WO 9421806 A1 WO9421806 A1 WO 9421806A1
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Prior art keywords
delivery system
hiv
cell
cells
gene
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PCT/GB1994/000568
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English (en)
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Elaine Anne Dzierzak
Hugh Joseph Martin Brady
Colin Graham Miles
Daniel John Pennington
David John Abraham
Roger Kingdon Craig
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Medical Research Council
Therexsys Limited
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Priority to JP6520813A priority Critical patent/JPH08508642A/ja
Priority to AU62614/94A priority patent/AU696399B2/en
Priority to EP94909987A priority patent/EP0689602A1/fr
Publication of WO1994021806A1 publication Critical patent/WO1994021806A1/fr

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    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
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Definitions

  • the present invention relates to a method for the prevention or therapy of AIDS.
  • the invention relates to methods for preventing the replication and spread of HIV and to agents for doing the same.
  • HIV Human immunodeficiency virus
  • AIDS human acquired immunodeficiency syndrome
  • Conventional therapeutic strategies have concentrated on antiviral drugs such as AZT and on the development of preventive vaccines.
  • antiviral drugs such as AZT
  • the intracellular immunisation approach (Baltimore, 1988) has lead to the development of molecular strategies for the inhibition of HIV replication (Malim et al . , 1989, Trono et al . , 1989, Sczakiel et al . , 1991, Sullenger et al . , 1990).
  • cytotoxic agents such as diphtheria toxin A or ricin A chain genes under the control of lens (Breitman et al . , 1987; Landel et al . , 1988) or pituitary (Behringer et al . , 1988) specific promoters. After icroinjection into mouse embryos and the production of transgenic animals, these constructs resulted in the destruction of either lens or pituitary cells.
  • these toxin genes are unsuitable for somatic therapy because of the constitutive cell lethality and the extreme sensitivity of mammalian cells to diphtheria and ricin toxins.
  • the Herpes Simplex Virus type 1 thy idine kinase (tk) gene product is a conditional cell lethal and has been shown to be toxic to mammalian cells only in the presence of nucleoside analogues such as acyclovir (ACV) or ganciclovir (GCV) . These analogues kill actively cycling cells because they possess high affinity for the tk gene product with little or no affinity for endogenous mammalian tk.
  • ACV acyclovir
  • GCV ganciclovir
  • Another potential system for use in anti-HIV therapy involves the expression in cells susceptible to HIV infection of a decoy gene.
  • Decoy genes encode proteins which act as antagonists to natural proteins involved in the replication of the HIV virus.
  • a decoy gene may encode a defective mutant of a transactivator protein which is capable of binding to the transactivator-responsive site on the host or viral genome, yet is incapable of activating transcription.
  • Transdominant mutations have been reported in a number of viral transactivators which abolish or attenuate the ability of the wild-type protein to transactivate the target gene. Examples include transdominant mutations of E1A (Glen et al . , 1987), tax ( achsman et al . , 1987) and VM65 (Friedman et al . , 1988). Similar mutations in HIV genes have been described for the Tat transactivator (Pearson et al . , 1990) and the Rev transactivator (Bevac et al , 1992).
  • a potential disadvantage of the use of decoy gene approaches is that when a decoy is expressed in the absence of the infecting virus a host immune response may result from the production of the decoy gene product, leading to destruction of the host cell.
  • promoter and enhancer elements which display at least some degree of tissue-specificity can be selected.
  • use of such elements in isolation in gene transfer approaches leads to uncertainty in the levels of expression of the anti-HIV agent which will be achieved, since transgenes are invariably expressed in an integration- site dependent manner.
  • any leaking of the gene in unsuitable tissue-types will be unacceptable.
  • Locus control regions are elements which confer position-independent, copy number-dependent expression of genes in gene transfer approaches. They have also been shown to permit high levels of expression of cloned genes and to possess tissue-specific properties. First discovered in globin genes (Grosveld et al , 1987) these elements are believed to direct the creation of independent regulatory domains within the chromatin structure of cell genomes, thereby ensuring the activity of a co-transferred gene.
  • LCRs other than those for globin genes have been described, for example in the CD2 gene in T-lymphocytes (Greaves et al . , 1989), the lysozyme gene in macrophages (Bonifer et al . , 1990) and Class II MHC genes in B cells (Carson and Wiles, 1993).
  • LCRs are known to be able to direct efficient tissue- specific expression of cloned genes. However, they are not known to be responsive to the infection status of a cell.
  • the HIV Long Terminal Repeat (LTR) promoter element is composed of cis-acting control sequences; enhancer, transcription start site and the Tat responsive region (TAR) (Rosen et al . , 1985, Jones et al . , 1986; Peterlin et al . , 1986; Garcia et al . , 1987; Muesing et al . , 1987; Siekevitz et al . , 1987; Feng and Holland, 1988; Harrich et al . , 1989). This basic structure is conserved for both HIV-1 and HIV-2 (Guyader et al . , 1987; Arya and Gallo, 1988; Markovitz et al . , 1990).
  • Tat trans-activation requires the presence of the TAR region which is situated 3' to the LTR and is strictly orientation and position dependent (Peterlin et al . , 1986; Sharp and Marcinial, 1989) .
  • a common mechanism of Tat trans- activation is shared by HIV-1, HIV-2 and SIV (Emerman et al . , 1987; Guyader et al . , 1987; Fenrick et al .
  • the three Tat gene products are not completely interchangeable in their effects.
  • the Tat gene product from both HIV strains and that of SIV effectively trans-activate the HIV- 2 promoter whereas the HIV-1 promoter and SIV promoter are only maximally trans-activated by their own specific Tat protein (Emerman et al . , 1987; Fenrick et al . , 1989; Shibata et al . , 1990).
  • These functional studies are supported by structural studies of the Tat proteins and stem-loop structures of the TAR regions (Fenrick et al . ,1989; Berkhout et al . , 1990b).
  • the HIV-2 promoter and TAR element function optimally in HIV-l or HIV-2 infected human cells or in SIV infected primate cells.
  • 5* HIV promoter deletion constructs containing the TAR element show that Tat trans-activation of the HIV promoter is retained while basal levels of transcription are dramatically decreased (Arya and Gallo, 1988; Berkhout et al . , 1990; Zeichner et al . , 1991; Berkhout et al . , 1992).
  • the 5* enhancer/promoter element of HIV-l which contains two NFcB binding sites and three Spi sites, plays a dominant role is basal transcription.
  • NF ⁇ B is a cellular transcription factor found in many cell types including mitogen induced T cells. It has been demonstrated that mitogen induction can increase HIV-l directed basal transcription in CD4+ human T cells (Nabel and Baltimore, 1987) .
  • the targets of HIV infection are primarily CD4+ T- lymphocytes, but also include macrophages, dendritic cells and some cells located in the brain (reviewed in McCune, 1991) . These cells share very few common features except for being derived from common hematopoietic stem cells and susceptibility of HIV infection. Hematopoietic stem cells are not infected by HIV (Molina et al . , 1990; Davis et al . , 1991) . Specific molecular ablation of only HIV infected cells within the hematopoietic system requires differential regulation of the HIV promoter-directed gene expression between HIV infected (Tat positive) and uninfected (Tat negative) cells.
  • HIV infection is limited at the level of viral entry by CD4, non-CD4+ human cells can actively transcribe HIV sequences, as demonstrated by transfection studies (Barry et al . , 1991). Since therapy may entail the transduction of antiviral sequences into hematopoietic stem cells (thus creating a complete blood system with all precursor and mature cell types carrying the antiviral sequence) , the absence of basal transcription from the promoter in uninfected (Tat negative) cell types is required.
  • a method for the prevention or treatment of a viral infection comprising administering to a subject an effective amount of a delivery system, comprising a vector effective to express in a cell susceptible to infection by the virus a gene encoding an agent which is effective, directly or indirectly, to prevent, eliminate or attenuate the infection, wherein the gene is subject to control between an operative state and an inoperative state through factors specifically associated with the virus and the cell.
  • the gene encoding the agent according to the method of the invention is subject to regulation between an inoperative state and an operative state.
  • inoperative state it is intended to signify that when the gene is in this state, the antiviral effects of the gene product are substantially absent.
  • the antiviral gene product is a protein
  • the absence of antiviral effects will normally be due to a lack of production of the protein, which prevents the overburdening of the protein expression machinery of the cell.
  • the antiviral agent is a cytotoxic agent in the inoperative state, not enough toxin will be produced to lead to cell death on a significant scale.
  • sufficient antiviral agent is produced to prevent, eliminate or attenuate the infection of the subject by the virus. It is to be understood, however, that regulation between the two states may be achieved by means other than levels of transcription. For example, the splicing of the gene transcript may be regulated.
  • the antiviral agent used in the method of the invention may be any agent capable of preventing, eliminating or attenuating a viral infection, whether alone or in combination with a second agent.
  • this second agent may be a drug such as a prodrug which is administered to the subject in a non-targeted manner.
  • This second agent may then be activated at the site of infection by the antiviral agent.
  • Suitable antiviral agents include toxins, inhibitors of gene transcription and protein synthesis, immunoactive agents or other drugs capable of directly influencing the metabolism and viability of a cell, as well as alternative factors capable of activating a second agent, such as a drug.
  • the agent could be an enzyme or a ribozyme.
  • antisense oligonucleotides or oligonucleotide analogues capable of disrupting the expression of a cellular or viral gene and decoy genes.
  • vector signifies the nucleic acid which comprises the gene of the invention
  • delivery system signifies the means used to introduce the vector to the target cell.
  • a vector may be any nucleic acid capable of expressing a gene in a cell.
  • nucleic acid vectors suitable for use in the present invention include circular and linear lengths of DNA which either integrate into the host genome or are maintained in episomal form, delivered by non-viral delivery systems such as naked DNA, liposomal or receptor-mediated delivery systems, or viral vectors such as retroviral vectors, adeno- associated or adnoviral vectors and other virus-based vectors known in the art.
  • targeting of the vectors may be achieved by designing delivery systems so that they are selectively taken up by cells susceptible to infection by the virus. This may be effected by targeting the delivery systems to specific receptors found on cells infected by the virus or by targeting to progenitor cells such that the delivered gene is subsequently expressed in cells of a particular lineage susceptible to infection by the virus.
  • the above-identified disadvantage is overcome by the use of genes which are under the control of tissue-specific factors.
  • the advantages of the invention may be combined with the use of targeted delivery systems to provide a second level of control in the treatment of viral infections.
  • tissue-specific expression of antiviral agents may be effectively achieved in vivo in hematopoietic cells by the use of LCR sequences.
  • the use of CD2 and macrophage LCRs is particularly preferred.
  • the use of tissue specific control elements is combined with the use of regulatory elements responsive to a virus associated factor. This will ensure that expression of the antiviral gene takes place only in cells actually infected by the virus. Also, the possibility of a host immune response to the product of the transfected gene, which may be expressed over a long period of time, will be significantly reduced.
  • Suitable decoy genes for anti-HIV therapy include derivatives of the HIV-encoded proteins Rev, Tat, Vpu, Vpr and Nef. Production of mutated forms of Rev and Tat inhibits infection by the HIV virus. It has been found that by using a tissue-specific LCR, elevated levels of expression of transfected mutant or wild-type proteins may be achieved in lymphoid tissue. Alternatively, agents which function to ablate the cell or arrest viral growth may be used. Such agents include cytotoxic polypeptides, ribozy es, proteolytic enzymes and nucleases.
  • regulatory elements responsive to virus associated factors allows the use of prodrug approaches for viral inhibition or selective cell ablation, because the antiviral gene will only be in an operative state in the presence of a viral infection.
  • virus associated factor it is intended to refer to a factor, capable of modulating the expression of a gene, which is dependent on the virus.
  • a factor capable of modulating the expression of a gene, which is dependent on the virus.
  • it may be expressed by the cell only in the specific presence (or absence) of a viral infection.
  • it may be encoded by the virus.
  • this factor will modulate the transcription of the gene of the invention.
  • the factor may be active by other means, for example by specifically activating a product of the gene of the invention or by acting in unison therewith to potentiate a biological effect.
  • the gene of the invention may be subject to control by a viral transactivator.
  • the gene of the invention is controlled by a viral promoter/enhancer which is transactivatable by the viral transactivator.
  • the promoter/enhancer elements responsible for the transactivation may be incorporated into other, non-viral or synthetic promoter/enhancers to confer the transactivatability thereon.
  • transactivatable viral promoters will be apparent to those skilled in the art and include the adenovirus E1A promoter/enhancer, the hCMV-MIE promoter/enhancer, retroviral LTR elements such as HIV-l and HIV-2 LTRs as well as promoter/enhancers from herpesviruses and poxviruses.
  • the Tat transactivator may be used.
  • This factor acts via the TAR element which is comprised in a Tat-responsive promoter, such as the LTR.
  • a Tat-responsive promoter such as the LTR.
  • Expression from promoters regulated by the Tar element is greatly increased in the presence of the HIV Tat protein. Accordingly, transcription from the gene of the invention will be greatly increased in a cell infected by HIV, in which the Tat protein will be present.
  • HIV-2 LTR promoter In order to effect regulation through HIV-associated factors, the use of an HIV-2 LTR promoter is particularly preferred.
  • HIV-2 promoter for cell-specific gene expression has been described previously. However, this promoter displays levels of basal transcription which, in combination with certain agents, may be excessive. Accordingly, the HIV-2 promoter is preferably modified in order to reduce the basal levels of transcription.
  • Modification may be accomplished according to a number of strategies.
  • the use of negative regulatory elements which decrease the level of transcription is envisaged.
  • the modification of the promoter sequences in order to reduce the basal levels of transcription.
  • Promoter sequences may be modified in order to remove regulatory elements which respond to cell-specific regulatory factors.
  • the HIV-2 promoter contains elements responsive to SPI and NFKB factors.
  • elements responsible for activation by cell-specific factors may be mutated or deleted .
  • modification of the HIV-2 promoter may not be necessary in association with all antiviral agents. For example, a small amount of leakage from the promoter when used to express a decoy gene will not be excessively detrimental to the cells of the subject. However, even a small amount of leakage when the promoter is used to express a cytotoxic agent may prove unacceptable.
  • the invention further provides a polynucleic acid sequence comprising a gene as described above.
  • vectors comprising the polynucleic acid sequences according to the invention as well as the use of polynucleic acid sequences and vectors according to the invention in therapy.
  • Figure 1 Proposed method of HIV-2 tk retroviral mediated HIV specific intracellular molecular ablation.
  • Figure 2 A: HIV-2 LTR promoter constructs.
  • NF/cB d and NF/cB refer to the distal and proximal NFcB sites respectively.
  • mSPl refers to a mutated SPI site.
  • the HIV-2 promoter sequences were generated by PCR and then cloned upstream of the human growth hormone (hGH) reporter gene to make pH2ghl to pH2gh8. The same sequences were also cloned upstream of the Herpes Simplex Virus-1 thymidine kinase gene to make plas ids pH2tkl to pH2tk8.
  • hGH human growth hormone
  • FIG. 3 Differential Tat trans-activated expression from HIV-2 promoter constructs in transiently transfected COS cells. pH2ghl, pH2tk3, pH2tk4, pH2gh5, pH2gh7 and pH2gh8 were co-transfected into COS cells with a human ⁇ globin expression plasmid, p/3BSV328, in the absence (-) or presence (+) of Tat. RNA from the transfected cells was hybridised to 3 P end labelled DNA probes specific for either the HIV-2tk or HIV-2hGH transcripts and to the 3 1 end of the human ⁇ globin gene. The arrows indicate the fragments protected by the correctly initiated HIV-2 hGH transcripts and HIV-2 tk transcripts and the 3 ' end of the ⁇ globin transfection control (212 bases) .
  • Figure 4 HIV-2 retroviral vector design and viral passage analysis of cells infected with HIV-2 tk containing retroviruses.
  • A. Schematic diagram of the pLA vectors showing the HIV-2 tk sequences cloned in the opposite orientation to the SV40 promoter and the 5' LTR. The Asp718 sites are as indicated.
  • B. Southern blot analysis on DNA from NIH 3T3 cells infected with retroviruses LAI, LA4 and LA5. Asp718 digested DNA was probed with a Bglll-BamHI fragment containing the tk gene. Plasmid control Asp718 digests are indicated by lanes marked pLAl, pLA4 and pLA5.
  • DNA from populations and clones of infected 3T3 cells are indicated as pop 1, 31.1 and 31.2 for cells infected with LAI, pop 4, 34.1 and 34.2 for cells infected with LA4 and pop 5, 35.1 and 35.2 for cells infected with LA5.
  • D. Southern blot analysis on DNA from LAI infected HeLa clones, H1.2-H1.6. Asp718 digested DNA was probed with the tk gene.
  • Figure 5 Differential Tat trans-activated expression of tk in cells infected with LAI.
  • A. SI analysis of 3T3 cells infected with LAI and containing Tat. RNAs from an LAI infected 3T3 clone without
  • Tat (31.1) and three subclones containing Tat, (31.1A, B and C) were analysed by SI nuclease protection after hybridisation to a HIV-2 tk specific DNA probe and ⁇ actin probe.
  • 30.ID is a Tat transfected, uninfected cell line.
  • the ⁇ actin probe is a normalisation control for the amount of RNA in each lane.
  • the RNA was hybridised to HIV-2 tk and ⁇ actin specific probes.
  • the ⁇ actin probe is used to normalise the amount of RNA in each lane.
  • FIG. 6 Differential Tat trans-activated ablation of HIV- 2 tk retrovirus-infected 3T3 and HeLa cells.
  • GCV treatment was performed on LAl-infected Tat- negative 3T3 cells (31.1), LAl-infected Tat- positive (31.1A and 31.IB), uninfected Tat- positive (30.ID), and 3T3 cells (A) and LAl- infected (31.1), LA4-infected (34.1), and 3T3 cells (B) .
  • GCV was also administered to LAl- infected HeLa cells without Tat (HI.
  • Figure 7 A construct comprising the CD2 LCR and a 650 bp Nef fragment, which was used to generate transgenic mice.
  • Figure 8 A) Southern blot of CD2-nef Transgenic mice.
  • FIG. 9 Distribution of CD4 and CD8 cell subsets in lymphoid tissues of CD2 Nef transgenic mice. Thymocytes, lymph node cells and spleen cells from transgenic, (A) line F, 1147 allele and (B) line B, 1191 allele, and non-transgenic littermates were stained for CD4 (PE ordinate) and CD8 (FITC abscissa) . 10 A cells were anlaysed in each sample using the Becton Dickinson FACScan. Relative fluorescence intensities are shown on a logarithmic scale, with percentages of double negative, double positive and CD4/CD8 single positive cells indicated.
  • FIG. 10 Levels of expression of CD4 and other T cell surface markers on Nef transgenic thymocytes. Thymus cells from non-transgenic and transgenic littermates from (A) line F, 1147 allele and (B) line B, 1191 allele were stained with anti-CD4, CD8, CD3 and Thy-1 antibodies. Histogram plots show number of cells (ordinate) and intensity of fluorescence (logarithmic scale, abscissa) for non-transgenic cells (solid line) , transgenic cells (thick line) and no stain controls (dotted line) .
  • FIG. 11 Thymocyte proliferation in Nef-transgenic mice. Varying numbers of thymocytes from non-transgenic (open symbols) and transgenic (solid symbols) littermates of Nef lines F, B, A and D were stimulated with anti-CD3e antibody and PMA and proliferation was measured via [ 3 H]thymidine incorporation. PMA alone produced no activation (not shown) . Activation is plotted as [ 3 H] c.p.m. incorporated versus cell number.
  • Figure 12 Construction of the HIV-2 TK CD2 transgene.
  • Figure 13 DNA slot blot showing HIV-2 TK CD2 transgenic mice generated by microinjection of embryos with the HIV-2 TK CD2 transgene.
  • Figure 14 S 1 analysis of HeLa cell RNA showing the expression of the HIV-2 TK CD2 transgene when transfected into HeLa cells.
  • Figure 15 A: Southern blot analysis of DNA from tissues derived from a 20-copy HIV-2 YK CD2 founder animal;
  • Table 2 Basal and Tat trans-activated levels of hGH expression after transient transfection in CEM cells.
  • CEM cells were co-transfected with pH2ghl to pH2gh8, a ⁇ galactosidase expression vector, to normalise transfection efficiency and where indicated the pSV2Tat expression vector. Each transfection was carried out in at least three independent experiments.
  • Table 3 CD4/CD8 T-cell subset changes in CD2-nef transgenic mice. Thymocytes from three transgenic and three non-transgenic mice from various aged litters were stained with anti-CD4 and anti-CD8 antibodies. 10 4 stained cells per thymus were analysed on a Becton Dickinson FACScan. Double negative, double positive, CD4 single positive and CD8 single positive populations were gated and displayed as a percentage of total thymocyctes. The mean value and standard deviation for non-transgenic and transgenic mice are shown. Table : CD4 and CD8 down-regulation on T-cell surfaces in CD2-nef transgenic mice.
  • Thymocytes from three transgenic and three non-transgenic mice from various aged litters were stained with anti- CD4 and anti-CD8 antibodies. 10* stained cells per thymus were analysed on a Becton Dickinson FACScan. DN, DP, CD4 SP and CD8 SP populations were gated and then displayed as a single parameter (CD4 or CD8) histogram with cell number (ordinate) against log of fluorescence intensity
  • HIV-2 sequences used were based on the sequence of the HIV-2 LTR as previously described (Guyader et al . , 1987) .
  • the regions of the HIV-2 LTR were generated by PCR amplification using the plasmid pHIV-2 LTR CAT (-556/+156) (Emerman et al . , 1987).
  • Oligonucleotide primers were synthesised incorporating restriction enzyme sites for ease of cloning.
  • the 3 1 end of each amplified fragment was from primer 9 (3 1 ) CGTGAACCGGCCACGACCCGTTCATGACCTAGGTG (5') which contains HIV-2 LTR sequences +96 to +116 and a Seal and BamHI restriction site on the end.
  • the 5' ends also contained restriction enzymes for Hindlll, Bgl II and Xhol in primers 1-5 and Hindlll plus Xhol in primers 6-8.
  • the HIV-2 LTR sequence in the primers was as follows (5'-3-):
  • Primer 7 AGAAGGGGCTGTAACCAACGTACGTATGCTTCGCCCACATATTCTCTG
  • Primer 8 AGAAGCGGCTGTAACCAACGTACGTATGCTTCGCCCACATATTCTCTG
  • PCR amplification was carried out in lOmM Tris (pH 8.3), 50 mM KCL, 5mM MgCl 2 and 200 ⁇ M dNTPs with 200ng pHIV-2 LTR CAT template and 4ul of Taq poly erase (Boehringer Mannheim) with 94°C denaturation, 55°C annealing and 72°C extension.
  • the amplified products were blunted using Klenow fragment then digested with Hindlll and BamHI before cloning into the Hindlll and BamHI sites of p ⁇ GH (Selden et al .
  • pH2ghl to pH2gh8.
  • the same PCR amplified sequences were also fused to the Herpes Simplex Virus-1 thymidine kinase (tk) gene.
  • pH2tkl, 3, 4 and 5 were made by ligating the HIV-2 LTR Bglll-Scal fragments from the respective pH2gh plasmids to BamHI linearlised pUC19 and a BamHI-Bglll (blunted with Klenow) fragment containing the tk gene (Wagner et al . , 1981).
  • pH2tk7 and 8 were made by ligating the same tk fragment to Hindlll-BamHI linearised pUC19 and the HIV-2 LTR containing Hinglll-Scal fragment from pH2gh7 and pH2gh8 respectively. The constructions were checked by dideoxy sequencing (Sanger et al . , 1977).
  • p/?BSV328 is a 4.9 kb Bglll fragment containing the human ⁇ globin gene cloned into the BamHI site of p/3BSV328 (Grosveld et al . , 1982).
  • HeLa cells were maintained in DMEM with 10% fetal calf serum. They were transfected by the calcium phosphate co-precipitation technique with 48 hr exposure of the cells to the DNA- calcium phosphate co-precipitate and then harvested.
  • CEM cells human leukemic CD4+ cell line
  • RPMI 1640 10% fetal calf serum
  • Reproducible CEM cell transfection was carried out using electroporation.
  • CEM cells at lx 10 7 cells/ml in 0.8 ml of serum-free RPMI 1640 were shocked at 500 ⁇ F and 300V (BioRad Gene Pulser) .
  • lOO ⁇ g DNA of each pH2gh plasmid, 50 ⁇ g pSV2Tat and 50 ⁇ g pCMV LacZ expression vector (Tassios and LaThangue, 1990) as a normalisation control were used.
  • the amphotropic retrovirus packaging cell lines PA317 (Miller and Buttimore, 1986) and AM12 (Markowicz et al . , 1988) were maintained in DMEM with 10% newborn calf serum.
  • the packaging cells were transfected with 12 ⁇ g of each pLA vector using the calcium phosphate co-precipitation technique.
  • NIH 3T3 cells were maintained in DMEM with 10% newborn calf serum.
  • Retrovirally infected 3T3 cells were transfected with pSV2Tat and a hygromycin expression plasmid (from R. Zamoyska) in a 10:1 ratio using the calcium phosphate co- precipitation technique.
  • 24 hr following transfection the cells were split and stably transfected clones isolated by selection in 120 ⁇ g/ml hygromycin B (Sigma) .
  • Human growth hormone reporter gene expression was measured 72 hr after transient transfection of COS and CEM cells. Supernatants were collected from the transfected cells and lOO ⁇ l used to assay for secreted human growth hormone in a radioimmunoassay using the Tandem-R HGH system (Hybritech) . From a standard curve produced in each assay the levels of hGH secreted were measured in ng/ml. The small background level in a mock transfected control was subtracted from the results of the transfected samples. The hGH levels were normalised by reference to a co-transfected LacZ expression vector. An actin promoter driving LacZ expression (Gunning et al .
  • the probe for the HIV-2 hGH transcript was a Pstl-Avall (dephosphorylated end) in pH2ghl and the probe for the HIV-2 tk transcript was a Xhol-Mlul (dephophorylated end) fragment from pH2tkl as shown in Figure 3.
  • the probe (700 bases) for the 3' end of the human ⁇ globin gene was an EcoRI-Sall fragment where the EcoRI is within the final exon and labelled by filling with reverse transcriptase in the presence of ⁇ 32 P dATP.
  • the protected fragment was 212 bases.
  • the same probe for ⁇ actin was used for mouse and human cell lines due to cross hybridisation.
  • a Xhol-Aval fragment from pHF/?A-l (Gunning et al . , 1983) was dephosphorylated and end labelled to give a protected fragment of 145 cases with RNA from human cells and 112 bases with RNA from mouse cells. Fold induction of transcripts was quantified by a phosphorlmager (Molecular Dynamics) or Joyce-Loebl Chromoscan densitometer.
  • Retroviral vectors were produced by cloning selected HIV-2 LTR sequences fused to the tk gene from the pH2tk plasmids into pLA.
  • a double stranded oligonucleotide adapter containing 5'-3* Smal, BamHI and Xhol restriction enzyme sites was synthesised.
  • the adapter contained a 5' overhang to anneal to a Xhol site and 3' overhand to anneal to a BamHI site, neither of which will reform the Xhol or BamHI site after ligation.
  • pLA was constructed by ligating Pvul- BamHI linearised pLJ to the adapter and a Pvul-Xhol (residues 419-1560) containing part of the gag region of the Mo-MLV genome.
  • the viruses were made by cloning the HIV-2 tk containing XhoI-BamHI fragment from pH2tkl, pH2tk4 and pH2tk5 and pLA5.
  • Packaging cell lines, PA317 and AM12 were transfected as described above. One day after transfection the packaging cells were split and grown for 10-14 days in 0.4 mg/ml G418 (Gibco-BRL) .
  • G418 resistant colonies were isolated and the higher titre clones identified by analysis of viral RNA in cell supernatants as previously described (Huszar et al . , 1989) .
  • NIH 3T3 and HeLa cells were infected with supernatants of cloned packaging cells in 8 ⁇ g/ml polybrene and were selected in 0.6 mg/ml G418.
  • CEM cells were infected by co-cultivation with packaging cells as previously described by Stevenson et al . (1988). Infected populations were isolated after 4 weeks in G418 1-1.5 mg/ml.
  • the cells infected by the HIV-2 tk vector were free of replication competent virus after extensive culture as determined by the method described by Markowicz et al . (1988) .
  • Restriction enzyme digested genomic DNA was transferred to Nytran nylon filters by the Southern procedure. Hybridisation and washing were as described in Sambrook et al . (1989). A 2.8 kb Bglll-BamHI fragment of the tk gene was used as an OLB labelled probe.
  • HIV-2 promoter was used because its TAR region is very responsive to HIV-l, HIV-2 and SIV derived Tat and can provide the widest range of application and testing in the future.
  • Eight minimal promoter constructs (Fig. 2) were generated using PCR, incorporating base substitutions, deletions and consensus sequences in the factor binding sites of the wild type HIV- 2 LTR.
  • constructs all contain the HIV-2 TATA sequence and TAR region and include the following 5* elements: 1.) the wild type HIV-2 promoter up to -230, 2.) a minimal TATA promoter, 3.) a promoter with 2 NF/cB sites, 4.) one with 3 Spi sites, 5.) another with a single Spi site, 6.) the same Spi site mutated, 7.) the proximal NFcB site and the mutated Spi site and 8.) a mutated proximal NFcB site and a mutated Spi site.
  • the spacing between the factor binding elements and the TATA sequence was maintained.
  • the ability of the HIV-2 promoters to direct basal transcription was tested by transient expression of the hGH reporter gene (Selden et al . , 1986) in transfected COS and CEM (CD4 positive human T) cells. As shown in Table 1, the basal levels of hGH expression were decreased in both cell lines with all of the modified promoter constructs. In COS cells the levels of hGH protein ranged from 0.52 to 1.2 ng/ml and are at least 4 fold lower than from the wild type promoter 1 (4.9 ng/ml). The basal levels of hGH protein produced in CEM cells from the modified constructs were 0.25 to 1.9 ng/ml, with the levels decreased 2 to 14 fold in comparison to the wild type promoter (3.5 ng/ml).
  • basal activity was found with construct 4. Deletion of either NF «B or Spi sites effectively decreased basal activity.
  • basal activity was measured by analysis of mRNA in an SI protection assay, low levels of reporter gene transcripts were found to correspond to the reduced basal levels of hGH protein.
  • HIV-2 promoter constructs can be trans-activated to varying levels
  • the HIV-2 promoter constructs were tested for their ability to be Tat trans-activated in transfected COS and CEM cells. Our results indicate that all the promoter sequences can be trans-activated by HIV-l Tat (but to varying degrees) in both cell lines (Table 1 and 2) .
  • the wild type promoter transfectant was optimally trans-activated for hGH expression by a factor of 20 while transfectants receiving constructs 3, 4, 5 and 7 were trans-activated 4 to 10 fold.
  • the levels of hGH protein were decreased from 100 ng/ml as produced by construct 1, to 3, 5, 12 and 3.4 ng/ml from constructs 3, 4, 5 and 7 respectively.
  • Tat trans-activation greatly increased hGH production.
  • Wild type promoter driven hGH production was increased to 426 ng/ml and the levels of hGH expression from constructs 3, 4 and 5 were raised to 32, 43 and 69 ng/ml respectively.
  • COS and CEM cells containing construct 5 produced higher levels of hGH than construct 4 (three Spi sites) .
  • the degree of trans-activation in CEM cells of construct 4 and 5 was higher than the Tat induced trans-activation produced by the wild type construct 1 (123-fold) .
  • Construct 3 with two NFcB sites was trans-activated only by a factor of 43.
  • promoter constructs 1, 4 and 5 were the most highly trans-activated whilst having decreased basal activity, these promoters were cloned with the tk gene in the reverse orientation into the retroviral vector pLA to produce pLAl, pLA4 and pLA5 respectively (Fig. 4A) .
  • Amphotropic producer clones LAI, LA4 and LA5 were found to have titers of 5 x 10 3 , 1 x 10 3 and 5 x 10 3 CFU/ml respectively, as assayed by G418 resistance of infected 3T3 cells.
  • Trans-activated tk expression occurs after retroviral mediated gene transduction.
  • the efficiency of uptake as measured by transfection with a CMV-LacZ plasmid and ⁇ -galactosidase staining was determined to be 2-6% (data not shown) . This indicates that the levels of trans-activation in these cells should be increased by an additional 16-50 fold.
  • ganciclovir ganciclovir
  • the basal expression of the HIV-2 tk construct 1 resulted in complete ablation of the 3T3 cells (31.1) at GCV concentrations greater than 4 ⁇ M (Fig 6A and B) whereas less than 0.3 ⁇ M GCV was completely cytotoxic to Tat transfected subclones 31.1A and 31.IB (Fig. 6A) .
  • HeLa cells (HI.4) infected with the same retrovirus were 40 fold more sensitive to GCV than the 3T3 clone and were killed at GCV concentrations greater than 0.1 ⁇ M (Fig 6C) .
  • LAI infected 3T3 cells As a functional test of diminished basal activity from the modified HIV-2 promoter constructs, we compared the GCV sensitivity of LAI infected 3T3 cells with LA4 infected cells (Fig.6B). LAI infected 3T3 cells (31.1) were completely ablated with greater than 4 ⁇ M GCV while LA4 infected 3T3 cells (34.1) were less sensitive to the pro-drug and required greater than 16 ⁇ M GCV for complete ablation. As shown in Figure 6D, we also compared the GCV sensitivity of LAI infected HeLa cells with LA4 and LA5 infected cells.
  • LAI infected HeLa cells were completely ablated with greater than 0.1 ⁇ M GCV but LA4 and LA5 infected cells required GCV concentrations of greater than 1 ⁇ M for cytotoxicity.
  • the cytotoxic effect of GCV on the basal expressing cells is greatly reduced by the use of the modified HIV-2 promoters in the LA4 and LA5 retroviruses.
  • Nef transgenic mice express Nef in thymocytes and peripheral T cells
  • mice Four transgenic lines of mice were produced with a construct containing the human CD2 promoter and LCR element and a 621 bp Nef fragment (Figure 7) .
  • Two different alleles of the HIV-l Nef gene were used to examine the effects of Nef in vivo, 1147 with threonine at amino acid position 15 and 1191 with an alanine at position 15.
  • In vitro studies show CD4 downregulation with both alleles (Guy et al . , 1990) but only the 1147 allele is phosphorylated at position 15.
  • DNA from the four lines; A (1147), F (1147), B (1191) and D (1191) was analyzed by Southern blot analysis and copy numbers were determined to be 6, 25, 48 and 26 respectively (Figure 8A) .
  • Thymocytes and peripheral T cells populations are altered in Nef transgenic mice
  • Nef expression results in a downregulation of CD4 on the surface of developing T cells
  • Mitogen induced or anti-CD3e mediated activation assays were performed to examine whether downregulation of CD4 or loss of CD4+ cells had negative effects on thymocyte or peripheral T cell activation.
  • Proliferation of thymocytes as measured by 3 H thymidine incorporation after activation via the calcium ionophore (ionomycin) and phorbol ester (PMA) revealed small differences between Nef transgenic and non-transgenic cells of both alleles, demonstrating that the total response of transgenic thymocytes to mitogen is not impaired.
  • nef is therefore responsible for downregulation of CD4 expression on thymocytes, which we have found to be due to intracellular sequestration of CD4.
  • Nef is accordingly not useful as a decoy protein when expressed indiscriminately of the infecion status of the cell.
  • transgenic mice were created carrying a transgene expressing the HSV-1 TK gene under the control of the HIV-2 promoter in the presence of the CD2 LCR.
  • Construction of an HIV-2 LTR driven transcriptional unit with the inclusion of the CD2 LCR element for high level expression Construction of an HIV-2 LTR driven transcriptional unit with the inclusion of the CD2 LCR element for high level expression.
  • the TK gene contains its own translational initiation codon, polyadenylation and termination sequences but no promoter.
  • a 5.5 kb BamHl-Xbal fragment containing the human CD2 LCR was ligated downstream of the HIV-2 TK sequences.
  • the complete construct (8.7 kb) can be isolated from plasmid sequences by digestion with Xhol.
  • Founder mouse 2A containing 20 copies of the HIV tk CD2 transgene ( Figure 13) was sacrificed and DNA extracted from various tissues for use in Southern blot analysis. DNAs were digested with Pstl and Xbal to liberate an internal fragment within the transgene and run on a 1% agarose gel. The DNA was transferred onto a nylon filter and hybridized with a CD2 probe. All tissues analysed, namely; thymus (T) , spleen (S) , lymph node (LN) , brain (Br) , heart (H) , kidney (K) , lung (Lg) , liver (Li) and muscle (M) were positive for the transgene indicating that this founder was not a mosaic. The slower migrating band in lane LN indicates only a partial digest of this DNA. Copy number controls 0, 1, 10 and 20 copies per cell are indicated ( Figure 15a) .
  • the HIV-2 TK CD2 transgene expresses highly in HeLa cells
  • HeLa cells were transiently transfected (24 hours) with the HIV-2 TK CD2 construct (HeLa 2) and with a HIV-2 TK construct (HeLa 1) as a control. At the same time, these cells were cotransfected with a CMV-globin quantiation control and pSV2-TAT for transactivated expression from the HIV-2 promoter.
  • Total RNA was isolated from mock transfected HeLa cells and two transfected populations (HeLa 1 and 2) . When SI nuclease RNA protection was performed, both transfected populations showed a high level of Tat- transactivated TK transgene expression. Cotransfected genes, TAT and globin were also expressed. No expression was observed in the mock transfected HeLa cells.
  • the HIV-2 TK CD2 construct is expressed in human cell line with no added transcriptional effect due to the presence of the CD2 LCR.
  • lymphoid tissues lymph nodes, spleen and thymus
  • T cell specific expression Although no expression is seen in the non-lymphoid tissues, some expression is seen in the brain. Basal expression is observed in the absence of tat. We would expect that transcription would increase 100-200 fold, particularly in the lymphoid tissues, with transactivation of the HIV promoter by tat.
  • the human 9-globin gene contains multiple regulatory regions: identification of one promoter and two downstream enhancers. EMBO J. 7: 377-384.
  • HIV-l tat trans- activation requires the loop sequence within tar. Nature 334: 165-167.
  • Thymidine kinase obliteration Creation of transgenic mice with controlled immune deficiency. Proc. Natl. Acad. Sci. USA 86: 2698-1702.
  • HIV-l Tat protein increases transcriptional initiation and stabilises elongation. Cell 59: 283-292.
  • HIVs human immunodeficiency viruses
  • RNA can account for the trans- activation of human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 83: 9734-9738.
  • HIV TAR an RNA enhancer? Cell 59: 229-230.
  • HIV-l gag mutants can dominantly interfere with the replication of the wild type virus.

Abstract

L'invention concerne un procédé servant à la prévention ou au traitement d'une infection virale et comprenant l'administration à un sujet d'une quantité efficace d'un système d'administration comprenant un vecteur capable d'exprimer dans une cellule susceptible d'être infectée par le virus un gène codant un agent présentant une efficacité directe ou indirecte de prévention, d'élimination ou d'atténuation de l'infection, ledit gène étant sujet à une régulation entre un état opérationnel et un état inopérationnel dans la cellule par l'intermédiaire de facteurs spécifiquement associés au virus et à la cellule. Le procédé permet de réguler efficacement des produits génétiques dans des cellules cibles.
PCT/GB1994/000568 1993-03-19 1994-03-21 Systeme d'administration commande par des facteurs associes au vih et a la cellule WO1994021806A1 (fr)

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AU697095B2 (en) * 1993-09-24 1998-09-24 Cobra Therapeutics Limited Expression of viral decoy proteins under the control of a locus control region and uses thereof
WO1995008635A1 (fr) * 1993-09-24 1995-03-30 Therexsys Limited Expression de proteines leurres virales soumises a une region de regulation de locus et leurs utilisations
WO1996009066A2 (fr) * 1994-09-23 1996-03-28 The Johns Hopkins University School Of Medicine Procede de traitement d'infections dues au virus de l'immunodeficience humaine (vih)
WO1996009066A3 (fr) * 1994-09-23 1996-05-23 Univ Johns Hopkins Med Procede de traitement d'infections dues au virus de l'immunodeficience humaine (vih)
EP0816507A1 (fr) * 1995-03-16 1998-01-07 Hisamitsu Pharmaceutical Co., Inc. Nouveau recombinant genique
EP0816507A4 (fr) * 1995-03-16 1998-07-01 Hisamitsu Pharmaceutical Co Nouveau recombinant genique
US5869306A (en) * 1995-03-17 1999-02-09 Hisamitsu Pharmaceutical Co., Inc. Gene transfer preparation
WO1996032489A1 (fr) * 1995-04-14 1996-10-17 Institut National De La Sante Et De La Recherche Medicale Vecteur viral recombinant inductible par le glucose
FR2732978A1 (fr) * 1995-04-14 1996-10-18 Inst Nat Sante Rech Med Vecteur viral recombinant, composition pharmaceutique le contenant et cellules transformees correspondantes
US6309878B1 (en) 1995-04-14 2001-10-30 Institut National De La Sante Et De La Recherche Medicale Glucose-inducible recombinant viral vector
AU715377B2 (en) * 1995-04-14 2000-02-03 Institut National De La Sante Et De La Recherche Medicale Glucose-inducible recombinant viral vector
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WO1997028818A1 (fr) * 1996-02-12 1997-08-14 Cobra Therapeutics Limited Nouveaux procedes de vaccination et vaccins associes comprenant un acide nucleique codant un premier epitope, ainsi qu'un peptide contenant un second epitope
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WO1999034006A3 (fr) * 1997-12-23 1999-09-02 Genera Spa Methode de selection positive de cellules t cd4+ transduites avec des genes anti-vih

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JPH08508642A (ja) 1996-09-17
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EP0689602A1 (fr) 1996-01-03
AU696399B2 (en) 1998-09-10
AU6261494A (en) 1994-10-11

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