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WO1999013064A2 - Infection virale de cellules - Google Patents

Infection virale de cellules Download PDF

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Publication number
WO1999013064A2
WO1999013064A2 PCT/GB1998/002753 GB9802753W WO9913064A2 WO 1999013064 A2 WO1999013064 A2 WO 1999013064A2 GB 9802753 W GB9802753 W GB 9802753W WO 9913064 A2 WO9913064 A2 WO 9913064A2
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Prior art keywords
cells
infected
infection
tnfα
viral vector
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PCT/GB1998/002753
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WO1999013064A3 (fr
Inventor
Marc Feldmann
Brian Maurice John Foxwell
Fionula Mary Brennan
Jan Bondeson
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The Mathilda And Terence Kennedy Institute Of Rheumatology
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Priority to EP98942892A priority Critical patent/EP1012321A2/fr
Priority to JP2000510853A priority patent/JP2001515716A/ja
Publication of WO1999013064A2 publication Critical patent/WO1999013064A2/fr
Publication of WO1999013064A3 publication Critical patent/WO1999013064A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This current invention relates to an improved in vitro method for infecting cells with a viral
  • the invention capable of transporting recombinant nucleic acid to those cells.
  • the invention also relates to cells infected
  • viruses such as human adenovirus or retroviruses such as Human Immunodeficiency Virus (HIV) derivatives as vectors to deliver exogenous or recombinant nucleic acid, such as foreign genes, into cells is well known.
  • Replication-defective forms of human adenovirus have been used to deliver foreign genes into diverse cells types including liver, muscle, nerve .and airway epithelial cells .
  • the success of adenoviruses as a gene delivery vector is based in part on their highly efficient mode of cell entry and their lack of requirement for host cell replication.
  • a major difficulty in using viral vectors to transport recombinant nucleic acid into cells has been the difficulty in achieving high levels of infectivity in some cells.
  • any effect of the transferred gene on the cells may be difficult to observe as a result of the background of non-infected cells.
  • a high level of gene transfer would allow the study of, for example, intracellular mechanisms , without any requirement to remove a non-infected population of cells. This would
  • monocytes When adenovirus is used to infect monocytes, for example, the maximum level of infection achieved has been ap O roximately 50% which required a multiplicity of infection (M.O.I. ), as plaque forming units per cell, of up to 1000 ( Huang, 1995 J. Virol. 69: 2257-63. Haddada , 1993 Bioche . Biophys . Res. Commun. 195:1174-83).
  • monocytes are prepared by isolating mononuclear cells from healthy adult donors using density gradien t s , washing the mononuclear cell fraction by low-speed centrifugation and resuspending the mononuclear ce_ls obtained in growth medium. The ceils are then allowed to
  • the adherent cell population comprises typically 90% monocytes.
  • monocytes obtained may then be treated with a cyto ine, such as a granuiocyte-macrophage colony stimulating factor (GM-C ⁇ F) or acropha ⁇ colony stimulating factor ( M-CSF) , prior to infection with the viral vector.
  • a cyto ine such as a granuiocyte-macrophage colony stimulating factor (GM-C ⁇ F) or acropha ⁇ colony stimulating factor ( M-CSF)
  • cytokines increase the expression of integrins ( ⁇ V ⁇ 3 and ⁇ V ⁇ 5) on the cell surface and are required for viral entry into cells.
  • the inventors have identified an improved method of infecting ceils with viral vectors which produces infectivity races of approximately 100% with considerably lower concentrations of viral vector. This removes the necessity to remove non-infected ceils and means that less recombinant viral-vector need be used. Consequently the regulation of cellular pathways can be studied.
  • the inventors realised that inflammatory diseases such as rheumatoid arthritis,
  • Adenovirus is
  • the invention provides an in vitro method
  • ⁇ cytokine infecting the col lected cel l s with a viral vector.
  • the cells are subjected to elutriation prior to collection.
  • centrifugal elutriation This combines centrifuging a sample to sediment out particles with elutriation, the process of separation by washing.
  • a suspension of cells is pumped into a funnel shaped chamber at a preset flow rate.
  • As fluid travels through the chamber its velocity decreases as the chamber gets wider thus creating a velocity gradient from the narrow end of the chamber to its widest part.
  • Cells migrate to positions in the velocity gradient where the effects of both the centrifugal force field and the fluid velocity are balanced. Smaller cells are at equilibrium at the elutriation boundary where the centrifugal force field and the velocity are low. Larger cells will remain near the inlet to the chamber where the centrifugal force field and the velocity are high.
  • By gradually increasing the flow rate cells can be washed out according co size.
  • Cytokines have been found to increase the concentration of integrins, such as ⁇ V ⁇ 3 or ⁇ V ⁇ 5,
  • viral vectors may,
  • a further aspect of the invention provides an in vitro method for infecting one
  • rheumatoid synovial cells with a viral vector capable of transporting exogenous or
  • asthma and chronic obstructive pulmonary disease C.O.P.D.
  • gut biopsies of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis
  • the viral vector is an adenovirus, especially a replication-deficient adenovirus.
  • the M.O.I. / expressed as plaque forming units per cell may be 10-1000, preferably 35-100, especially 50-100.
  • the cells to be infected are monocytes. Such cells may be isolated from, for example, single donor plateletphoresis blood residues . The cells may be partially purified by density centrifugation to provide the suspension of cells prior to elutriation.
  • the isolated cells may be cultured in a suitable growth medium, but are preferably treated with cytokine substantially immediately after collection from the elutriator with cytokine.
  • Cytokine is preferably added to the medium in which the cells axe cultured and may be incubated with the cells for between 24 and 96 hours, typically 65-75 hours, preferably about 72 hours.
  • the cytokine may be M-CSF.
  • the concentration of cytokine used is preferably 100ng-l ⁇ g/ml-
  • the exogenous or recomhi ⁇ ant nucleic acid within the viral vector is DNA.
  • the recombinant nucleic acid preferably comprises a sequence encoding a gene product of interest operably linked to suitable regulatory sequences to enable the gene to be expressed within the infected cell.
  • the foreign gene encodes a modulator for one or more biochemical pathways within the cell to allow the cell pathways to be studied.
  • a modulator is the I / cB protein, an endogenous regulator, especially I ⁇ B ⁇ , an inhibitor of NF K B.
  • a mutated enzyme such as one in which catalytic activity has been destroyed,
  • a single chain antibody or antibody fragment, or antisense DNA may be
  • the invention also relates to cells infected by virus by means of the methods of the invention.
  • monocytes by GM-CSF and M-CSF monocytes by GM-CSF and M-CSF.
  • Monocytes were untreated or treated with GM-CSF (500U/ml) , or M-CSF (l ⁇ g/ml) or both cytokines for 48 hours. The cells were then
  • FIG. 2 The effect of cytokine treatment on adenoviral infection of primary human monocytes .
  • Monocytes were treated with GM-CSF (500U/ml) or M-CSF ( l ⁇ g/ml ) for 72 hours followed by infection with 0-galactosidase (shown by dotted line) containing adenovirus or control virus (solid line) at 100 m.o.i. (a) for various m.o.i. (b) .
  • 0-galactosidase shown by dotted line
  • adenovirus or control virus solid line
  • FIG. 3- M-CSF treatment is not required for infection of R A W 264.7 cells.
  • RAW 264.7 cells were incubated with or without M-CSF (lug/ml) for 48 hours followed by infection with control adenovirus or a vector containing ⁇ -galactosidase. Following culture for 4 days the cells were assayed for ⁇ -galactosidase expression by FACS. Key: control virus, no M-CSF (thick line) , control virus + M-CSF (thin line), ⁇ -galactosidase virus, no M-CSF
  • RAW 264.7 cells infected monocytes and RAW 264.7 cells.
  • RAW 264.7 cells (a) or M-CSF treated monocytes (b) were untreated (un) or infected with IkB ⁇ adenoviral vector at the given m.o.i.
  • IkB ⁇ expression was determined as above: equivalent amounts of protein were loaded on each track.
  • FIG. 5 Expression of NFkB and IkB proteins in virus infected monocytes .
  • Monocytes were treated with M-CSF for 48 hours. The cells were then infected with a control virus or an IkB ⁇ containing adenoviral vector at
  • monocytes were uninfected or infected with IkB ⁇ or control adenovirus followed by LPS
  • M-CSF treated cells were untreated or
  • Dat a is given as percentage TNF production of non- infected control cells .
  • IL-l ⁇ A
  • IL-8 B
  • IL-6 Q, expressed as a percentage of production of the cytokine
  • Figure 9 Effect of exogenous TNF on IL-1/? and IL-6 production. Pretreatment with
  • TNF ⁇ -induced (20 ng/ml) IL-6 is potently inhibited by
  • Fig isure 11 Effect of I ⁇ B ⁇ infection on IL-lra production. Effect of infection of human
  • TNF ⁇ mRNA expression (C) is unaffected by infection with 80:1 of AdvI B ⁇ . Lower
  • paneis contain GADPH expression. This is a representative of three complete experiments.
  • TNF ⁇ Effect of the prote ⁇ ome inhibitor PSl on LPS-induced ( ⁇ ) and zymosan-induced
  • Figure 15 In excess of 90% of rheumatoid synovial cells can be infected with -adenovirus.
  • AdvO f 111 ed line
  • Adv ⁇ gal solid linei A representative of five experiments.
  • FIG. 17 I B ⁇ overexpression permanently inhibits TNF ⁇ .and IL-6 production. Effect of infection of rheumatoid synovial ceils with 40: 1 of either AdvO or AdvI B ⁇ on die spontaneous production of TNF ⁇ (A) or IL-6 (B) over time. Symbols denote uninfected Q, AdvO-infected () or AdvI ⁇ B ⁇ -infected () ceils. A representative of three independent experiments.
  • Figure 19 I ⁇ B ⁇ overexpression potently inhibits MMP-1 .and MMP-3 production, but slightly potentiates the production of TIMP-1.
  • Fisure 20 Effect of AdvI ⁇ B infection on the balance between MMP production versus the production of TEMP-l over time. Effect of infection of rheumatoid synovia! cells with 40: 1 of either AdvO or AdvI B ⁇ on the spontaneous production MMP-1 (A), MMP-3 (B) and TTMP-l (C) over time. Symbols denote uninfected—, AdvO-infected ⁇ or AdvI ⁇ B ⁇ -infected ⁇ cells. A representative of three independent experiments.
  • Cells were usually collected from the Ficoll gradient resuspended in Hanks and centrifuged at ⁇ 2000 rpm in a centrifuge for 10 minutes. The pellets were resuspended to 50ml in a Falcon tube and respun ⁇ lOOOrpm for 15 minutes .
  • the resulting pellet was resuspended in RPMI with 10% FCS up to 50ml before injecting into the elutriator. A maximum of 1000xl0 6 cells were loaded into the chamber. Typically the cells were elutriated according to the manufacturers instructions .
  • the elutriator head was assembled and 200ml 1% Etoxaclean was flushed through. This was followed by 1.5L of sterile distilled water. 300ml of elutriation buffer (RPMI with P/S and 1% low endotoxin FCS) was then pumped through. The sample containing cells to be purified was then aspirated through the elutriator at 2000rpm and 10' C. Fractions containing the cells of interest were collected in Falcon tubes .
  • Monocyte purity was assessed by flow cytometry using directly conjugated anti-CD45 and anti-CD14 antibodies (Leucocyte, Becton Dickinson, UK) and was routinely greater than >90%. All media used in separation and culture of monocytes was tested for endotoxin using the Limulus amoebocyte lysate test (Bio Whittaker Inc. Bethesda MD) and were rejected if endotoxin contamination exceeded 0.1 unit/ml.
  • the recombinant replication-deficient adenovirus vectors encoding E.coli ⁇ -galactosidase or having no insert (rAd ⁇ ) was provided by Dr. A. Byrnes (Oxford UK) .
  • a second virus encoding E.coli ⁇ -galactosidase gene (Ad/ ⁇ -gal) [Watanabe, 1996, Blood 87:5032-9] was generously provided by Canji, Inc. (San Diego CA) and the vector encoding IkB ⁇ (rAD
  • viruses were prepared, purified, titered as previously described [Watanabe, supra] .
  • Virus was produced in the 293 human embryonic kidney cell line and purified by ultracentrifugation through two caesium chloride gradients.
  • the titers of viral stocks were determined by a limiting dilution plaque assay on 293 cells, and were 2.9 to 5.8 x 10 10 infectious units/ml as measured before dilution for use.
  • Viruses were suspended in a buffer solution of 2% sucrose and 2mmol/L MgCl 2 and stored at -70OC.
  • monocytes were washed in RPM 1640 and resuspended at 5 x 10 5 cells/ml.
  • the cells were infected with virus at the indicated plaque forming units per cell (multiplicity of infection - m.o.i.) .
  • ⁇ -galactosidase expression in individual cells was measured by FACS .
  • cvtosolic proteins 4 x 10 6 cells were plated on petri dishes (Nunc) , and cultured overnight. They were stimulated, washed in ice cold PBS, removed from dishes by scraping, and lysed (20mM Tris pH 8.0, 137mM NaCl, ImM MgCl 2 0.1% NP-40 10 minutes at 40C) . Lysates were spun (120Ox g 10 minutes 4 ⁇ C) and the supernatant was retained and assayed for protein concentration by the Commercially available BCA method.
  • Nuclear extracts were prepared as previously described [Whiteside, 1992, Nuc . Acids Res. 20:1531-8 ]. Briefly cells were washed with ice cold PBS then spun (1200x g 30 second 4 ⁇ C) and the aspirated pellet was resuspended in low salt lysis buffer (lOmM Hepes, 1.5mM MgCl 2 lOmM KCl 0.5 mM DTT ImM PMSF lO ⁇ g/ml aprotinin 30 ⁇ g/ml leupeptin) at 4 ⁇ C for 5 minutes, NP-40 was added to a final concentration of 0.125% and cells were vortexed immediately.
  • low salt lysis buffer lOmM Hepes, 1.5mM MgCl 2 lOmM KCl 0.5 mM DTT ImM PMSF lO ⁇ g/ml aprotinin 30 ⁇ g/ml leupeptin
  • the samples were spun (120Ox g 10 minutes 4 ⁇ c) , the cytosolic fraction was removed and the nuclear pellet was resuspended in nuclear extraction buffer (5mM Hepes 25% w/v glycerol, 0.4M NaCl 1.5ml MgCl 2 0.2mM EDTA ImM DTT ImM PMSF lO ⁇ g/ml aprotitin, lO ⁇ g/ml pepstatin 30 ⁇ g/ml leupeptin) and left at 4 ⁇ C for 1 hour.
  • the samples were spun (120Ox g 10 minutes 4 ⁇ c)and the supernatant was
  • Membranes were blocked with a 5% milk powder solution (20mM Tris 150mM NaCl pH 8.0.0.1% Tween 20) and then proteins were detected with rabbit polyclonal antibodies and horseradish peroxidase conjugated donkey anti-rabbit F(ab) 2 fragments (Amersham UK), both diluted in milk powder, and visualised by enhanced che iluminescence (Amersham UK) .
  • Electrophoretic mobility shift assays were performed as previously described (Clarke, 1995, Eur. J. Immunol. 25: 2961-6). Data was analysed using a ⁇ iorad GS-670 densito eter.
  • cytosolic mRNA 15x10 6 cells were plated overnight, stimulated for 2 hours with LPS and cytokine, washed in ice cold PBS and resuspended in 400 ⁇ l lysis buffer (lOmM tris pH 7.9 150mM NaCl 1.5mM MgCl 2 0.65% NP-40 10 minutes 4 ⁇ c. Lysates were spun (1200Ox g, 5 minutes, room temp.). EDTA (l ⁇ l 0.25M) and SDS (20 ⁇ l 10% in H 2 0) were added to the required supernatant and the tube was gently mixed.
  • mRNA was purified by extraction, with 1:1 phenol chloroform, and precipitation (-20OC, 30 minutes) with sodium acetate (pH 5.2) to a final concentration of 0.3M and 2 columns of ethanol.
  • the precipitate was spun down (120Ox g, 15 minutes, 40C) , washed with freezing 70% ethanol, then re-dissolved in water.
  • Northern blottin ⁇ mRNA (lO ⁇ l, I ⁇ g/ ⁇ l) was mixed with 2 ⁇ l H0, 5 ⁇ l lOx northern buffer (200mM MOPS, 50mM sodium acetate), lOmM EDTA pH 7.0), and 25 ⁇ l deionised formamide and heated to 60OC for 5 minutes.
  • lO ⁇ l 6x loading buffer 0.25% bromophenol blue, 0.25% xylenol cyanol 25% glycerol
  • was added and the samples were run on an agarose gel 1% agarose, 20mM MOPS, 5mM sodium acetate, ImM EDTA, 6.5% formaldehyde pH 7.0, 100mA 4 hours).
  • mRNA was transferred to a Hybond-N membrane (Amersham UK) by capillary action and fixed by baking (80oc, 2 hours) .
  • the membrane was pre-incubated in hybridisation solution (50% formamide, 5x SSC, 0.05M sodium phosphate pH 6.6 0.5x Denhardts solution O.lmg/ml salmon sperm DNA) for 4 hours at 42 ⁇ C and hybridised (12 hours, 420C) with l-3x 10 6 cpm/ml of 32 P labelled cDNA probe dissolved in hybridisation solution.
  • the membrane was washed in 0.2xSSC 0.1% SDS at 42oc and exposed to hyperfilm MP at -70OC for 1-3 days.
  • the murine macrophage cell line RAW 264.7 was infected by adenovirus ⁇ -galactosidase to a degree similar to human monocytes at 100 m.o.i. ( Figure 4), and still showed >60% infection at m.o.i. of 10 (results not shown) .
  • These cells did not require prior exposure to M-CSF ( Figure 4) .
  • IkB is known to inactivate NFkB a transcription factor within cells. Inhibition of NFkB allows the suppression of proteins whose expression is dependent on the factor to be studied. Infection of both RAW 264.7 cells of M-CSF treated human primary macrophages result in the prominent over-expression of IkB ⁇ ( Figure 5) . No changes in IkB ⁇ expression were evident following
  • bind activity was also investigated by EMSA ( Figure 6) . Infection of monocytes with the virus resulted in total ablation of nuclear NFkB DNA binding activity. No such effect was observed in uninfected or control virus treated cells. The activity of AP-1 was also investigated. Unlike NFkB, AP-1 appeared to be constitutively active in the M-CSF treated cells and no LPS induced activity was detectable. However it was noted that there was a some low level reduction in nuclear AP-1 binding activity in IkB ⁇ virus infected
  • NFkB Activation of NFkB is thought to be essential for the
  • synoviocytes obtained from rheumatoid joints. These cells constitutively express TNF and other cytokines . Infection with virus resulted in the inhibition of TNF production by these rheumatoid synoviral cells (Figure 7C).
  • NFkB has been reported to be a key anti-apoptotic mechanism and that inhibition of NFkB induced by TNF resulted in cell death, it was possible that cell depletion may account for the inhibitory effects of the IkB virus .
  • the inventors have demonstrated approximately 100% infection with m.o.i. of 50-100. Previous studies [Haddada 1993 supra and Huang, 1995 supra] only obtained approximately 50% infection which required a m.o.i. of 1000.
  • the inventor's observation that high levels of gene transfer can be achieved with the conditions of the invention has important consequences for the study of intracellular mechanisms of macrophages as it precludes any requirement to remove a non-infected population and obviates the need for cloning . This allows the regulation of endogenous chromosomal genes to be studied and precludes the need to use ectopically expressed reporter constructs which may be regulated differently.
  • Adenoviral Vectors Recombinant, replication-deficient adenoviral vectors encoding E.
  • nuciear localization sequence (AdvI ⁇ B ⁇ ) was provided by Dr R. de Martin (Vienna.
  • Viruses were propagated in the 293 human embryonic kidney cell line and purified by ultracentrifugation through two cesium chloride gradients. The titers of viral stocks were determined through a plaque assay on 293 cells, as described (24).
  • the elutriated human monocytes were incubated at approximately 2 x lO'/ml in RPMI 1640 with 25 mM HEPES and 2 mM L-glutamine, supplemented with 5% (v/v) heat inactivated fetal calf serum and 10 units/ml penicillin/streptomycin.
  • M-CSF 100 ng ml; obtained from the Genetics Institute. Boston MA
  • LPS is capable of inducing all the cytokines (TNF ⁇ , IL-l ⁇ , IL-6, IL-8) and inhibitors (IL-
  • IL-6 was induced equally well by LPS and zymosan.
  • IL-8 could be induced by the entire array of stimuli. Zymosan and. LPS were die only stimuli to induce IL-10 and the p55 soluble TNF receptor (Table I) whereas
  • PMA could induce IL-lra and p75 soluble TNF receptor.
  • adenovirus produced high levels of I ⁇ B ⁇ expression. This resulted in a potent inhibition
  • TNF ⁇ can induce the synthesis of other cytokines, e.g. IL-l ⁇ , IL-6 and IL-8, the
  • TNF ⁇ -driven IL-l ⁇ might be less NF- B dependent than the IL-l ⁇ response
  • TNF ⁇ is a weaker induc ⁇ r
  • the major anti-inflammatory cytokine produced by macrophages is IL-10 .
  • TNF ⁇ mRNA expression in response to PMA and UV light was ablated by I ⁇ B ⁇ over-
  • AdvI ⁇ B ⁇ were stimulated with PMA or UV light (Table II). Ionomycin, a stimulus that
  • adenoviral gene transfer into macrophages provides a reliable, reproducible and convenient method of studying intracellular signalling
  • IL-6 was most potently inhibited (> 85%) by over-
  • I ⁇ B ⁇ may reflect the observation that this cytokine was the most potently
  • IL-10 is under complex control, and in LPS-stimuiated cells, it appears to be at least partially driven via LPS-
  • ther ⁇ is some effect also on IL-10, but never, even with 80: 1 of the virus, exceeding 30%.
  • IL-l ⁇ is also, although to lesser extent than IL-10, driven partly by LPS-
  • the model of human macrophages infected with Adv cB ⁇ has, from a
  • NF- ⁇ B is an important therapeutic target in chronic infI.ammatory diseases
  • Rheumatoid synovial cells produce high levels of cytokines and express high levels of integrins on their surface (Feldman M. et al Ann. Rev. Immunol (1996) vol. 14). They have been used to demonstrate mat in vivo activated cells, with spontaneous high levels of integrins and cytokines allow improved viral infection rates to be .achieved. A use of such a system is also demonstrated.
  • Synovium from patients with rheumatoid arthritis undergoing joint surgery was dissociated by cutting into small pieces, and digested with collagenase and DNAse().
  • the total cell mixture was cultured at 37°C in RPMI 1640 with 25 mM HEPES and 2 M L-glutamine. supplemented with 5% heat inactivated fetal bovine serum, on 24-well or 48-well plates.
  • adenovirus encoding porcine -B ⁇ with a CMV promoter and a nuclear localization sequence was provided as above.
  • Viruses were propagated in the 293 human embryonic kidney cell line and purified by ultracentrifugation through two cesium chloride gradients. The titers of viral stocks were determined through a plaque assay on 293 cells. All viruses used were plaque purified from a master stock, in order to prevent contamination with wild form adenovirus.
  • rheumatoid synovial cells were resuspended in 1 ml serum-free RPMI 1640 on a 12- well plate at 2.5-4 million cells per well. After incubation for lh. they were either left uninfected, or infected with Adv B ⁇ or AdvO at a multiplicity of infection of 40: 1. After 2h, the supernatants were removed and replaced with 0.5 ml RPMI 1640 supplemented with 5% heat inactivated fetal bovine serum.
  • the nonadherent cells were carefully spun down and reintroduced to the rheumatoid cell coculture in 0.5 ml RPMI 1640 supplemented with 5% heat inactivated fetal bovine serum (total volume thus 1 ml).
  • rheumatoid synovial cells were infected as above, but at a density of 0.4 million cells per well in a final volume of 0.2 ml, on a 96-well plate.
  • Four wells of cells from each cathegory - uninfected, AdvO-infected and AdvI ⁇ B ⁇ -infected - were plated.
  • cells were resuspended in 0.6 ml serum-free RPMI 1640 on a 24-well plate at 1 million cells per well. After incubation for lh, they were either left uninfected, or infected with Adv gal or AdvO at a multiplicity of infection of 40: 1.
  • AdvO-infected or Adv gal-infected cells were then resuspended in 25 ⁇ l staining solution and incubated with 125 ng of anti-CD3 PerCP and 500 ng of anti-CD 14 PE (both from Becton Dickinson, San Jose CA), in a total volume of 45 ⁇ l for 45 min at 4°C. They were then incubated at 37°C for 10 min, before 45 ⁇ l of a 2 mM solution of Fluorescein di-(/?-D-galactopyranoside (Sigma) was added for 1 min. Addition of
  • TNF ⁇ and IL-6 is produced.
  • IL-1 receptor antagonist are unaffected by overexpression of B ⁇ ( Figure 18A-B ).
  • NF- ⁇ B is a key therapeutic target in rheumatoid arthritis, and probably other inflammatory diseases.
  • the unique spectrum of effects achieved by inhibiting this transcription factor includes a potent inhibition of
  • proinflammatory cytokines particularly TNF ⁇ and IL-6, but no effect on the key

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Abstract

L'invention porte sur un procédé in vitro visant à infecter une ou plusieurs cellules avec un vecteur viral capable de transporter l'acide nucléique exogène ou recombinant dans ces cellules. Ce procédé consiste à: a) recueillir les cellules devant être infectées; b) traiter les cellules infectées avec au moins une cytokine; et c) infecter les cellules recueillies avec un vecteur viral tel qu'un adénovirus. L'invention porte également sur un procédé visant à infecter des cellules synoviales rhumatoïdes et d'autres cellules comportant des taux élevés d'intégrines à leur surface.
PCT/GB1998/002753 1997-09-11 1998-09-11 Infection virale de cellules WO1999013064A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP98942892A EP1012321A2 (fr) 1997-09-11 1998-09-11 Infection virale de cellules
JP2000510853A JP2001515716A (ja) 1997-09-11 1998-09-11 細胞のウイルス感染

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9719238.9A GB9719238D0 (en) 1997-09-11 1997-09-11 Viral infection of cells
GB9719238.9 1997-09-11

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US09508350 A-371-Of-International 2000-04-07
US10/033,267 Continuation US20020177572A1 (en) 1997-09-11 2001-10-25 Viral infection of cells

Publications (2)

Publication Number Publication Date
WO1999013064A2 true WO1999013064A2 (fr) 1999-03-18
WO1999013064A3 WO1999013064A3 (fr) 1999-06-17

Family

ID=10818857

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1998/002753 WO1999013064A2 (fr) 1997-09-11 1998-09-11 Infection virale de cellules

Country Status (4)

Country Link
EP (1) EP1012321A2 (fr)
JP (1) JP2001515716A (fr)
GB (1) GB9719238D0 (fr)
WO (1) WO1999013064A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001021834A1 (fr) * 1999-09-22 2001-03-29 The Mathilda & Terence Kennedy Institute Of Rheumatology Etude de polymorphisme
US7915009B2 (en) 1999-12-24 2011-03-29 The Mathilda And Terence Kennedy Institute Of Rheumatology Trust Activation and inhibition of the immune system

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7105336B2 (en) * 2002-10-07 2006-09-12 Biogaia Ab Selection and use of lactic acid bacteria for reducing inflammation caused by Helicobacter

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0779361A3 (fr) * 1995-12-15 1999-11-10 F. Hoffmann-La Roche Ag Forme tronquée de la protéine inhibitrice kappa B, production récombinante et utilisation

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001021834A1 (fr) * 1999-09-22 2001-03-29 The Mathilda & Terence Kennedy Institute Of Rheumatology Etude de polymorphisme
US7915009B2 (en) 1999-12-24 2011-03-29 The Mathilda And Terence Kennedy Institute Of Rheumatology Trust Activation and inhibition of the immune system

Also Published As

Publication number Publication date
EP1012321A2 (fr) 2000-06-28
JP2001515716A (ja) 2001-09-25
WO1999013064A3 (fr) 1999-06-17
GB9719238D0 (en) 1997-11-12

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