WO2001021823A2 - Vecteurs adenoviraux - Google Patents
Vecteurs adenoviraux Download PDFInfo
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- WO2001021823A2 WO2001021823A2 PCT/GB2000/003645 GB0003645W WO0121823A2 WO 2001021823 A2 WO2001021823 A2 WO 2001021823A2 GB 0003645 W GB0003645 W GB 0003645W WO 0121823 A2 WO0121823 A2 WO 0121823A2
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- regulatory region
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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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
- 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
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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
- C12N2830/00—Vector systems having a special element relevant for transcription
Definitions
- the invention relates to adenoviral vectors, and in particular to adenoviral vectors comprising a mammalian regulatory region, such as a promoter, and a reporter gene operatively linked to the cytokine promoter.
- the invention also relates to cells containing the adenoviral vectors and to methods of using the adenoviral vectors.
- Adenoviral vectors which are capable of carrying foreign DNA into cells are known in the art.
- Adeno viruses exist as non-enveloped double-stranded DNA viruses.
- the adeno virus enters cells by a receptor-mediated endocytosis pathway.
- the adenovirus binds specific receptors present on the cell surface via fibres on its outer surface.
- the receptors with bound adenovirus cluster in coated pits, the virus is internalised within a clathrin-coated vesicle and, subsequently, into an endosomal vesicle, known as a receptosome or endosome.
- the adenovirus is ultimately transported to the nucleus where it directs synthesis of new nucleic acid and hence virus.
- Adenovirus has been used as means of transporting DNA into cells. There are two means by which such transfer has been affected. Firstly, the adenovirus has been employed to facilitate the transfer of non- viral DNA either linked to the surface of the adenovirus or where the molecule is internalised, taken along within the adenoviral receptor-endosome complex. Secondly, the adenovirus has- been employed to transfer non-viral molecules which are packaged within the adenovirus either in place of, or in addition to normal adenoviral genome. All systems have been used to take in exogenous nucleic acid placed within the double-stranded DNA genome of the virus. Such exogenous DNA has been placed under the control of a promoter to allow the exogenous nucleic acid to be transcribed.
- adenoviral vectors comprising a mammalian regulatory sequence such as a cytokine promoter.
- Cytokines are peptide regulators of inflammatory and immune reactions. Substances considered as cytokines include, for example, interleukins 1-18, interferons, tumour necrosis factors (TNF), transforming growth factors (TGF), platelet-derived growth factors, chemokines and colony-stimulating factors. Some compounds were sometimes previously known by the term "lymphokine".
- Cytokines act in a complex interacting network on leucocytes, vascular endothelial cells, mast cells, haemopoietic stem cells, fibroblasts and osteoclasts, controlling proliferation, differentiation and/or activation through autocrine or paracrine mechanisms.
- cytokines for example TNF- ⁇ and IL-1 are important inflammatory mediators and have been implicated in several chronic inflammatory and autoimmune diseases such as rheumatoid arthritis.
- the cytokines are being increasingly studied because of their importance in the formation of inflammatory and autoimmune diseases. Because the cytokines are involved in complex interconnecting reactions it is difficult to investigate the control of regulatory regions such as promoters or enhancers associated with the cytokines.
- cytokine promoter could be used to control a readily detectable reporter gene and allow the promoter to be studied.
- adenovirus is a suitable vector because it enables the promoter-reporter gene construct to be inserted into a wide variety of cells including cultured cell-lines, normal cells such as macrophages, diseased tissue and primary cell-lines.
- the ability to insert a construct into primary cells is of particular importance because it allows the control of the promoter to be studied within, for example, diseased cells.
- adenovirus also had the unexpected benefit of allowing unexpectedly high levels of regulatory region-controlled reporter gene function and behaviour to be observed that were similar to the behaviour of the endogenous gene. These levels were far in excess of levels observed with other methods of inserting a cytokine regulatory region-reporter gene construct into cells. Furthermore, it has the advantage that, for the first time, cytokine promoters, such as TNF- ⁇ promoters, have been able to be studied in monocytic cells for the first time. It is expected that the high levels of expression observed means that such promoters can be studied in a variety of cells.
- TNF ⁇ promoter- luciferase vectors have been produced by Udalova et al (J. Biol. Chem., 1998, Vol. 273, pages 21178-21186).
- the vector containing the construct, pXP-1 can only be used in a limited number of cell types, and with low levels of expression of the promoter.
- the invention provides an adenoviral vector comprising: a) a mammalian regulatory region; and b) a reporter gene operatively linked to the cytokine regulatory region (a).
- mammalian regulatory region is intended to mean a region of DNA which, in its natural state, is upstream and/or downstream of a gene which it regulates. For example, it may be the promoter for the gene encoding a human oestrogen receptor.
- the regulatory region may be a promoter or an enhancer region or a fragment of a region, such as an NF- ⁇ B binding site.
- the regulatory region may regulate the expression of a cytokine gene.
- the cytokine promoter is an interleukin promoter such as IL-10.
- the IL-10 promoter has the sequence from NT 7 to NT 4046 of Genbank sequence number X78437, where NT 4023 is the transcription start site, as shown in Annex 2.
- the promoter may be derived from other genes that have application in inflammatory disease, such as IL-6 and MMP.
- the cytokine promoter may also be the promoter for TNF ⁇ . More especially, the TNF ⁇ receptor is a human TNF ⁇ receptor, especially the TNF ⁇ promoter identified in Genbank Accession No. Ml 6441. This is enclosed as Annex 1. This is also the promoter used in the Udilova paper (Supra). Most preferably the sequence used is encompassed within -1173 to +130 of the human TNF gene, where 1 is the transcriptional start site. The transcriptional start site is nucleotide 4094 in the Genbank Accession No. Ml 6441. Hence, preferably the promoter sequence starts from 2921 of Ml 6441.
- adenoviral vector Any suitable adenoviral vector can be utilised in the present invention.
- a "vector” is a molecule that serves to transfer nucleic acid of interest into a cell.
- the adenoviral vector utilised in accordance with the present invention can encompass any adenoviral vector that is appropriate for the introduction of nucleic acids into eukaryotic cells and is capable of functioning as a vector as that term is understood by those of ordinary skill in the art.
- the adenovirus can be any serotype of adenovirus and, preferably, is a serotype that can transduce and/or infect a human cell.
- the adenovirus may comprise a complete adenoviral virion consisting of a core of nucleic acid and a protein capsid. Alternatively, it may comprise a naked adenoviral genome or any other manifestation of adenovirus as described in the art which can be used to transfer a cytokine regulatory region operatively linked to a reporter gene. Alternatively the virion may contain minimal (packaging signal) adenoviral genome sequences. These "gutless" viruses are produced by methods known in the art.
- the adenovirus employed for transfer of the regulatory region-reporter gene construct can be wildtype, that is replication-competent. However, it is not necessary that the genome of the employed adenovirus be intact. Indeed, the adenovirus can be rendered replication-deficient, by techniques known in the art.
- the reporter gene is operatively linked to the mammalian regulatory region.
- the reporter gene may be any nucleic acid sequence encoding a detectable gene product.
- the gene product may be an untranslated RNA product, such as mRNA or antisense RNA. Such untranslated RNA may be detected by techniques known in the art such as PCR, Northern or Southern blots.
- the reporter gene may encode a polypeptide, such as protein or peptide, product. Polypeptide may be detected immunologically or by means of its biological activity.
- the reporter genes used may be any known in the art.
- the reporter gene is luciferase.
- Luciferase reporter genes are known in the art. They are usually derived from firefly (Photinus pyralis) or seapansy (Renilla reniformis). The luciferase enzyme catalyses a reaction using D-luciferin and ATP in the presence of oxygen and Mg 2 " resulting in light emission. The luciferase reaction is quantitated using a luminometer which measures light output. The assay may also include coenzyme A in reaction which provides a longer, sustained light reaction with greater sensitivity.
- CAT chloramphenicol acetyltransferase
- This is a reporter gene which is well known in the art.
- CAT catalyses the transfer of the acetyl group from acetyl-CoA to the substrate chloramphenicol.
- the enzyme reaction can be quantitated by incubating cells or cell lysates with [ 14 C] chloramphemcol and following product formation by physical separation with, for example, thin layer chromatography or organic extraction.
- the CAT protein can be quantitated using an enzyme-linked immunosorbent assay. Such an assay is available from, for example, Promega Corporation, Victoria, United Kingdom.
- a further reporter system which may be used is lacZ gene from E.coli. This encodes the ⁇ -galactosidase enzyme. This catalyses the hydrolysis of ⁇ -galactoside sugars such as lactose.
- the enzymatic activity in cell extracts can be assayed with various specialised substrates, for example x-gal, which allow enzyme activity quantitation using a spectrophotometer, fluorometer or a luminometer.
- the reporter gene may also be gfp (green fluorescent protein) or -globin, which are also known in the art.
- the adenoviral vector preferably includes suitable termination sequences known in the art to allow the transcription of the reporter gene to be successfully terminated.
- the vector also comprises a polyadenylation sequence.
- sequences may be derived from the gene from which the regulatory region is derived or alternatively from other sources such as an SV40 late polyadenylation signal to allow secretion of the reporter gene product.
- the termination sequences and or polyadenylation sequences may be operatively linked 3' to the reporter gene.
- the adenoviral vector may additionally comprise, 3' to the reporter gene, a 3'-UTR (untranslated region).
- a 3'-UTR is from a cytokine.
- the 3'-UTR is from the human TNF gene.
- the 3'-UTR may be from sequence identified as Genbank Accession No. M16441, especially the sequences encoding 1041NT to the 3' of
- the adenoviral vector may additionally comprise one or more sequences to enable it to be maintained or replicated within a suitable bacterial host vector, such as Esherichia coli.
- a suitable bacterial host vector such as Esherichia coli.
- sequences are well known in the art and include, for example, a bacterial origin or replication such as fl ori. It may also include one or more genes to enable selection of bacteria comprising the adenoviral vector, such as ampicillin or tetracycline resistance genes known in the art.
- the vector may additionally comprise a further reporter gene under the control of a constuitive or unregulated promoter such as CMV promoter.
- the further reporter gene will be different to the first reporter gene. It acts as an internal control, allowing variations, such as the amount of vector within each cell, to be taken into account.
- a further aspect of the invention provides a method of studying mammalian regulatory region activity comprising inserting an adenoviral vector according to the invention into a cell and measuring the amount of reporter gene product produced by the mammalian regulatory region encoded by the adenoviral vector.
- the cell into which the vector has been inserted may be exposed to one or more inhibitors or activators of the mammalian regulatory region. Cells may or may not be activated with a stimulus such as lipopolysaccharide
- the activity in a diseased cell may be compared with the activity of the regulatory region within a non-diseased cell. This allows a better understanding of the diseased state to be studied.
- the cell into which the adenoviral vector is inserted may be selected from normal cells, macrophages. dendritic cells, phagocytes, epithelium, endothelium, tissue or cell lines
- Adenoviral vectors of the invention can be introduced into cells using the natural capability of the viruses and centre cells and to mediate uptake of macromolecules, that is by receptor-mediator endocytosis.
- the vectors can be introduced by any other suitable means such as transfection, calcium phosphate-mediated transformation, microinjection, electroporation, osmotic shock and the like.
- the invention also provides a mammalian cell, especially a human cell, containing an adenoviral vector according to the invention.
- the type of cell may be selected from those listed above.
- kits comprising vectors according to the invention.
- kits may also include instructions for the use of the vector.
- the invention also provides a method of screening a compound as an enhancer or an inhibitor of a mammalian regulatory region, comprising the use of an adenoviral vector according to the invention, the vector may be placed into a suitable cell, and the effect of the compound on the amount of reporter gene product produced observed.
- the compound may act directly on the regulatory region or indirectly via, for example, a cascade pathway.
- FIG. 1 Schematic representation of TNF reporter constructs.
- the TNF promoter lying before the 5'UTR of TNF is represented as a thin line.
- the 5' and 3'UTR are represented by hatched bars.
- the luciferase coding sequence is shown as a solid bar. Locations of the start codon, stop codon and TATA box are indicated for each construct.
- SV40 poly(A) sequence is represented by an open bar.
- the AU-rich element (ARE) sequences are indicated as a grey box. Both constructs are produced in vector pGL3 and pAdeasyl for transfection and adenovirus infection experiments, respectively.
- Figure 2 Activity of TNF based reporter constructs when transfected or infected into RAW 264.7 and human macrophages.
- A, B - cells were either transfected (A) or infected with adenoviral constructs at m.o.i. 40:1 (A, B). Following LPS (10 ng/ml.) stimulation (4h), cells were then harvested and assayed for luciferase.
- Figure 3 Comparison of the kinetics of LPS-induced TNF production and luciferase expression in human macrophages.
- Cells were either uninfected (A) or infected with Advp5' (B) or Advp5'3'UTR (C) and either untreated (O). or activated with 10 ng/ml. LPS (•). At the indicated times, cell tysates or culture supernatants were harvested to assay luciferase or TNF production respectively. In the absence of LPS, TNF production was undetectable.
- Figure 3(D) Cells were infected with Advp5' (D) or Advp5'3'UTR (O) and activated with LPS for four hours.
- Actinomycin D was added to stop any further mRNA synthesis and the cells incubated for a further 0, 15, 30, 45 or 60 minutes, after which time they were harvested for RPA analysis of luciferase or GAPDH mRNA. The results are shown normalised to 100% at the 0 minute point. Each experiment was repeated at least two (D) or three (A-C) times with blood from distinct donors.
- Advp5' A or Advp5'3'UTR (B) and activated simultaneously with LPS (10 ng/ml.) and various concentrations of IL-10.
- Luciferase activities •
- TNF production O
- Ordinates represent the percentages of luciferase activation and TNF production induced by LPS, in the absence of IL-10.
- Data are mean values ⁇ S.E.M. from 4 separate experiments conducted with blood from diffeerent donors.
- FIG. 5 Kinetics of IL-10 inhibitory activity on TNF production and luciferase expression.
- Human monocytes-derived macrophages were uninfected (A) or infected with Advp5'3'UTR
- Figure 6 Effect of IL-10 on TNF and Luciferase mRNA levels.
- Advp5' Human macrophages infected with either Advp5', Advp5'3'UTR or AdvO were treated with IL-10 (10 ng/ml.) simultaneously or 24h before LPS.
- IL-10 10 ng/ml.
- IL-10 can inhibit TNF expression even when added post-LPS stimulation.
- Figure 8 Effect of prolonged exposure to IL-10 prior to LPS stimulation on luciferase expression.
- Figure 9 Prolonged exposure to IL-10 prior to LPS-stimulation results in the inhibition of luciferase expression in the absence of 3'UTR.
- Infected cells Advp5' (A)
- Advp5'3'UTR (B) were pre-treated for 24h with various concentrations of IL-10 before LPS exposure.
- the experiment was harvested after 4h LPS stimulation and luciferase activities (•) and TNF production (O) were assayed. Data are expressed as percentages of luciferase activation and TNF production induced by LPS in absence of IL-10. Data are mean values ( ⁇ SEM) from four experiments done with different donors.
- Figure 10 In murine cells, IL-10 inhibition of luciferase expression requires the 3'UTR regardless of length of exposure to the cytokine.
- Infected cells (m.o.i. 40:1) were incubated in presence of LPS (10 ng/ml.) without ( ⁇ ), or with IL-10 ( 10 ng/ml.) added simultaneously (D) or 24h before LPS addition (D). After LPS stimulation (4h), mTNF production and luciferase activities were assayed. Ordinates represent the percentages of luciferase activation and TNF production induced by LPS in the absence of cytokine. The results are representative of three (RAW264.7 cells) or two (primary murine macrophages) experiments.
- Human TNF promoter (-1173bp) with 3 'untranslated region of the human TNF gene (pGL3-7N -3'UTR) or without the 3' UT region (pGL3-rNF) (Udalova et al, 1998) and the human IL10 promoter construct (-4080bp) (pGL3-IL10) were subcloned into the pAdTrack vector (He et al., 1998) to generate pAdTrack- 7/NF-Luc-3' UTR, pAdTrack- TNR- c and pAdTrack-/ZJ0-Luc.
- Kpnl/Sall fragments containing the human T ⁇ F or IL10 promoter, the luciferase reporter gene and the SV40 late poly (A) signal were derived from their pGL3 respective constructs and cloned into Kpnl/Sall sites of AdTrack vector.
- the 3'UTR construct was obtained by substituting a Xbal/BamHI fragment containing the SV40 late poly(A) signal in the pGL3-7NF plasmid for approximately lkbp of 3'UTR amplified by PCR with corresponding primers: 3'UTR-F (Xbal): aattctagaGGAGGACGAACATCCAAC; 3 'UTR-R(BamHI): aatGeATcCCCAGAGTTGGAAATTC.
- the Kpnl/Sall fragments were subsequently cloned into the pAdTrack vector.
- the pAdEasy-1 adenoviral plasmid was provided by B. Vogelstein (The Howard Hughes Medical Institute, Baltimore, MD) and disclosed in He et al. Recombinant viruses were generated in BJ5183 bacterial cells transformed by the heat-shock method with l ⁇ g of linearised modified AdTrack constructs and lOOng of replication-deficient adenoviral vector pAdEasy-1. Positive recombinant clones were selected through their resistance to kanamycin. Following selection D ⁇ A extracted was used for virus propagation in the 293 human embryonic kidney cells. Viruses were purified by ultracentrifugation through two cesium chloride gradients as described in He et al, 1998.
- Titres of viral stocks were determined by plaque assay in 293 cells after exposure for 1 hour in serum free DMEM medium (Gibco BRL) and subsequently overlayed with 1.6% agarose/2xDMEM with 4% FCS mixture v/vl :1 and incubated for 10-14 days (He et al, 1998).
- Mononuclear cells were isolated from single donor plateletphoresis residues by Ficoll-Hypaque centrifligation proceeding monocyte separation in a Beckman JE6 elutriator (High Wycombe. UK). Monocyte purity was assessed by flow cytometry with CD 14 and was approx. 80% (Williams et al, 1996).
- the elutriated human monocytes were incubated at 1 x lOVml in RPMI 1640 with 25 mM HEPES and 2 mM L-glutamine supplemented with 10% (v/v) heat-inactivated FBS and 10 U/ml penicillin streptavidin.
- purified human monocytes were pretreated with M-CSF at lOOng/ml (Genetics Institute, Boston, MA) for 48h to allow up-regulation of integrin ⁇ v ⁇ s, which has previously been shown to be essential for adenovirus infection of monocytes (Huang et al., 1995).
- RAW 264.7 mouse macrophages were maintained in DMEM supplemented with 10% FCS and 10 U/ml penicillin/streptavidin.
- RAW 264.7 cells/well were plated in growth medium (6 wells/plate). The following day. cells were transfected with 100 ng of DNA by using Superfect (Qiagen, Germany). For infection, these cells were plated at 0.1 x lOVwell (48 wells/plate). After M-CSF treatment human macrophages were replated at 0.2 x lOVwell (96 wells/plate). RAW 264.7 and M-CSF treated macrophages were then exposed to virus for lh in serum free medium followed by washing and reculturing in growth medium with 2% FCS for 24h.
- RNA samples were infected with a multiplicity of infection (MOI) of 40: 1. Infected or transfected cells were then stimulated with LPS (10 ng/ml) for 4h. Infection of M-CSF treated macrophages by Adv ILIO-Luc was efficient at MOI of 80: 1 and cells were stimulated for 18h with LPS (Salmonella typhimorium. Sigma, Poole, UK.). Luciferase assay
- TNF levels were measured in cell supernatants by sandwich enzyme-linked immunosorbant assay (ELISA).
- ELISA sandwich enzyme-linked immunosorbant assay
- a polyclonal rabbit anti-mouse TNF antibody for coating and the same biotinylated polyclonal antibody were used to detect mTNF.
- ELISA for hTNF was performed as previously reported (Engelberts, l, et al. 1991, Lymphokine Cytokine Res. 1-2, 69).
- RPA Ribonuclease protection assay
- Advp5' and Advp5'3'UTR infected cells Luciferase and GAPDH mRNAs were detected by ribonuclease protection assay by using luciferase and GAPDH riboprobes, respectively.
- TNF and GAPDH mRNAs were detected in AdvO-infected cells.
- Riboprobe vectors were constructed as follows.
- a 352-bp HincII-Xbal luciferase fragment was cloned from pGL3c (Promega) into pBluescript KS that had been digested with EcoRV and Xbal.
- a 268-bp TNF gene fragment was amplified by PCR from human genomic DNA, and subcloned into Spel site of pBluescript KS + (kindly provided by Dr A Clark, Kennedy Institute, London, UK.) that had been digested with Spel.
- Riboprobe template constructs were linearized by appropriate restriction and purified by phenol-chloroform extraction and ethanol precipitation.
- Luciferase and GAPDH riboprobes were synthesized using T7 polymerase and TNF riboprobe by using T3 polymerase (Boehringer Mannheim) in the presence of 50 ⁇ Ci of [ ⁇ -32P]UTP (800 mCi/mmol; Amersham). The final concentration of unlabelled UTP in in vitro transcription reactions was 12 ⁇ M, except in the case of Luciferase where it was 2.4 ⁇ M. Ribonuclease protection assays were carried out using the Direct Protect kit (Ambion). Under the conditions of hybridization DNA-RNA heteroduplexes were not detected.
- RNA fragments were resolved by electrophoresis on denaturing 6% polyacrylamide gels and were visualized and quantified by phosphorimaging (Fuji FLA 2000) and autoradiography. Each experiment was performed twice, and serial dilutions of lysates were used to check that quantitations were within the linear range of the assay.
- TNF gene reporter constructs used in this study are schematically illustrated in Figure 1.
- the 5'promoter and 5'promoter-3'UTR constructs were incorporated into the pGL3 vector for transient transfection experiments (using Lipofectin), or recombinant adenoviruses (reporter viruses, Advp5' and Advp5'3'UTR respectively) for infection studies.
- Initial studies compared the response of the reporter gene to LPS when delivered by transfection, with infection.
- the RAW 264.7 murine macrophage cell line was used for these studies, as our attempts to transfect primary human monocytes or macrophages have proved unsuccessful.
- LPS stimulation of transfected cells gave a modest absolute signal nd resulted in an approximate 20-fold response by both 5' and 5'3' constructs. This level of response was not dissimilar to other reported studies with the same type of construct and technique in this cell line (Hacker et al.
- IL-10 requires the 3'UTR to inhibit TNF production.
- the 5' construct was only weakly inhibited by IL-10, approximately 10%), whereas the 5'3'UTR construct showed a dose response profile similar to the endogenous TNF, although the maximum inhibition attained was less, 60% at 10 ng/ml
- the IC 5 o for IL-10 on TNF protein was 0.2-0.3 ng/ml, compared with 2-3 ng/ml. for the reporter gene.
- the concentration of half-maximal inhibition is calculated, then the activity of IL-10 is similar for the endogenous gene (-0.1 ng/ml) and the 5'3'UTR construct (0.2-0.3 ng/ml). This suggests that aspects of the inhibitory activity of IL-10 on the reporter gene and the endogenous gene are similar.
- luciferase mRNA levels were analysed by RPA. As shown in Figure 6, simultaneous IL-10 addition caused a marked reduction of luciferase mRNA from the 5'3'UTR construct, whereas, there was only a marginal effect on mRNA from the 5' construct. These data indicates that IL-10 causes a potential destabilization/enhanced destruction of mRNA via the 3'UTR. Data obtained with TNF mRNA showed identical results to the 5'3'UTR construct ( Figure 6).
- IL-10 mediates its activity on the post-transcriptional mechanism associated with TNF production. If this is so, IL-10 should still be able to inhibit TNF production, at least for a period, if added after LPS. Reporter virus infected macrophages were stimulated with LPS and IL-10 was added for periods of up to 2 hours post-activation; as before the experiment was harvested at 4 hours for assay. As expected, IL-10 had little effect on the activity of the 5' construct, regardless of when it was added (Figure 7).
- Pre-incubation of human macrophages with IL-10 before LPS stimulation reveals a second mechanism of inhibiting TNF production through the 5' promoter.
- IL-10 also inhibited the expression of the 5'3'UTR reporter to an identical degree to the endogenous gene (Figure 9B).
- pre-incubation for 24 hours with IL-10 now produced a dose-dependent inhibition of the 5'UTR construct that showed a maximum inhibition of 60% at 10 ng/ml.
- Figure 9A The IC50 for IL-10 on the 5' construct was 5 ng/ml. but this reduced to 0.5 ng/ml. if the half-maximal inhibition was again calculated.
- IL-10 cannot inhibit TNF gene by the 5' promoter in primary murine macrophages and cell lines.
- IL-10 was an optimal concentration for the inhibition of TNF production by the murine cells (23). For simplicity the data with IL-10 from each study are shown as a comparison with the LPS only control that is given as 100%) ( Figure 10). In RAW 264.7 cells, IL-10 inhibited TNF production and 5'3' luciferase construct to a similar degree when added with LPS.
- constructs have also been demonstrated to be able to be used to study regulatory region, such as cytokine, activity with primary macrophages which has previously been difficult to undertake.
- LOCUS HUMTNFAB GenBank (R) GenBank ACC. NO. (GBN): Ml 6441 CAS REGISTRY NO. (RN): 140752-05-2 SEQUENCE LENGTH (SQL): 7112 MOLECULE TYPE (CI): DNA; linear DIVISION CODE (CI): Primates DATE (DATE): 14 Jan 1995 DEFINITION (DEF): Human tumor necrosis factor and lymphotoxin genes, complete cds.
- KEYWORDS lymphotoxin
- tumor necrosis factor SOURCE Human placenta DNA, clone pTNFl 86.
- ORGANISM ORGN
- Eukaryotae mitochondrial eukaryotes; Metazoa;
- AUTHOR Nedospasov, S. A.; Shakhov,A.N.; Turetskaya,R.L.;
- TITLE Tandem arrangement of genes coding for rumor necrosis factor (TNF-alpha) and lymphotoxin
- LOCUS LOCUS
- R GenBank ACC. NO. (GBN) ** + X78437*** CAS REGISTRY NO. (RN) : 156795-75-4
- SEQUENCE LENGTH SQL: 4181 MOLECULE TYPE (CI) : DNA; linear DIVISION CODE (CI) : Primates DATE (DATE) : 27 Jan 1997 DEFINITION (DEF) : H. sapiens IL-10 gene.
- KEYWORDS ST) IL-10 gene; interleukin 10 SOURCE : human.
- ORGANISM ORGN
- Eukaryotae mitochondrial eukaryotes; Metazoa; Chordata; Vertebrata; Eutheria; Primates; Catarrhini; Hominidae ; Homo
- NUCLEIC ACID COUNT (NA) 1181 a 1046 c 961 g 993 t REFERENCE : 1 (bases 1 to 4181)
- AUTHOR Kube,D.; Platzer,C; von Knethen,A.; Straub,H.; Bohlen,H.; Hafner,M.; Tesch,H.
- TITLE Isolation of the human interleukin 10 promoter. Characterization of the promoter activity in
- AUTHOR Kube , D . TITLE (TI) : Direct submission JOURNAL (SO) Submitted (26-APR-1995) D. Kube, Camillische
- AUTHOR Kube, D. TITLE (TI) : Direct submission JOURNAL (SO) Submitted (25-JAN-1996) D. Kube, Medizinische
- AUTHOR Eskdale,J.; Kube,D.; Gallagher, G. TITLE (TI) : A second polymorphic dinucleotide repeat in the 5' flanking region of the human IL10 gene
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AU74355/00A AU7435500A (en) | 1999-09-22 | 2000-09-22 | Adenoviral vectors |
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GBGB9922283.8A GB9922283D0 (en) | 1999-09-22 | 1999-09-22 | Adenoviral vectors |
GB9922283.8 | 1999-09-22 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2001021823A2 true WO2001021823A2 (fr) | 2001-03-29 |
WO2001021823A3 WO2001021823A3 (fr) | 2001-10-11 |
Family
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB2000/003645 WO2001021823A2 (fr) | 1999-09-22 | 2000-09-22 | Vecteurs adenoviraux |
Country Status (3)
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AU (1) | AU7435500A (fr) |
GB (1) | GB9922283D0 (fr) |
WO (1) | WO2001021823A2 (fr) |
Cited By (14)
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---|---|---|---|---|
EP1558292A2 (fr) * | 2002-11-01 | 2005-08-03 | Mayo Foundation For Medical Education And Research | Procedes et vecteurs de maitrise d'expression genique |
EP2042517A1 (fr) | 2002-09-27 | 2009-04-01 | Xencor, Inc. | Variantes FC optimisées et leurs procédés de génération |
EP2053062A1 (fr) | 2004-03-24 | 2009-04-29 | Xencor, Inc. | Variantes d'immunoglobine en dehors de la région Fc |
US7587286B2 (en) | 2003-03-31 | 2009-09-08 | Xencor, Inc. | Methods for rational pegylation of proteins |
US7610156B2 (en) | 2003-03-31 | 2009-10-27 | Xencor, Inc. | Methods for rational pegylation of proteins |
US7642340B2 (en) | 2003-03-31 | 2010-01-05 | Xencor, Inc. | PEGylated TNF-α variant proteins |
US7657380B2 (en) | 2003-12-04 | 2010-02-02 | Xencor, Inc. | Methods of generating variant antibodies with increased host string content |
EP2325206A2 (fr) | 2004-11-12 | 2011-05-25 | Xencor, Inc. | Variants de FC avec une liaison altérée à FCRN |
EP2368911A1 (fr) | 2003-05-02 | 2011-09-28 | Xencor Inc. | Variantes FC optimisées et leurs procédés de génération |
EP2444423A1 (fr) | 2007-10-31 | 2012-04-25 | Xencor Inc. | Variants de Fc dont la liaison à FcRn est altérée |
EP2471813A1 (fr) | 2004-07-15 | 2012-07-04 | Xencor Inc. | Variantes optimisées de Fc |
EP2808343A1 (fr) | 2007-12-26 | 2014-12-03 | Xencor Inc. | Variantes Fc avec liaison altérée en FcRn |
US9416171B2 (en) | 2011-12-23 | 2016-08-16 | Nicholas B. Lydon | Immunoglobulins and variants directed against pathogenic microbes |
US9988439B2 (en) | 2011-12-23 | 2018-06-05 | Nicholas B. Lydon | Immunoglobulins and variants directed against pathogenic microbes |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1999009045A1 (fr) * | 1997-08-20 | 1999-02-25 | Somagenics, Inc. | Produits therapeutiques antisens a proprietes de liaison ameliorees et leurs methodes d'utilisation |
-
1999
- 1999-09-22 GB GBGB9922283.8A patent/GB9922283D0/en not_active Ceased
-
2000
- 2000-09-22 WO PCT/GB2000/003645 patent/WO2001021823A2/fr active Application Filing
- 2000-09-22 AU AU74355/00A patent/AU7435500A/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999009045A1 (fr) * | 1997-08-20 | 1999-02-25 | Somagenics, Inc. | Produits therapeutiques antisens a proprietes de liaison ameliorees et leurs methodes d'utilisation |
Non-Patent Citations (5)
Title |
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BONDESON ET AL: "Adenoviral transfer of the IkB gene inhibits the induction of proinflammatory cytokines" ARTHRITIS & RHEUMATISM, vol. 40, September 1997 (1997-09), page S322 XP000981222 * |
KAWASAKI ET AL.: "Selection of the best target site for ribozyme-mediated cleavage within a fusion gene for adenovirus E1A-associated 300 kDa protein (p300) and luciferase" NUCLEIC ACIDS RESEARCH, vol. 24, 1996, pages 3010-3016, XP002162887 * |
NETTELBECK ET AL.: "GENE THERAPY / Designer promoters for tumour targeting" TIG, vol. 16, April 2000 (2000-04), pages 174-181, XP002162888 * |
ROBERT ET AL.: "An adenoviral vector-based system to study neuronal gene expression: Analysis of the rat tyrosine hydroxylase promoter in cultured neurons" JOURNAL OF NEUROCHEMISTRY, vol. 68, 1997, pages 2152-2160, XP000989957 * |
UDALOVA ET AL: "Complex NF-kB interactions at the distal tumor necrosis factor promoter region in human monocytes" JOURNAL OF BIOLOGICAL CHEMISTRY , vol. 273, 1998, pages 21178-21186, XP002162886 cited in the application * |
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Also Published As
Publication number | Publication date |
---|---|
AU7435500A (en) | 2001-04-24 |
GB9922283D0 (en) | 1999-11-17 |
WO2001021823A3 (fr) | 2001-10-11 |
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