WO1997012048A1 - Virus recombines comprenant une proteine pouvant etre clivee par une protease - Google Patents

Virus recombines comprenant une proteine pouvant etre clivee par une protease Download PDF

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WO1997012048A1
WO1997012048A1 PCT/GB1996/002381 GB9602381W WO9712048A1 WO 1997012048 A1 WO1997012048 A1 WO 1997012048A1 GB 9602381 W GB9602381 W GB 9602381W WO 9712048 A1 WO9712048 A1 WO 9712048A1
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viral
protease
die
heterologous polypeptide
particle
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PCT/GB1996/002381
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English (en)
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Stephen James Russell
François-Loic COSSET
Frances Joanne Morling
Bo Harald Kurt Nilson
Kah - Whye Peng
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Medical Research Council
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Priority claimed from GBGB9519691.1A external-priority patent/GB9519691D0/en
Priority claimed from GBGB9523225.2A external-priority patent/GB9523225D0/en
Priority claimed from GBGB9604562.0A external-priority patent/GB9604562D0/en
Application filed by Medical Research Council filed Critical Medical Research Council
Priority to AU70911/96A priority Critical patent/AU716466B2/en
Priority to EP96931908A priority patent/EP0854929A1/fr
Priority to JP9513224A priority patent/JPH11513249A/ja
Publication of WO1997012048A1 publication Critical patent/WO1997012048A1/fr

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    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
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    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates to recombinant viral particles incorporating protease cleavable proteins and to various applications of the recombinant particles.
  • Retroviral envelope glycoproteins mediate specific viral attachment to cell surface receptors and subsequently trigger fusion between the viral envelope and die target cell membrane.
  • All retroviral envelope spike glycoproteins examined to date are homooligomers containing two to four heterodimeric subunits (Doms et al. 1993 Virology 193. 545). Each subunit comprises a large extraviral glycoprotein moiety (SU) noncovalently attached at its C-terminus to a smaller transmembrane polypeptide (TM) that anchors the complex in the viral membrane.
  • SU extraviral glycoprotein moiety
  • TM transmembrane polypeptide
  • SU comprises two domains connected by a proline-rich hinge, the N-te ⁇ ninal domain conferring receptor specificity and exhibiting a high degree of conservation between murine leukemia viruses (MLVs) with different host ranges (Battini et al. 1992 J. Virol 66, 1468-1475).
  • MLVs murine leukemia viruses
  • Moloney MLV envelopes confer an ecotropic host range because they attach selectively to a peptide loop in the murine cationic amino acid transporter (CAT-1), found only on cells of mouse and rat origin (Albritton et al. 1989 Cell 57, 659- 666).
  • 4070A MLV envelopes attach to an epitope on the ubiquitous RAM-1 phosphate symporter that is conserved throughout many mammalian species, and confer an amphotropic host range (Miller et al. PNAS 91,, 78-82; VanZeijl et al. 1994 PNAS 9_i, 1168-1172).
  • retroviral vectors with 4070A envelopes infect human cells promiscuously, whereas vectors wid Moloney envelopes fail to infect human cells.
  • the SU and TM polypeptides are derived from a single chain precursor glycoprotein that undergoes proteolytic maturation in the Gogi compartment during its transport to the cell surface.
  • Uncleaved envelope precursor glycoproteins can be incorporated into viruses but are unable to trigger membrane fusion.
  • the requirement for proteolytic maturation/activation is a feature common to the fusogenic membrane glycoproteins of many virus families and is most commonly mediated by the ubiquitous Golgi compartment serine protease, furin.
  • a general method has been disclosed which allows the display of a (glyco)polypeptide on the surface of a retroviral vector as a genetically encoded extension of the SU glycoprotein (WO 94/06920, Medical Research Council).
  • the polypeptide is fused (by genetic engineering) to the N-terminal part of the SU glycoprotein such that the envelope protein to which it has been grafted remains substantially intact and the fused nonviral polypeptide ligand is displayed on die viral surface.
  • a virus displaying such a chimaeric envelope protein might be capable of multivalent attachment both to the natural virus receptor (via the N-terminal domain of SU) and to the cognate receptor for the displayed polypeptide.
  • EGF epidermal growth factor
  • the displayed polypeptide may sterically hinder the interaction between the N-terminal domain of SU and the natural virus receptor.
  • ecotropic and amphotropic vectors displaying EGF could bind to EGF receptors but were thereafter sequestered into a non-infectious entry pathway, giving greatly reduced titres on EGF receptor-positive cells, but normal titres on EGF receptor- negative cells.
  • EGF receptor-negative cells which were fully susceptible to the engineered retroviral vector, showed reduced susceptibility when they were genetically modified to express EGF receptors. The reduction in susceptibility was in proportion to the level of EGF receptor expression.
  • soluble EGF was added to competitively inhibit virus capture by the EGF receptors, gene transfer was restored.
  • the engineered vector is capable of binding to the natural virus receptor or to the receptor for EGF; attachment to the natural virus receptor leads to infection of the target cell, whereas the attachment to the EGF receptor does not lead to infection of the target cell.
  • the two binding reactions (4070A envelope protein to RAM-1, and EGF to EGF receptor) proceed in competition and the infectivity of the virus for the target cells is reduced in proportion to the efficiency with which the EGF-EGF receptor binding reaction competes virus away from RAM-1.
  • the degree to which gene transfer can be inhibited by this mechanism depends on die relative affinities of the two binding reactions (envelope protein to natural receptor and non-viral ligand to its cognate receptor), die relative densities of die two receptors on the target cell surface, and die relative densities of ie nonviral ligand and the intact envelope protein on the viral surface. Inhibition of gene transfer is additionally influenced by intrinsic properties of die receptor for the non-viral ligand, such as d e distance it projects from die target cell membrane, its mobility widiin die target cell membrane and its half life on the cell surface after engagement of ligand.
  • chimaeric envelopes displaying die N-terminal domain from 4070A MLV SU as an N-terminal extension of Moloney MLV SU can apparently bind to RAM-1 (the receptor for 4070A SU) but not to ecoR (the receptor for Moloney SU); it may be possible that the displayed domains from 4070A SU may form a trimeric cap over the Moloney SU trimer, completely masking its receptor binding sites.
  • the invention provides a recombinant viral particle capable of infecting a eukaryotic cell, the viral particle comprising: a substantially intact viral glycoprotein fused, via an intervening linker region, to a heterologous polypeptide displayed on the surface of the particle, which heterologous polypeptide modulates the ability of the viral panicle to infect one or more eukaryotic cell types and is cleavable from the viral glycoprotein by a protease acting selectively on a specific protease cleavage site present in the linker region, such that cleavage of the heterologous polypeptide from the viral glycoprotein allows the glycoprotein to interact normally with its cognate receptor on the surface of a target cell.
  • Such a panicle is of considerable benefit in die targeted delivery of nucleic acid sequences, which may be present within the panicle, to specific desired target cells, such as is required for gene dierapy.
  • the invention provides a nucleic acid construct, comprising a sequence encoding a fusion protein, the fusion comprising a substantially intact viral glycoprotein fused, via an intervening linker region, to a heterologous polypeptide, wherein the fusion protein is capable of being incorporated into a viral particle capable of infecting an eukaryotic cell, and further wherein the heterologous polypeptide modulates the ability of the viral particle to infect one or more eukaryotic cell types, but cleavage of the heterologous polypeptide from the fusion protein allows the viral glycoprotein to interact normally with its cognate receptor on the surface of the eukaryotic cell.
  • the invention provides a nucleic acid sequence library comprising a plurality of the nucleic acid constructs defined above, wherein at least part of the sequence encoding the intervening linker region is randomised in each construct, such diat each construct comprises one of a plurality of different linker regions which are represented in the library.
  • the invention also provides a library of the viral panicles defined above, each panicle comprising a single nucleic acid construct from the nucleic acid library defined above.
  • substantially intact as used herein is intended to refer to a viral glycoprotein which retains all of its domains so as to conserve post-translational processing, oligomerisation (if any), viral incorporation and fusogenic properties.
  • certain alterations e.g. point mutations, deletions, additions
  • the gluycoprotein may lack a few (e.g. about 1 to 10) amino acid residues, especially at the N terminus, but will otherwise be generally the same size as the wild-type protein and possess substantially the same biological properties as the wild-type protein.
  • the intervening linker region will preferably be quite short, typically comprising from 4 to 30 amino acid residues, more typically 5 to 10 residues.
  • a short linker is preferred, because this will tend to maximise the modulation of infection effected by the heterologous polypeptide.
  • a suitable linker region may be present as a natural part of the heterologous displayed polypeptide.
  • the viral particle may be any virus capable of infecting one or more eukaryotic cell types, but conveniently will be a viral particle suitable for use in gene therapy, such as an adenovirus or a retrovirus (especially a C-type retrovirus).
  • the viral glycoprotein will typically comprise a viral envelope glycoprotein, or may be a chimeric polypeptide comprising sequences conesponding to different viral glycoproteins but which, in total, consitute a substantially intact, functional protein.
  • the heterologous polypeptide may be a short amino acid sequence (say, a peptide of about 10-20 residues, especially if d e sequence undergoes oligomerisation, e.g. a leucine zipper peptide sequence) but more typically will comprise 30 or more amino acid residues. Generally, but not essentially, the polypeptide will comprise a functional binding domain.
  • the heterologous polypeptide when fused to die viral glycoprotein via the linker region, modulates die ability of the viral particle to infect one or more eukaryotic cell types. Specifically, the presence of the heterologous polypeptide serves to inhibit the process of infection of a eukaryotic target cell mediated by the viral glycoprotein.
  • heterologous is intended to refer to any polypeptide which is not naturally fused or otherwise bound to d e viral glycoprotein.
  • the heterologous polypeptide may or may not possess specific binding affinity for a surface component of a target cell.
  • die heterologous polypeptide has affinity for a cell surface component, binding to which will not lead to infection of the cell by the virus.
  • a variety of different examples can be envisaged.
  • a eukaryotic cell expresses a receptor for the viral glycoprotein (binding to which allows the virus to infect the cell) and a non-permissive receptor for the heterologous polypeptide, with inhibition of infection resulting simply from competition between the viral glycoprotein and the heterologous polypeptide for binding to their respective receptors on the target cell.
  • the conformational arrangement of the respective receptors and their ligands is such that binding of the heterologous polypeptide to its receptor causes steric hindrance, such that binding of the viral glycoprotein to its receptor, or fusion of the virus and die cell, is blocked.
  • the heterologous polypeptide does not bind to a non-permissive receptor on the target cell, but the presence of the heterologous polypeptide serves to create steric hindrance sufficient to prevent binding of the viral glycoprotein to its receptor, or may allow binding to occur but inhibits subsequent fusion of the viral particle with the target cell, such that infection of the cell by the viral particle is inhibited at the binding and/or fusion stage.
  • the heterologous polypeptide is capable of forming oligomers when displayed on the surface of the viral particle.
  • the oligomer will be a dimer or, more preferably, a trimer.
  • Such oligomerisation may allow for efficient inhibition of the interaction between the substantially intact viral glycoprotein and its receptor, which inhibition may be removed by proteolytic cleavage of the oligomerised heterologous polypeptide from the viral glycoprotein.
  • the intervening linker may also undergo oligomerisation.
  • the heterologous polypeptide oligomerises with the same stoichiometry as that of the viral glycoprotein.
  • Vascular endothelial growth factor (VEGF) and rumour necrosis factor (TNF) are both proteins which are known to oligomerise and have high affinity for cell surface ligands. Effective (oligomer-forming, and preferably ligand-binding) portions of these proteins may be particularly suitable for use as heterologous polypeptides in accordance with the present invention.
  • the heterologous polypeptide is cleavable from the viral glycoprotein by the selective action of a protease (i.e. a molecule capable of cleaving a peptide bond) which cleaves the linker region at a protease cleavage site.
  • the cleavage site represents a unique peptide sequence not present, or at least not accessible to the protease, in the viral glycoprotein, although a similar site may be present in the heterologous polypeptide (this is generally preferably avoided, as proteolytic attack on the heterologous polypeptide may affect its functioning).
  • the size, and number, of the protease cleavage sites in the linker region may be varied with advantage. Thus, for example, the presence of two or more cleavage sites, recognised by the same or by respective proteases could facilitate cleavage, whilst the use of one long cleavage site will tend to enhance specificity of cleavage.
  • proteases are involved in a number of physiological and/or pathological processes, such as tissue remodelling, wound healing, inflammation and tumour invasion, and such proteases would be of use in the present invention.
  • Specific classes of protease which would be of use include: serine proteases (such as plasminogen/plasmin enzymes); cysteine proteases; and matrix metalloproteinases (MMPs) of various types, (such as Gelatinase A and membrane-type MMP [or MT-MMP]).
  • the protease which serves to cleave the heterologous polypeptide from the viral glycoprotein is preferably selectively secreted by die cell to which it is desired to target the viral particle, or at least the tissue in which the target cell is located. It is preferred that die protease will be secreted only by cells of the target cell type or, less preferably, only by cells (other than the target cells) remote from die tissue containing die target cell. This confers an extra degree of specificity, which is desirable when die particle is used for targeted gene delivery.
  • die present invention allows for two-step targeting, in which a first level of specificity may be imposed by the heterologous polypeptide (e.g.
  • a second level of specificity may be imposed by selective cleavage of d e heterologous polypeptide by proteases secreted by, or in the same tissue as, the target cell.
  • the relevant protease may be added exogenously, such d at if the viral particle is used for targeted gene delivery in a patient, the protease may be administered (e.g. by injection) to die tissue in which the target cell is located.
  • Accessibility of the protease cleavage site to the relevant protease may also be varied. It has been found by the present inventors that use of a short intervening linker region (e.g. 5 amino acid residues) tends to restrict accessibility of the cleavage site, and use of a larger linker region (e.g. 15 to 20 residues) tends to increase accessibility of the cleavage site. This phenomenon is presumably due to seric hindrance of the cleavage site due to die proximity of the viral glycoprotein and or die heterologous polypeptide. Accordingly, it should also be possible to modify accessibility of the cleavage site, as desired, by varying the size of the heterologous polypeptide.
  • a short intervening linker region e.g. 5 amino acid residues
  • a larger linker region e.g. 15 to 20 residues
  • the cleavage site is accessible to die relevant protease before the viral particle becomes bound to an eukaryotic cell, whilst in an alternative embodiment die cleavage site is inaccessible to the protease until the viral particle has become bound to a eukaryotic cell.
  • die cleavage site may be made accessible by a conformational change occurring as a result of binding of the heterologous polypeptide to its cognate receptor.
  • the viral glycoprotein binding to its cognate receptor may make die cleavage site accessible, cleavage of the heterologous polypeptide then allowing fusion of the viral particle to the eukaryotic target cell.
  • the invention provides for a method of selectively delivering a nucleic acid to a target eukaryotic cell present among non-target cells, comprising: administering to die target and non-target cells a recombinant viral particle capable of infecting eukaryotic cells, the particle comprising die nucleic acid to be delivered, and a fusion protein comprising a substantially intact viral glycoprotein fused, via an intervening linker region, to a heterologous polypeptide displayed on the surface of the particle, which heterologous polypeptide modulates die ability of the particle to infect one or more eukaryotic cell types and being cleavable from die glycoprotein by a protease acting selectively on a specific protease cleavage site present in the linker region, such that cleavage of the heterologous polypeptide from the glycoprotein occurs preferentially at, or in the vicinity of, the target cell and allows d e viral glycoprotein to interact normally wid its cognate receptor on the surface of
  • the method may be performed in vitro, for example to deliver a ledial nucleic acid to fibroblasts in tissue culture, which cells often outgrow a slower-growing, more differentiated cell type in culture.
  • the mediod may be performed as a method of gene therapy, in vivo or may be performed ex vivo, on cells which are then re- introduced into a human or animal subject.
  • Preferential cleavage of the protease cleavage site may occur only when the viral particle is bound to die target cell, or when the viral particle is adjacent to die target cell and dius exposed to a protease secreted by die target cell.
  • protease may well be preferred to add the relevant protease exogenously, after administration of die viral particle, so as to ensure sufficient concentration of the protease and as anodier aid to specificity of delivery (by local administration of the protease). It is already known that some proteases may be safely given in vivo (e.g. those enzymes, such as urokinase, streptokinase and tPA, given to patients with myocardial infarcts).
  • those enzymes such as urokinase, streptokinase and tPA, given to patients with myocardial infarcts.
  • the invention also provides, in a further aspect, a method of screening nucleic acid sequences for diose which encode an amino acid sequence which may or may not be cleaved by a protease.
  • a method of screening nucleic acid sequences for diose which encode an amino acid sequence which may or may not be cleaved by a protease.
  • many viral envelope glycoproteins are processed through d e cellular export pathway of the eukaryotic cell in which they are synthesised, generally leading to cleavage, which cleavge is essential for production of an infectious viral particle.
  • the invention d erefore provides a method of screening nucleic acid sequences for those which encode an amino acid sequence which may or may not be cleaved by a protease present in the export pathway of an eukaryotic cell, comprising: causing die expression of a plurality of nucleic acid sequences in eukaryotic cells, each sequence encoding a substantially intact viral glycoprotein fused to a heterologous polypeptide via a randomised intervening linker region, the presence of the heterologous polypeptide serving to inhibit die (binding or fusion) interaction of the viral glycoprotein with its cognate receptor, and wherein each nucleic acid sequence further comprises a packaging signal allowing for viral incorporation, such that those intervening linkers which are recognised by a protease present in die export padiway of the eukaryotic cells will allow for cleavage of the heterologous polypeptide from die viral glycoprotein, resulting in the production of an infectious viral particle; and recovering those nucleic acid sequences directing the expression
  • Nucleic acid sequence determination may optionally be performed, to deduce diose amino acid sequences which are recognised by an export protease.
  • a modification of the above method will allow for die screening of nucleic acid sequences for diose which encode an amino acid sequence which may or may not be cleaved by a protease present in die eukaryotic cell import pathway.
  • the presence of a heterologous polypeptide may, in some embodiments, still allow for binding of the viral glycoprotein to its cognate receptor, but will prevent fusion of the viral particle with d e eukaryotic cell to which it is bound. Cleavage of die heterologous polypeptide by a protease in the cellular import pathway will then allow infection of the cell.
  • the invention provides for a method of screening nucleic acid sequences for those which encode an amino acid sequence which may or may not be cleaved by a protease, comprising: causing the expression of a plurality of nucleic acid sequences in eukaryotic cells, each sequence encoding a substantially intact viral glycoprotein fused to a heterologous polypeptide via a randomised intervening linker region, the presence of the heterologous polypeptide serving to inhibit the fusion of a viral particle with a eukaryotic cell to which it is bound, and wherein each nucleic acid sequence further comprises a packaging signal allowing for viral incorporation; enriching the viral particles so produced for those which retain the heterologous polypeptide (and so are non-infectious); and contacting the enriched particles with a susceptible eukaryotic cell comprising, or in the presence of, a protease such that diose intervening linkers which are recognised by the protease will allow for cleavage of
  • the enrichment step is required because of die possibility that die heterologous polypeptide may be cleaved from d e viral glycoprotein by an export pathway protease during syndiesis of the panicles.
  • a number of possible enrichment techniques will be readily apparennt to those skiled in the an widi die benefit of the present teaching.
  • die viral particles prior to infection of the susceptible cells, die viral particles could be subjected to an affinity enrichment technique - die particles could be passed dirough an antibody affinity column, wherein the antibody has affinity for the heterologous polypeptide.
  • Those particles which retain die heterologous polypeptide will be bound to ie column, whilst those in which the heterologous polypeptide was cleaved during export from die producing cell will pass straight through the column.
  • the bound particles may be eluted (e.g. by competition widi free heterologous polypeptide, or the part d ereof recognised by die antibody, or by alteration of pH or other factors) and then used to infect d e susceptible "indicator" cells.
  • Figure 1 is a schematic representation of retroviral vector constructs coding for chimeric envelopes
  • Figures 2 and 3 are photographs of Western blots demonstrating viral incorporation of certain chimeric polypeptides and dieir sensitivity to Factor Xa protease;
  • Figure 4 is a photograph showing d e infectivity of various /3-galactosidase transducing viruses on target cells with or without Factor xa treatment, as judged by assay on X-gal containing plates;
  • Figure 5 is a schematic representation of how two-step targeting of gene delivery might be achieved using die present invention
  • Figure 6A is a photograph of a Western blot demonstrating viral incorporation of certain chimeric polypeptides and dieir sensitivity to Factor Xa protease;
  • Figure 6B is a bar chart illustrating die infectivity of certain recombinant viruses in the presence or absence of Factor Xa;
  • Figure 7 is a schematic representation of retroviral vector constructs coding for chimeric envelopes
  • Figure 8A is a photograph of two Western blots, the upper one comparing electrophoretic mobility of various chimeric polypeptides, die lower one comparing the amount of protein present;
  • Figure 8B is a photograph of a Western blot comparing the sensitivity to Factor Xa protease of various chimeric polypeptides
  • Figure 8C is a photograph of a Western blot comparing processing of certain chimeric polypeptides
  • Figure 9 is a panel of photographs comparing the growth of of a recombinant virus on NIH 3T3 and A431 cells, widi or widiout Factor Xa treatment;
  • Figure 10 is a schematic representation of retroviral vector constructs coding for chimeric envelopes
  • Figure 11 shows three Tables, A, B and C, illustrating die titre (in enzyme forming units, "e.f.u. ") of various recombinant viruses on NIH 3T3 or A431 cells in the absence (-) or presence ( +) of Factor Xa protease;
  • Figure 12 is a schematic representation of retroviral vector constructs coding for chimeric envelopes
  • Figure 13 A is a photograph of a Western blot demonstrating viral incorporation of various chimeric polypeptides
  • Figure 13B is a photograph of a Western blot comparing the sensitivity of various chimeric polypeptides in die presence (+) or absence (-) of pro-gelatinase A, with (+) or without (-) pre-activation of die protease by p-aminopheny .mercuric acetate (APMA);
  • Figure 14 is a bar chart showing how infectivity of a recombinant virus is dependent upon concentration of pro-gelatinase A;
  • Figure 15 is a bar chart comparing die infectivity of three different recombinant viruses on HT 1080 or A431 cells;
  • Figure 15 A is a photograph comparing the growth of a recombinant virus on HT 1080 or A431 cells;
  • Figure 16 is a panel of four photographs (I, II, III and IV) comparing the infectivity of various viruses on HT 1080 (H) or A431 (A) cells;
  • Figure 17 is a photograph of a gel for detection of gelatinolytic activity.
  • Figures 18 and 19 are schematic representations of retroviral vector constructs coding for chimeric envelopes.
  • Tropism-modifying binding domains were anchored to murine leukaemia virus (MLV) envelopes via factor Xa-cleavable linkers to generate retroviral vectors whose tropism could be regulated by factor Xa protease.
  • the binding domains could not be cleaved from vector particles by factor Xa when the linker was fused to amino acid +7 of Moloney MLV SU but could be efficiently cleaved when fused to amino acid + 1 of Moloney or 4070 A MLV SU glycoproteins.
  • Vectors displaying a cleavable EGF domain were selectively sequestered on EGF receptor-expressing cells, but their infectivity was fully restored when the EGF domain was cleaved from die vector particles widi factor Xa. Partial restoration of infectivity was observed when only a fraction of the envelope proteins were cleaved. Conversely, vectors that displayed a cleavable RAM-1 binding domain fused to Moloney MLV SU had an expanded host range that was reversible upon treatment with factor Xa. It is suggested diat retroviral vectors with engineered binding specificities whose tropism is regulated by exposure to specific proteases may facilitate novel strategies for targeting retroviral gene delivery.
  • MLV-derived retroviral vectors are versatile gene delivery vehicles whose host range can be varied by incorporation of different envelope spike glycoproteins (Miller, 1992 Curr. Top. Microbiol. Immunol. 158, 1; Vile & Russell, 1995 British Medical Bulletin. 51, 12; Weiss, in Retroviridae, J. Levy, Ed. (Plenum Press, 1993), pp. 1-108). Retroviral envelope spike glycoproteins mediate virus attachment to specific receptors on the target cell surface and subsequently trigger fusion between the lipid membranes of virus and host cell.
  • the envelope spike glycoproteins of murine leukaemia viruses are homotrimers in which each of the three heterodimeric subunits comprises a large extraviral glycoprotein moiety (SU) attached at its C-terminus to a smaller transmembrane polypeptide (TM) that anchors the complex in the viral membrane (March et al. , 1974 Virology 60, 595; Ikeda et al. , 1975 J. Virol. 16, 53; Kamps et al., 1991 Virology 184, 687).
  • SU extraviral glycoprotein moiety
  • TM transmembrane polypeptide
  • SU consists of two domains connected by a proline-rich hinge, the N-terminal domain conferring receptor specificity and exhibiting a high degree of conservation between MLVs with different host ranges (Battini et al. , 1992 J. Virol. 66, 1468; Morgan et al. , 1993 J. Virol. 67, 4712; Battini et al. , 1995 J. Virol. 69, 713).
  • Moloney MLV envelopes confer an ecotropic host range because they attach selectively to a peptide loop in d e murine cationic amino acid transporter (CAT-1), found only on cells of mouse and rat origin (Albritton et al.
  • 4070A MLV envelopes attach to an epitope on the ubiquitous RAM-1 phosphate symporter that is conserved diroughout many mammalian species and confer an amphotropic host range (Miller et al. , 1994 Proc. Natl. Acad. Sci. U.S.A. 91, 78; VanZeijl et al. , 1994 Proc. Natl. Acad. Sci. U.S.A. 91, 1168; Kavanaugh et al. , 1994 Proc. Natl. Acad. Sci. U.S.A. 91, 7071).
  • retroviral vectors with 4070A envelopes infect human cells promiscuously whereas vectors with Moloney envelopes fail completely to infect human cells.
  • ecotropic vectors displaying a RAM-1 receptor-binding domain from 4070 A SU were able to infect RAM-1 -positive human cells whereas amphotropic vectors displaying epidermal growth factor (EGF) could bind to EGF receptors but were thereafter sequestered into a noninfectious entry pathway, giving greatly reduced titres on EGF receptor-positive cells, but normal titres on EGF receptor- negative cells.
  • EGF epidermal growth factor
  • each construct is a schematic representation of the N-terminal region of the expressed envelope glycoprotein monomer; Open circles indicate N-terminal receptor-binding domain of die (ecotropic) Moloney MLV SU glycoprotein, filled squares indicate die N-terminal receptor binding domain of die (amphotropic) 4070 A MLV SU glycoprotein, grey triangles represent EGF, and factor Xa cleavage sites are denoted with arrows.
  • LTR long terminal repeat
  • L envelope signal peptide
  • p polyadenylation sequence. The Notl cloning site is also shown.
  • chimaeric envelopes and a control ecotropic (Moloney) envelope were expressed in TELCeB ⁇ cells which express MLV gag-pol core particles and an nlsLacZ retroviral vector (Cosset et al., 1995 J. Virol. 69, 7430-7436).
  • Virus-containing supernatants from the transfected TELCeB ⁇ cells were harvested, filtered (0.45 ⁇ m), digested widi 0 or 4 ⁇ g/ml factor Xa protease for 90 minutes and ultracentrifuged to pellet the viral particles.
  • Retroviral particles incorporating chimaeric envelopes were analyzed by Western immunoblotting ( Figure 2) before (-) or after (+) treatment with factor Xa protease. Lanes A, B and C were loaded widi pelleted retroviral vectors incorporating Mo, EXMol, and EXMo7 envelopes, respectively. The different envelope expression constructs were transfected (as described in Sambrook et al., Molecular cloning, A laboratory manual, (Cold Spring Habour, N.Y., 1989) pp. 16.33-16.36) into TELCeB ⁇ packaging cells and stable phleomycin (50 ⁇ g/ml) resistant colonies were expanded and pooled.
  • EGF was not cleaved from the EXMo7 envelope by factor Xa (Fig. 2, lane C), suggesting diat die cleavage site was not accessible to the protease when inserted in diis position.
  • EMol and EXMol coding for chimaeric envelopes in which EGF is fused to amino acid + 1 (rather dian +7) of Moloney SU by a linker comprising 3 alanines, or 3 alanines and the IEGR factor Xa cleavage site (see Figure 1).
  • EMol and EXMol chimaeric envelopes were incorporated into virions and analysed on immunoblots after treatment with 0 or 4 ⁇ g/ml factor Xa protease for 90 minutes.
  • FIG. 2 shows that EXMol envelopes were cleaved by factor Xa to yield an SU cleavage product whose mobility was indistinguishable from unmodified Moloney SU. Control EMol envelopes which lack the factor Xa cleavage site were not cleaved.
  • Retroviruses displaying these chimaeric envelopes could bind to EGF receptors but were thereafter sequestered into a noninfectious entry pathway, giving greatly reduced titres on EGF receptor-positive cells, but near-normal titres on EGF receptor- negative cells.
  • the expression plasmids FBMoS ALF and FB4070ASALF (described by Cosset et al. , 1995 J. Virol. 69, cited above) coding for unmodified Moloney and 4070A MLV envelopes are referred to in the text as Mo and A respectively. Construction of EA, EMo7 (previously called EMO) and AMO expression plasmids was also described by Cosset et al., (cited above).
  • PCR primers NotXMo7Back, NotMolBack and NotXMolBack were used widi primer envseq7 to amplify modified envelope fragments from Mo (FBMoSALF) which were digested widi Notl and BamHI and cloned into the N_>fI/_3 ⁇ mHI-digested backbone of EMo7.
  • PCR primers NotAlBack and NotXAlBack were used widi primer 4070Afor to amplify modified envelope fragments from A (FB4070ASALF) which were digested widi Notl and BamHI and cloned into die Notl/BamHl-digested backbone of EA.
  • AMol and AXMol constructs were generated by cloning the Ndel-Notl fragment from AMO into the N_f__/N_».l-d_gested backbones of EMol and EXMol, respectively. The correctness of all constructs were confirmed by DNA sequencing.
  • Oliogonucleotides used were:
  • NotXMo7Back 5'-GCA AAT CTG CGG CCG CAA TCG AGG GAA GGC CTC ATC AAG TCT ATA ATA TCA CC (Seq ID No. 1);
  • NotXMolBack 5'-GCA AAT CTG CGG CCG CAA TCG AGG GAA GGG CTT CGC CCG GCT CCA GTC C-3' (Seq ID No. 3); NotAlBack 5'GCA AAT CTG CGG CCG CAA TGG CAG AGA GCC CCC ATC-3' (Seq ID No. 4);
  • NotXAlBack 5'-GCA AAT CTG CGG CCG CAA TCG AGG GAA GGA TGG CAG AGA GCC CCC ATC-3' (Seq ID No. 5);
  • Figure 3 shows diat the IEGR sequence in die interdomain linker of the expressed EXA1 envelopes was correctly recognized and cleaved by factor Xa whereas there was no cleavage of control EAl envelopes.
  • Figure 3 an immunoblot of the recombinant amphotropic retroviral particles before (-) or after (+) treatment with factor Xa protease: lanes A, B and C were loaded widi pelleted retroviral vectors incorporating A, EAl and EXA1 envelopes, respectively. The analysis was performed as described above for Figure 2.
  • EGF receptor-expressing cell lines A431 ATCC CRL1555), HT1080 (ATCC CCL121), and EJ (Bubenik, et al., 1973 Int. J. Cancer 11, 765) were grown in DMEM supplemented with 10% fetal calf serum (Gibco-BRL) at 37°C in an atmosphere of 5 % CO.
  • Jurkat T cells ATCC CRL8805 were grown in RPMI supplemented widi 10% fetal calf serum at 37°C in an atmosphere of 5% CO 2 .
  • target cells were seeded at 2 x 10 5 cells/well in six-well plates and incubated at 37 °C overnight.
  • Producer cell supernatants containing / 3-galactosidase-transducing retroviruses were filtered (0.45 ⁇ m) after overnight incubation at 32 C C in serum free medium.
  • Supernatant dilutions in 2.5 ml serum-free medium were incubated with target cells for 2 hours in die presence of 8 ⁇ g/ml polybrene.
  • the retroviral supernatant was then removed and the cells were incubated with regular medium for 48-72 hours.
  • X-Gal staining for detection of j3-galactosidase activity was performed as previously described (Takeuchi et al. , 1994 J. Virol. 68, 8001).
  • Viral titre (enzyme forming units/ml) was calculated by counting blue stained colonies microscopically wid die use of a grid place underneath the 6 well plates.
  • Botii vectors incorporating EAl or EXAl envelopes could infect EGF receptor-negative Jurkat cells but were selectively sequestered on EGF receptor-expressing human cells, although EXAl was sequestered less completely than EAl (Table 1).
  • EXAl was sequestered less completely than EAl (Table 1).
  • soluble EGF was added as competitor to prevent the vectors from binding to EGF receptors their infectivity on EGF receptor positive cells could be fully restored (Table 1), confirming that sequestration was mediated specifically dirough binding of the engineered envelopes to EGF receptors.
  • Factor Xa protease is capable of binding directly to procoagulant phospholipid on die surface of an enveloped virus (Pryzdial & Wright, 1994 Blood 84, 3749-3757) and might therefore go on to become stably associated widi phospholipid in the engineered vector particles after cleaving their EXAl envelopes.
  • a control experiment was therefore performed to confirm that the restoration of infectivity of vectors incorporating EXAl envelopes on A431 cells was due to cleavage of EGF, and not mediated by panicle-associated factor Xa protease.
  • t + indicates incubation of cells with retroviral vectors in the presence of 1 ⁇ M human EGF (R&D systems, UK).
  • Retroviral vectors with engineered binding specificity whose tropism is regulated by exposure to specific proteases may facilitate novel strategies for targeting retroviral gene delivery.
  • Vectors incorporating EXAl envelopes were therefore treated widi factor Xa and titrated on EGF receptor-expressing A431 cells.
  • Complete cleavage of the fused EGF domain with 4 ⁇ g/ml factor Xa for 90 minutes completely restored die infectivity of vectors with EXAl envelopes but had no effect on the infectivity of vectors carrying EAl envelopes (Figure 4).
  • Figure 4 illustrates factor Xa-mediated infection of A431 cells with chimaeric EGF-4070A MLV vector particles.
  • Filtered supernatants containing /3-galactosidase-transducing retroviruses were preincubated with 0 (-) or 4 (+) ⁇ g/ml concentrations of factor Xa (Promega) for 90 minutes at 37°C with 2.5 mM added CaCl 2 .
  • the treated supernatants were then used for target cell transduction, as described above.
  • X-gal-stained plates were photographed widiout magnification.
  • A431 and EJ cells were incubated with 2 ml of filtered superna ⁇ tant containing ⁇ -galactosidase-transducing retroviruses for 1 hr at 4°C. Cells were then washed two times with cold serum-free medium and incubated with 0 or 4 ⁇ g/ml of factor Xa (Promega) for 2 hrs at 37°C in serum-free medium. After incubation for 48 hrs with medium supplemented with 10% fetal calf serum the viral titres were determined as described in Table 1.
  • proteases that may be of interest in this respect such as the proteases that co-operate in degrading die extracellular matrix during tumour invasion (Poustis-Delpont et al., 1992 Cancer Research 52, 3622-3628; Vassalli & Pepper, 1994 Nature 370, 14-15; Sato et al. , 1994 Nature 370, 61-65; and Chen et al., 1995 Breast Cancer Res. Treat. 31, 217-226); haematopoietic differentiation antigens that are also membrane proteases (Shipp & Look, 1993 Blood 82, 1058-1070) or the membrane protease that has been implicated in the entry pathway of HIV (Murakami et al., 1991 Biochim. Biophys. Acta 1079, 79-284).
  • MLV-derived retroviral vectors are versatile gene delivery vehicles whose host range properties are determined by membrane glycoproteins which mediate dieir attachment to specific receptors and subsequently trigger fusion.
  • the envelope glycoproteins of the murine leukaemia virus (MLV) are displayed as a homotrimeric complex on the surface of the virus (Fass et al. , Nature Structural Biology 5:465-469; Kamps et al., Virology 754:687-694).
  • Each subunit of the trimer consists of two parts, SU and TM.
  • the SU (surface) component is entirely extraviral and is attached to die retrovirus via die smaller TM component, which anchors the complex in die viral membrane (Pinter et al., Virology 9 :345-351).
  • the N-te ⁇ ninal domain of the SU glycoprotein confers receptor specificity and exhibits a high degree of conservation between MLVs with different host ranges (Battini et al., J. Virol. 69:713-719).
  • Moloney MLV envelopes confer an ecotropic host range because they bind to a murine cationic amino acid transporter (Albritton et al., J. Virol. 67:2091-2096; Albritton et al. , Cell 57:659-666).
  • 4070A MLV envelopes attach to the RAM-1 phosphate transporter which is conserved diroughout many mammalian species, to confer an amphotropic host range (Kavanaugh et al. , Proc. Nad. Acad. Sci. USA 97:7071-7075). After binding to target cell receptors has occurred, die trimeric SU-TM complex is thought to undergo a large conformational rearrangement which triggers the process of fusion between the viral and target cell membranes.
  • step one the retroviral vector attaches to the target cell via an engineered binding domain
  • step two the engineered linker that tethers the virus to the binding domain is cleaved by a specific protease
  • the uncleaved vector therefore retains the ability to infect non target cells through die Ram-1 receptor.
  • envelope modifications diat would completely inhibit die infectivity of uncleaved vectors but would permit full restoration of infectivity upon exposure to a selected protease.
  • the unmodified envelopes of 4070A MLV and Moloney MLV were encoded by d e expression plasmids FB4070ASALF (A) and FBMoSALF (Mo), respectively (Cosset et al., 1995 J. Virol. 69, 7430-7436)).
  • the constructs AMol and AXMol which code for chimaeric envelopes in which the RAM-1 receptor binding domain from 4070A SU is fused to amino acid + 1 of Moloney SU by a factor Xa protease-cleavable (AAAIEGR) or non-cleavable (AAA) linker have been described previously (Nilson et al. , Gene Ther. 5:280-286).
  • EAl and EXAl coding for chimaeric envelopes in which EGF is fused to amino acid + 1 of 4070A SU by a linker comprising three alanines, or three alanines and die IEGR factor Xa cleavage site, have also been described (Nilson et al., Gene Ther. 5:280-286).
  • plasmids pEGSlXAl and pEGS3XAl were first produced in which there is a 12 amino acid (AAAGGGGSIEGR, Seq ID No. 8) or 22 amino acid (AAAGGGGSGGGGSGGGGSIEGR, Seq ID No. 9) linker, respectively, between the 4070A MLV envelope and die displayed EGF domain.
  • PCR primers NotGSlXAlback and NotGS3XAlback (respectively) were used widi primer 4070Afor to amplify modified envelope fragments from EXAl which were digested with Notl and BamHI and cloned into die Notl/BamHI-digested backbone of EAl.
  • Figure 7 is a diagramatic representation of plasmid constructs coding for chimaeric envelope glycoproteins in which the helical peptides AA, VL and II were fused to residue + 1 of the 4070A MLV SU.
  • the general format is shown diagramatically and die amino acid sequence (single letter code) of die helical peptides and the linkers between these peptides and die SU protein are shown in detail.
  • LTR long terminal repeat;
  • L envelope signal peptide.
  • Amino acid residues at die a and d positions of the heptad repeat are shown in bold.
  • PCR primers Gal4 VLback and Gal4 VLfor were used to produce PCR fragments by priming off each other and dien outer primers Gal4back and Gat ⁇ for were used to amplify the fragment further.
  • the PCR products were digested widi Sfil and Notl and cloned into the Sfil/Notl-digested backbones of EXAl, pEGSlXAl and pEGS3XAl.
  • PCR primers Gal4 AAback and Gal4 AAfor were used to produce PCR fragments by priming off each other and dien outer primers Gal4back and Gal4for were used to amplify the fragments further.
  • the PCR products were digested widi Sfil and Notl and cloned into die Sfil/Notl-digested backbones of EXAl and pEGS3XAl .
  • PCR primers Gal4 Ilback and Gal4 Ilfor were used to produce PCR fragments by priming off each other and then outer primers Gal4back and Gal4for were used to amplify die fragments further.
  • the PCR products were digested widi Sfil and Notl and cloned into d e 5/z7/N ⁇ r7-digested backbones of EXAl, pEGSlXAl and pEGS3XAl. The correct sequence of all constructs was verified by DNA sequencing.
  • NotGSlXAlback 5'-GCA AAT CTG CGG CCG CAG GTG GAG GCG GTT CAA TCG AGG GAA GGA TGG CAG AG-3' (Seq ID No. 10);
  • NotGS3XAlback 5'-GCA AAT CTG CGG CCG CAG GTG GAG GCG GTT CAG GCG GAG GTG GCT CTG GCG GTG GCG GAT CGA TCG AGG GAA GAA TGG CAG AG-3' (Seq ID No. 11);
  • Gal4 VLback (containing Sfil site), 5'-GGC ATT CAT GCG GCC GCG GCC CAG CCG GCC ATG AAG CAA CTA GAA GAC AAG GTG GAG GAA CTC CTT AGC AAG GTA TAC C-3' (Seq ID No. 12);
  • Gal4 VLfor (containing Notl site), 5'-GCA AAT CTG CGG CCG CCT CTC CAA CAA GCT TCT TCA GTC GAG CGA CTT CGT TCT CAA GAT GGT ATA CCT TGC TAA GGA G-3' (Seq ID No. 13);
  • Gal4 AAback (containing Sfil site), 5'-GGC ATT CAT GCG GCC GCG GCC CAG CCG GCC ATG AAG CAA GCA GAA GAC AAG GCA GAG GAA GCT CTT AGC AAG GCT TAC C-3' (Seq ID No. 14);
  • Gal4 AAfor (containing Notl site), 5'-GCA AAT CTG CGG CCG CCT CTC CAG CAA GCT TCT TTG CTC GAG CAG CTT CGT TCT CTG CAT GGT AAG CCT TGC TAA GAG C-3' (Seq ID No. 15);
  • Gal4 Ilback (containing Sfil site), 5'-GGC ATT CAT GCG GCC GCG GCC CAG CCG GCC ATG AAG CAA ATC GAA GAC AAG ATA GAG GAA ATT CTT AGC AAG ATC TAC C-3' (Seq ID No. 16);
  • Gal4 Ilfor (containing Notl site), 5'-GCA AAT CTG CGG CCG CCT CTC CTA TAA GCT TCT TGA TTC GAG CAA TTT CGT TCT CTA TAT GGT AGA TCT TGC TAA GAA TTT C-3' (Seq ID No. 17);
  • Gal4 for, 5'-GCA AAT CTG CGG CCG CCT CTC-3' (Seq ID No. 19); and 4070Afor (described above).
  • GP+Env AM12 cells (Markowitz et al., Virology 767:400-406) were derived from the murine cell line NIH 3T3 and express the MLV- A envelope which blocks the RAM-1 receptor by interference.
  • NIH 3T3, GP+Env AM 12 and the human cell line A431 (Giard et al., J. Natl. Cancer Inst. 57, 1417-1421), were grown in DMEM supplemented widi 10% fetal calf serum.
  • the different envelope expression constructs were transfected into TELCeB ⁇ packaging cells (Cosset et al., J. Virol. 69:7430-7436) by calcium phosphate precipitation (Takeuchi et al., J.
  • Virol. 65:8001-8007) and stable phleomycin (50mg/ml) resistant colonies were expanded and pooled.
  • Cells were grown in DMEM supplemented widi 10% fetal calf serum and when confluent transferred from 37 * C to 32 * C and incubated for 72hrs.
  • Supernatants containing retroviral particles were harvested after overnight (l ⁇ hrs) incubation at 32 * C in lOmls serum-free DMEM for infections, or DMEM supplemented with 2% fetal calf serum for immunoblots. All supernatants were filtered (0.45 ⁇ m) before use.
  • Virus producer cells were lysed in a 20mM Tris-HCl buffer (pH 7.5) containing 1 % Triton X-100, 0.05% SDS, 5mg/ml sodium deoxycholate, 150mM NaCl and ImM PMSF. Lysates were incubated for 10 mins at 4 ' C and were centrifuged for 10 mins at 10,000 x g to pellet die nuclei. Virus samples were obtained by ultracentrifugation of filtered viral supernatants (10ml) at 30 000 ⁇ m in a SW40 rotor (Beckman, USA) for 1 hr at 4 * C. The pelleted viral particles were resuspended in lOO ⁇ l PBS.
  • RLV Rausher leukaemia virus
  • CA RLV p30 capsid protein
  • Blots were developed with horseradish peroxidase-conjugated rabbit anti-goat antibodies (DAKO, Denmark) and an enhanced chemiluminescence kit (Amersham Life Science, UK).
  • Target cells were seeded at 2 x 10 5 cells/well in six- well plates and incubated at 37 * C overnight.
  • Producer cell supernatants containing /3-galactosidase-transducing retroviruses were filtered (0.45 ⁇ m) after overnight incubation at 32 "C in serum-free medium.
  • the harvested supernatants were incubated with 0 or 4 ⁇ g/ml of factor Xa (Promega) for 90 minutes at 37 ' C in the presence of 2.5mM CaCl,.
  • Supernatant dilutions in 2ml serum-free media were incubated with target cells for 6 hrs in the presence of 8 ⁇ g/ml polybrene.
  • the retroviral supernatant was then removed and the cells were incubated widi regular medium for 48-72 hrs.
  • X-Gal staining for detection of /3-galactosidase activity was performed as previously described (Tatu et al. , EMBO J. 74: 1340-1348).
  • Viral titre (enzyme forming units/ml) was calculated by counting blue stained colonies microscopically with die use of a grid placed underneath the 6 well plates.
  • AMol and AXMol are previously described chimaeric envelopes in which the RAM-1 receptor binding domain from 4070A SU is fused to aminoacid + 1 of Moloney SU by a noncleavable (AAA) or factor Xa-cleavable (AAAIEGR) linker (Nilson et al., Gene Ther. 5:280-286).
  • Viruses inco ⁇ orating the AMol and AXMol envelopes were pelleted, cleaved widi 0 or 4 ⁇ g/ml factor Xa protease and dien analysed on immunoblots using an anti-envelope antiserum as a probe.
  • Figure 6 A is an immunoblot of pelleted recombinant retroviral particles inco ⁇ orating Mo, AMol or AXMol envelopes before (-) or after (+) treatment with factor Xa protease, probed widi antiserum to the SU glycoprotein.
  • Figure 6B shows die results when the target cell line GP+Env AM12 was infected with harvested producer cell supernatants containing /3-galactosidase-transducing retroviruses (AMol , AXMol , Mo and A) widi or without treatment with factor Xa protease. Detection of /3-galactosidase activity was performed by X-gal staining and titres were expressed as e.f.u./ml.
  • Such a block would be expected to be reversible by cleaving the Ram-1 binding domain from the vector and, in keeping with this prediction, die infectivity of the AXMol vector was fully restored on Rec-1 positive, Ram-1 deficient cells when die Ram-1 targeting domain was cleaved from its surface with factor Xa protease (Fig. 6B).
  • the helical peptides diat were chosen for diese studies were variants of the dimeric GCN4 leucine zipper peptide widi systematic V, L, I or A (single letter aminoacid code) substitutions in the a and d positions of the heptad repeat that are known to force the formation of trimeric coiled coils (VL and II peptides) or to prevent oligomerisation (AA peptide) (Harbury, et al., Science 262: 1401-1407).
  • VL and II peptides trimeric coiled coils
  • AA peptide oligomerisation
  • the spacing between the 4070A SU glycoprotein and the displayed peptide motifs was dierefore varied by insertion of linkers comprising amino acids AAAIEGR, Seq ID No. 20), AAAGGGGSIEGR (Seq ID No. 8) or AAAGGGGSGGGGSGGGGSEEGR (Seq ID No. 9), where the highlighted sequence is known to be recognised and cleaved by Factor Xa (Nilson et al., Gene Ther. 5:280-286).
  • AA, VL and II chimaeric envelopes and a control amphotropic (4070A) envelope were stably transfected into TELCeB ⁇ cells which express MLV gag-pol core particles and an nls LacZ retroviral vector (Cosset et al., J. Virol. 69:7430-7436).
  • Virus-containing supernatants were harvested from these stably transfected TELCeB ⁇ cells and ultracentrifuged to pellet the viral particles. Pellets were than analysed on immunoblots for the presence of viral core proteins and envelope proteins (Fig. 8A).
  • Figure 8 illustrates the viral inco ⁇ oration and cleavage of chimaeric envelopes expressing factor Xa-cleavable helical peptides as N-terminal extensions of the 4070 A MLV SU.
  • Figure 8A is an immunoblot of pelleted retroviral particles inco ⁇ orating chimaeric envelopes. The lane contents are as follows: 1 :VLXA1, 2:VLGS1XA1, 3:VLGS3XA1, 4.AAXA1, 5:AAGS3XA1, 6:IIXA1, 7:IIGS1XA1, 8:IIGS3XA1, and 9:A.
  • the top immunoblot was probed widi an anti-SU antiserum and die lower one widi an anti-p30 antiserum to detect d e p30 CA protein.
  • Figure 8B shows die Factor Xa-mediated cleavage of chimaeric envelopes and takes the form of an immunoblot of pelleted recombinant amphotropic retroviral particles inco ⁇ orating A, VLXA1, AAXA1 , IIXA1 or EXAl envelopes before (-) or after (+) treatment with factor Xa protease, probed widi anti-SU antiserum.
  • Figure 8C is an immunoblot of cell lysates prepared from the virus producing TELCeB ⁇ transfectants A, VLXA1, AAXA1, IIXA1 and the control, untransfected TELCeB ⁇ , probed widi anti-SU antiserum.
  • Envelopes displaying die control monomeric peptide (AA) were inco ⁇ orated almost as efficiently as wild type 4070A envelopes whereas envelopes displaying die VL peptide were inco ⁇ orated much less efficiently and tiiere was no visible inco ⁇ oration of envelopes displaying die II peptide.
  • viral pellets were digested widi 0 or 4 ⁇ g/ml factor Xa protease and then analysed on immunoblots as before.
  • Figure 8B shows that there is a mobility shift when expressed envelopes VLXA1, AAXA1 and die control EXAl, have been cleaved widi factor Xa protease, indicating diat die helical peptides are indeed cleaved from the SU. Due to d e low levels of inco ⁇ oration of the IIXAl chimaeric envelope, cleavage can not be seen for this vector. This immunoblot also indicates diat d e chimaeric envelope AAXA1 was inco ⁇ orated 10 times more efficiently than VLXA1.
  • FIG. 9 shows the reversible inhibition of infection by cleavage of the chimaeric envelope, VLXA1 , expressing a factor Xa-cleavable, N-terrninal oligomerizing peptide and is a magnified view of virally infected cells after X-gal staining.
  • Chimaeric envelope VLXA1 shows strong inhibition of infectivity on NIH 3T3 and A431 cells, which is reversible on addition of factor Xa.
  • control vectors displaying the AA peptide gave titres comparable to that of the wild type amphotropic vector and the titres did not change after factor Xa cleavage indicating diat die AA peptide does not significantly interfere with die functions of the underlying 4070A envelope.
  • the vectors displaying die trimerising VL and II helical peptides gave greatly reduced titres on both cell lines which were enhanced as much as 2000-fold by factor Xa cleavage.
  • the Ram-1 binding domain from the homotrimeric 4070A SU glycoprotein can inhibit Rec-1 mediated infection by the homotrimeric Moloney SU glycoprotein when grafted to its N-terminus.
  • short trimeric leucine zipper peptides but not a monomeric helical peptide, can inhibit Ram-1 mediated infection by the 4070A envelope when fused to its N-terminus.
  • factor Xa protease to cleave the trimeric N-terminal extensions from the virally inco ⁇ orated envelopes, it was possible to reverse the block to Rec-1 or Ram-1 mediated infection.
  • the masking of envelope functions by these inhibitory N-terminal extensions is a consequence of their assembly into a trimeric complex at the tip of the SU glycoprotein trimer to which they are grafted.
  • VL, II and AA peptides diat we fused to the 4070A envelope are mutants of the GCN4 leucine zipper in which the conserved, buried residues diat direct dimer formation have been substituted widi valine, leucine, isoleucine or alanine residues (Harbury et al., Science 262: 1401-1407).
  • the VL mutant oligomerises to form extremely stable (T m 95 * C) two- and three-stranded alpha-helical coiled coil structures whereas the II mutant forms exclusively three-stranded coiled coils which are even more stable (T ra > 100 * C) than the VL structures.
  • T m 95 * C two- and three-stranded alpha-helical coiled coil structures
  • T ra > 100 * C stable
  • all of the hydrophobic core residues of the GCN4 leucine zipper were substituted widi alanines to prevent oligomerisation of the mutant peptide
  • Retroviral inco ⁇ oration of chimaeric envelopes displaying die VL and II peptides was significandy impaired relative to chimaeric envelopes displaying die control AA peptide, which showed only a slight reduction in inco ⁇ oration compared to unmodified 4070A envelopes.
  • the VL chimaeric envelopes were approximately ten-fold less abundant in viral pellets than the AA chimaeric envelopes, and the II chimaeric envelopes were so poorly inco ⁇ orated that they were not visible on immunoblots of pelleted virions.
  • All vectors carrying the VL or II chimaeric envelopes showed inhibition of infection on NIH3T3 and A431 cells, which was reversible on cleaving the peptides from the vectors with factor Xa protease. Titres were not restored completely to wild type levels due to die reduced levels of inco ⁇ oration of these envelopes.
  • the VL and II peptides dierefore function as oligomerising peptide adaptors which mask die functions of the retroviral envelope glycoprotein to which they are fused.
  • the inhibition of infection may be as a result of the oligomerizing peptide blocking binding of the vector to its target cells by masking the underlying binding domain.
  • the presence of an oligomerizing peptide may prevent dissociation of the envelope trimer, blocking fusion.
  • binding studies were uninformative so we are unable to determine which of these mechanisms is more dominant.
  • Vectors pEGF LVA1 and pEGF LVXA1 display an oligomerising peptide, LV, fused to residue + 1 of 4070A SU widi a non cleavable (SAA) or factor Xa protease-cleavable (SAAIEGR, Seq ID No. 21) linker and also display die EGF binding domain.
  • SAA non cleavable
  • SAAIEGR factor Xa protease-cleavable
  • PCR primers Gal4 LV, Gal4 LVbak and Gal4 LVfor were used for assembly of the PCR fragment coding for the oligomerising peptide, LV (Harbury et al., 1993 Science 262, 1401-1407).
  • the PCR product was digested with Notl and Eagl and cloned into die N ⁇ tf-digested backbones of EAl and EXAl.
  • Figure 10 shows a diagramatic representation of the two constructs. The correct sequence of the constructs was verified by DNA sequencing.
  • Gal4 LV 5'-GAC AAG CTA GAG GAA GTA CTT AGC AAG CTC TAC CAT GTC GAG AAC GAA CTT GCT CGA GTT AAG AAG-3' (Seq ID No. 22);
  • Gal4 LVback (containing Notl site), 5'-GGC ATT CAT GCG GCC GCA ATG AAG CAA GTG GAA GAC AAG CTA GAG GAA GTA C-3' (Seq ID No. 23);
  • Gal4 LVfor (containing Eagl site), 5'-GCA AAT CTG CGG CCG ACT CTC CCA GAA GCT TCT TAA CTC GAG CAA GTT C-3' (Seq ID No. 24).
  • the murine cell line NIH 3T3, and d e human cell line A431 were grown in DMEM supplemented widi 10% fetal calf serum.
  • the envelope expression constructs were transfected into TELCeB ⁇ packaging cells by calcium phosphate precipitation and stable phleomycin (50mg/ml) resistant colonies were expanded and pooled.
  • Cells were grown in DMEM supplemented with 10% fetal calf serum and when confluent transferred from 37°C to 32°C and incubated for 72hrs.
  • Supernatants containing retroviral panicles were harvested after overnight (l ⁇ hrs) incubation at 32 °C in lOmls serum-free DMEM for infections. All supernatants were filtered (0.45 ⁇ m) before use.
  • Target cells were seeded at 2 x 10' cells/well in six-well plates and incubated at 37 * C overnight.
  • the harvested supernatants containing /3-galactosidase-transducing retroviruses were incubated widi 0 or 4 ⁇ g/ml of factor Xa (Promega) for 90 minutes at 37' C in die presence of 2.5mM CaCL.
  • Supernatant dilutions in 2ml serum-free media were incubated widi target cells for 6 hrs in the presence of 8 ⁇ g/ml polybrene.
  • the retroviral supernatant was dien removed and die cells were incubated widi regular medium for 48-72 hrs.
  • X-Gal staining for detection of /3-galactosidase activity was performed and viral titre (enzyme forming units/ml) was calculated by counting blue stained colonies microscopically with the use of a grid placed underneath die 6 well plates.
  • MMPs matrix metalloproteinases
  • Matrix metalloproteinases are important for angiogenesis, tissue remodelling, inflammation and wound healing, and diey play a crucial role in various pathological processes including cancer invasion and metastasis and the destruction of articular cartilage in rheumatoid arthritis (Liotta et al., 1991 Cell 64, 327; Woessner Jr. , 1991 FASEB J.
  • MMPs include matrilysin, collagenasesl-3, stromelysinsl-3, gelatinases A and B and a group of 4 membrane-type MMPs (MT-MMP) which are anchored to cell membranes (Sato et al. , 1994 Nature 370, 61; Takino et al.,
  • MMPs are secreted as zymogen forms and require activation before diey can exert dieir proteolytic activities.
  • the net activities of the enzymes are also regulated by die tiiree tissue inhibitors of MMPs (TTMPs).
  • die MMPs co-operate widi one another in a cascade padiway to cause degradation of the extracellular matrix.
  • Gelatinase A GLA; MMP-2
  • MMPs are of special interest with respect to tumour invasion.
  • Pro-GLA is secreted by stromal fibroblasts and concentrated on tumour cell membranes, especially at the invasive front of the tumour (Afzal et al., 1996 Lab. Invest. 74, 406; Nomura et al. , 1996 Int. J.
  • GLA is often found to be elevated in invasive or metastatic tumours.
  • MMPs as promising targets for novel therapeutic agents and there are several general or specific MMP-inhibitors that are currently being tested for their usefulness in treatment of MMP-linked diseases in a number of clinical trials (Hodgson 1995 Biotech. 75, 554; Eccles et al. , 1996 Can. Res.
  • This example describes die generation of targeted retroviral vectors whose infectivity for human EGF receptor-expressing cancer cells is strongly activated by membrane-associated MMPs.
  • chimaeric envelope expression constructs were generated in which a cDNA coding for the 53 amino acid receptor binding domain of EGF was linked to die N- terminal codon of the 4070A murine leukemia virus (MLV) SU envelope glycoprotein via short non-cleavable or protease-cleavable linkers.
  • MMV murine leukemia virus
  • a and E.X.A have an EGF cDNA, flanked by Sfil and Notl restriction sites, inserted at codon + 1 of die N-terminus of wild type 4070A MLV SU (surface protein gp 70) envelope, widi a linker of either 3 alanines (E.A.) or 3 alanines and die IEGR Factor Xa cleavage sequence (E.X.A.) between die domains.
  • Figure 12 is a schematic representation of the chimaeric envelope expression constructs, E.A, E.G 4 S.A, E.X.A and E.MMP.A.
  • the envelope constructs were transfected into TELCeB ⁇ complementing cells, virus-producing clones were pooled and expanded in 10% FCS-DMEM selection medium containing 50 ⁇ g/ml phleomycin. Arrows indicate potential site of cleavage by respective proteases.
  • the E.A and E.G 4 S.A chimaeric envelopes contained non-cleavable linkers AAA and AAAGGGGS (Seq ID No. 25) respectively (single letter amino acid code), the E.X.A envelope contained a Factor Xa-cleavable linker AAAIEGR and die E.MMP.A envelope contained die linker AAAPLGLWA (Seq ID No. 26) in which the highlighted sequence is known to be recognised and cleaved by GLA and by MT1-MMP (Ye et al. , 1995 Biochem. 34, 4702; Will et al. , J. Biol. Chem. 277, in press) ( Figure 12).
  • the chimaeric envelope constructs and a wild type 4070A envelope expression construct were stably transfected into TELCeB ⁇ complementing cells which express Moloney MLV gag-pol proteins and die nlsLacZ retroviral vector, as described in the preceding examples.
  • infectious enveloped vector particles capable of transferring the lacZ marker gene are rescued into die culture supernatant.
  • Viral supernatants were harvested from confluent plates of pooled transfected TELCeB ⁇ cells and die viral particles were pelleted by ultracentrifugation and immunoblotted using an anti-envelope antiserum as probe. Immunoblotting was performed as described in die preceding examples. The results are shown in Figure 13 A, B.
  • Figure 13B is an immunoblot demonstrating cleavage of MMP-cleavable linker in E.MMP.A by purified p-aminophenylmercuric acetate (APMA)-activated gelatinase A (GLA).
  • APMA p-aminophenylmercuric acetate
  • E.X.A, E.G 4 S.A or E.MMP.A viral pellets were incubated with PBS, APMA (final concentration 2 mM) or APMA-activated GLA (32 ⁇ g/ml) for 30 min at 37°C].
  • E.MMP. A-SU On treatment of E.MMP. A-SU with activated GLA, a band with the same mobility as the wild type 4070A-SU was recovered, indicating diat the EGF domain could be efficiently cleaved from this chimaeric envelope without further GLA-mediated degradation (Fig. 13B).
  • the E.G 4 S.A and E.X.A chimaeric envelopes were unaffected by treatment with GLA indicating diat cleavage was specific for the MMP-sensitive linker (not shown).
  • E.A, E.G 4 S.A and E.MMP.A vectors on A431 cells were low between 10 2 -10 3 efu/ml and were not greatly increased by treatment with Factor Xa protease (not shown).
  • A431 cells in 10% FCS-DMEM were seeded, at a density of 3 x 10 4 per individual well, in a 24- well tissue culture plate (Corning, New York) overnight at 37°C. The media were removed the next day and the cells were washed once in serum- free DMEM. Varying amounts (final concentration 2-40 ⁇ g/ml) of pro-GLA were mixed widi 200 ⁇ l of filtered E.MMP.A viral supernatant after which the mixture was added to A431 cells and incubated at 37°C for 6 h. At die end of 6 h, die media was removed and cells were washed once in serum-free DMEM.
  • the cells were then incubated in 10% FCS-DMEM for 72 h at 37°C before they were washed once in cold PBS, fixed in 0.5 % gutaldehyde-PBS for 15 min, washed once again widi PBS and incubated widi X-gal overnight at 37°C.
  • the number of colonies transduced widi die vector blue colonies were counted and die titre expressed as efii/ml viral supemantant.
  • Figure 14 is a graph showing that increase in titre (efu x lO'Vml) of the E.MMP.A MMP-sensitive vector on A431 cells is correlated widi die amount of pro-gelatinase A (pro-GLA) added onto die cells.
  • HT1080 is a human fibrosarcoma cell line that constitutively produces MT1-MMP and pro-GLA (Okada et al. , 1995 Proc. Natl. Acad. Sci. 92, 2730).
  • Figure 15 is a graph showing die titre of EGF chimaeric vectors on A431 and HT1080 cells.
  • Figure 15A shows the high infectivity of E.MMP.A vector on HT 1080 cells compared to on A431 cells as indicated by the number of blue /3-galactosidase positive colonies.
  • One ml out of 10 ml filtered E.MMP.A viral supernatant was incubated widi
  • the infectivity of the vectors on A431 cells was low in die absence of exogenous pro-GLA.
  • the infectivity of the MMP-cleavable vector E.MMP.A was activated by two orders of magnitude compared to the MMP-resistant control vectors E.G 4 S.A and E.X.A (Fig. 15, 15A).
  • die higher titre of the MMP-dependent E.MMP.A vector must be due to its cleavage by MMPs produced endogenously by HT1080 cells.
  • the MMP-activatable E.MMP.A vector could selectively target the MMP- expressing HT1080 cells in preference over A431 cells, we allowed the vector to infect both cell types on the same petri dish simultaneously.
  • the coverslips coated widi die cells were placed in a 10 cm petri dish (Falcon) and E.G 4 S.A (1: 1.5 dilution), E.MMP.A (1:1.5) or 4070A (1:20) supernatants were added onto die petri dishes widi 8 ⁇ g/ml polybrene for 6 h at 37°C. At the end of the incubation period, die media was removed and die cells were incubated in 10% FCS-DMEM for 72 h before X-gal staining.
  • E.MMP.A vector grown on HT1080 (H) cells and A431 (A) cells is shown in I and II, widi die control E.G 4 S.A vector in III and die wild type 4070 A vector in IV.
  • E.MMP.A infected HT1080 cells preferentially over A431 cells.
  • TIMP-1 and TIMP-2 a synthetic inhibitor
  • CT 1339 a synthetic inhibitor
  • TIMP-1 at a final concentration of 10 ⁇ g/ml, TIMP-2 (5 ⁇ g/ml) or CT 1339 (1 mM) was used.
  • the inhibitors were added to 200 ⁇ l of diluted (1:10) E.MMP.A or undiluted E.G 4 S.A viral supernatants.
  • the mixture was then added onto A431 or HT1080 cells, which had been washed once in serum free DMEM, and die cells were incubated for 6 h at 37°C.
  • die cells were washed once in serum free DMEM, incubated for 72 h in 10% FCS-DMEM after which they were stained with X-gal.
  • the E.MMP.A supernatant was diluted to obtain a titre that would allow accurate counting of the number of transduced colonies.
  • Inhibition studies on A431 cells were performed with 200 ⁇ l undiluted E.MMP.A or E.G 4 S.A in presence of 16 ⁇ g/ml pro-GLA.
  • Table 5 Influence of MMP inhibitors on the titre of vectors on A431 and HT1080 cells.
  • MMP-dependent E.MMP.A vector was added to A431 cells in presence of 16 ⁇ g/ml pro- GLA or to HT1080 cells in the absence of exogenous pro-GLA, with or without the addition of natural MMP inhibitors TIMP-1, TIMP-2 or synthetic inhibitor, CT 1339.
  • TIMP-1 could not efficiently block the activation of E.MMP.A by endogenous MMPs on HT1080 cells (Table 5).
  • An important difference between TIMP-1 and d e odier inhibitors is that it displays only weak activity against the MT1-MMP expressed on HT1080 cells (Fig. 17. described below). These experiments therefore point to a central role for the MT-MMP in HT1080-mediated activation of the E.MMP.A vector.
  • Figure 17 is a gelatin zymogram showing the effect of TIMP-1 or a synthetic MMP- inhibitor, CT 1339 on cellular activation of endogenous pro-GLA on HT 1080 cells.
  • the E.MMP.A viral supernatant was incubated on HT 1080 cells for 6 h at 37°C in die absence of any inhibitors (lane 1), in die presence of 10 ⁇ g/ml (lane 2) or 30 ⁇ g/ml TIMP-1 (lane 3), and 1 ⁇ M (lane 4) or 10 ⁇ M (lane 5) CT 1339.
  • the targeting strategy diat we have pursued may have interesting parallels with d e mechanism of HIV entry in which primary virus attachment to CD4 leads to a conformational rearrangement or proteolytic cleavage in gpl20, and secondary virus attachment to one of the recently characterised HIV co-receptors (Feng et al. , 1996 Science 272, 872; Deng et al., 1996 Nature 557, 661; Handley et al., 1996 J. Virol. 70, 4451).
  • C-type retroviral vectors with engineered SU glycoproteins could dierefore be developed as model systems to probe die entry mechanisms that are employed by naturally occurring viruses, such as HIV.
  • Retroviral display of trimeric binding domains, TNF alpha and CD40 ligand Retroviral display of trimeric binding domains, TNF alpha and CD40 ligand.
  • the TELCeB ⁇ cell line has been described in die preceding examples.
  • the NIH 3T3, A431 (human squamous carcinoma; ATCC CRL1555) and HT1080 (human fibrosarcoma; ATCC CCL121) cell lines were grown in DMEM (Gibco-BRL, UK) supplemented with 10% fetal calf serum (FCS; PAA Biologicals, UK), benzylpenicillin (60 mg/ml) and streptomycin (100 mg/ml) at 37 C C in an atmosphere of 5% CO 2 .
  • the human tumour necrosis factor-alpha (TNF-a)-4070A SU chimaeric envelope expression vectors TNF-a.A. TNF-a.GS.A, TNF-a.X.A, TNF-a.XA, andTNF-a.MMP.A have an TNF-a cDNA (Wang et al. , 1995 Science, 225: 149-154), flanked by Sfil and Notl restriction sites, inserted at codon + 1 of the N-terminus of wild type 4070 A MLV SU envelope by different linkers (Fig. 18).
  • the TNF-a.A vector is linked via a 3 alanine (AAA) linker; TNF-a.GS.A via a non-cleavable AAAG 4 S linker; TNF-a.X.A via Factor Xa protease cleavable linker (AAAIEGR) and TNF-a.MMP.A via an MMP-cleavable linker (AAAPLGLWA) (single letter amino acid code).
  • the Factor Xa protease cleaves IEGR after die arginine residue and die PLGLWA linker is susceptible to gelatinase A (MMP-2) and MT-MMP between the giycine and leucine residues.
  • the CD40L-4070A SU chimaeric envelope expression vectors have part of the CD40L cDNA, flanked by Sfil and Notl restriction sites, inserted at codon + 1 of the N-terminus of 4070A MLV by the 4 different linkers as mentioned above.
  • the vectors are termed CD40L.A, CD40L.GS.A, CD40L.X.A and CD40L.MMP.A (Fig. 19).
  • a PCR derived Sfil-Notl DNA fragment encoding the 155 amino acids of d e trimeric human TNF-a was generated using a cDNA template and two primers, sTNFback
  • the Sfil-Notl PCR fragment encoding d e 145 amino acids of the soluble extracellular domain of the trimeric CD40L was generated using a cDNA template (ATCC 79813) and two primers: sCD40Lb (5' > CCG GTA CCG GCC CAG CCG GCC GGT GAT CAG AAT CCT CAA ATT GC, Seq ID No. 31) widi a Sfil site and nCD40Lf (5 * > AAG TCT TAG CGG CCG CGA GTT TGA GTA AGC CAA AGG, Seq ID No. 32) with a Norl site.
  • TNF-a.GS.A or CD40L.GS.A and TNF-a.MMP.A or CD40L.MMP.A
  • Sfil-Notl digested TNF-a or CD40L PCR fragments were cloned into Sfil-Notl digested E.GS.A or E.MMP.A backbones, respectively (Peng et al., A gene delivery system activatable by disease-associated matrix metalloproteinases, submitted). The sequences of the constructs were checked and verified by DNA sequencing.
  • TNF-a and CD40L envelope expression plasmids were stably transfected by calcium phosphate precipitation (Sambrook et al. , 1989, Molecular cloning: A laboratory manual) into the TELCeB ⁇ packaging cells.
  • Transfected cells grown in 10% FCS- DMEM at 37°C, were selected widi 50 ⁇ g/ml phleomycin (Sigma, Poole, Dorset, UK). Resistant colonies were pooled and expanded, and before harvest, the confluent cells were tranfened to 32 °C for 72 h.
  • the viral supernatants were dien harvested and filtered (0.45 ⁇ m, Acrodisc, Gelman Sciences MI, USA) after overnight incubation of the confluent cells with serum free DMEM at 32 °C. These filtered supernatants were then used eidier for immunoblotting, binding or infection assays.
  • the viral particles were pelleted by ultracentrifugation of the filtered viral supernatant (Beckman, USA) at 30,000 ⁇ m for 1 h at 4°C in a SW 40 rotor. The pellet was then resuspended in 100 ⁇ l cold PBS and stored at -70°C till further analysis.
  • the viral complementing cells were grown to confluency on petri dishes (10 cm in diameter), washed once in cold PBS and dien incubated for 10 min at 4°C widi cell lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 % Triton-X, 0.05% sodium dodecyl sulfate, 5 mg/ml sodium doxycholate and 1 mM PMSF.
  • the lysed cells were scraped from die plates and die suspension centrifuged at 10,000 xg for 20 min to pellet the nuclei. Thirty ⁇ l of the supernatant was used for electrophoresis and immunoblotting.
  • Infection Assays i. Infection of cells by chimaeric vectors-blocking infectivity of 4070A by trimeric ligand
  • the TNF-a and CD40L chimaeric vectors were tested for infectivity on NIH 3T3, A431 and HT1080 cells.
  • the cells were seeded overnight at 37°C at a density of approximately 1 x lO 5 cells per individual well in a 6-well tissue culmre plate (Corning, New York). The medium was removed die next day and cells were washed once in serum- free DMEM. An aliqout (1 ml) of the filtered viral supernatant was used to infect the cells in die presence of 8 ⁇ g/ml polybrene. At the end of die 6 h incubation period, die medium was removed and the cells washed once in serum-free DMEM and 10% FCS-DMEM was added.
  • the cells were then incubated for 72 h at 37° C before they were stained widi X- gal.
  • the cells were washed once in cold PBS, fixed in 0.5% glutaldehyde-PBS for 15 min, washed once in cold PBS and incubated widi X-gal overnight at 37 °C.
  • Number of colonies transduced widi die /3-galactosidase gene (blue colonies) were counted and die titre expressed as enzyme forming units (efu)/ml viral supernatant.
  • the titre of the TNF-a-4070A vectors on NIH3T3 and HT1080 cells were low (Table 6). This low level of infectivity could be due to die low level of chimaeric envelope expression. However, it could also be due to die display of the trimeric TNF-a on the 4070A-SU.
  • the trimer was able to block the infectivity of the amphotropic vector, which would be odierwise be highly infective on the murine NIH 3T3 cells, which do not bear die human TNF-a receptor.
  • the infectivity of the CD40L-4070A chimaeras are significandy lower than diat of the wild type on NIH 3T3, A431 and HT1080 cells (Table 8), indicating that the display of CD40L on the envelope is blocking die infectivity of the vector.

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Abstract

On décrit une particule virale recombinée pouvant infecter une cellule eucaryote et comprenant: une glycoprotéine virale, sensiblement intacte, fusionnée, via une région intermédiaire de liaison, à un polypeptide hétérologue présenté sur la surface de la particule, lequel polypeptide module la capacité de la particule virale à infecter un ou plusieurs types de cellules eucaryotes et peut être clivé à partir de la glycoprotéine virale par une protéase agissant de manière sélective sur le site de clivage spécifique des protéases, présent dans la région de liaison, de telle manière que le clivage du polypeptide hétérologue à partir de la glycoprotéine virale permette à cette dernière d'interagir normalement avec son récepteur correspondant sur la surface d'une cellule cible.
PCT/GB1996/002381 1995-09-27 1996-09-27 Virus recombines comprenant une proteine pouvant etre clivee par une protease WO1997012048A1 (fr)

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DE19737442A1 (de) * 1997-08-22 1999-03-04 Olfert Landt Tib Molbiol Synth Proteinumhüllte Polyribonukleinsäuren, ein Verfahren zu ihrer Herstellung und ihre Verwendung
WO1999060135A1 (fr) * 1998-05-21 1999-11-25 Cancer Research Ventures Limited Generation de produits geniques multiples a partir de proteines chimeres de fusion, par clivage a l'aide d'endoproteases ubiquistes
WO2001062980A1 (fr) * 2000-02-25 2001-08-30 Cambridge Drug Discovery, Ltd. Methodes d'identification d'un inhibiteur de protease
EP1224301A2 (fr) * 1999-10-08 2002-07-24 Biofocus Discovery Limited Procede et compositions de ciblage d'une cellule
EP1266036A1 (fr) * 2000-02-25 2002-12-18 Biofocus Discovery Limited Procedes et compositions permettant d'identifier une protease
JP2004520031A (ja) * 2001-01-09 2004-07-08 クイーンメリー アンド ウエストフィールド カレッジ 薬学的活性物質に潜在性を提供する方法
EP1791975A1 (fr) * 2004-08-05 2007-06-06 Biosite Incorporated Compositions et methodes d'expression a la surface des phages de polypeptides
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US10098943B2 (en) 2014-09-11 2018-10-16 Vlp Therapeutics, Llc Flavivirus virus like particle
US10385101B2 (en) 2014-08-08 2019-08-20 Vlp Therapeutics, Llc Virus like particle comprising modified envelope protein E3

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EP1224301A2 (fr) * 1999-10-08 2002-07-24 Biofocus Discovery Limited Procede et compositions de ciblage d'une cellule
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EP1266036A4 (fr) * 2000-02-25 2003-05-21 Biofocus Discovery Ltd Procedes et compositions permettant d'identifier une protease
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EP1791975A4 (fr) * 2004-08-05 2007-11-07 Biosite Inc Compositions et methodes d'expression a la surface des phages de polypeptides
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US11345726B2 (en) 2012-02-16 2022-05-31 VLP Theranentics. Inc. Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV) virus-like particles comprising heterologous antigens inserted into the envelope protein
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US10098943B2 (en) 2014-09-11 2018-10-16 Vlp Therapeutics, Llc Flavivirus virus like particle

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AU7091196A (en) 1997-04-17
AU716466B2 (en) 2000-02-24

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