WO2008014547A1 - Cellules génétiquement modifiées pour exprimer des marqueurs de fenestration et leurs utilisations - Google Patents

Cellules génétiquement modifiées pour exprimer des marqueurs de fenestration et leurs utilisations Download PDF

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WO2008014547A1
WO2008014547A1 PCT/AU2007/001066 AU2007001066W WO2008014547A1 WO 2008014547 A1 WO2008014547 A1 WO 2008014547A1 AU 2007001066 W AU2007001066 W AU 2007001066W WO 2008014547 A1 WO2008014547 A1 WO 2008014547A1
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cell
genetically modified
fenestration
homologue
variant
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PCT/AU2007/001066
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English (en)
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David Le Couteur
Victoria Cogger
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David Le Couteur
Victoria Cogger
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Priority claimed from AU2006904109A external-priority patent/AU2006904109A0/en
Application filed by David Le Couteur, Victoria Cogger filed Critical David Le Couteur
Publication of WO2008014547A1 publication Critical patent/WO2008014547A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5064—Endothelial cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5067—Liver cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates generally to a population of cells genetically modified to express a detectable marker of fenestration and uses thereof.
  • the cells of the present invention are useful in a wide variety of applications, in particular in the context of in vitro based screening systems for testing the effectiveness and/or toxicity of potential therapeutic or prophylactic treatment regimes.
  • Liver sinusoidal endothelial cells occupy a critical and strategic position in the hepatic sinusoid because they facilitate the bi-directional transfer of substrates between blood and hepatocytes (Fraser et al. 1995, Hepatology 21 :863-874; Le Couteur et al. 2005, Clinical Pharmacokinetics 44: 187-200; Wisse et al 1985, Hepatology 5 :683-692; McCuskey et al. 1993, Semin Liver Dis 13:1-12).
  • the remarkable vascularity of the sinusoidal network also generates a huge surface area for LSEC interactions with blood cells and particulate substrates for endocytosis. LSECs have many important physiological roles.
  • the LSEC acts as a sieve that filters lipoproteins (Fraser et al. 1995 supra; Le Couteur et al. 2005, supra; Hilmer et al. 2005, Hepatology 42:1349-1354).
  • Fenestrations are pores in LSECs approximately 50-200 nm in diameter that permit the passage of macromolecules, such as lipoproteins, into the extravascular space for subsequent hepatocellular uptake and metabolism.
  • one of the most striking functional characteristics of LSECs is their very high endocytic activity (Smedsrod et al. 1994, Gut 35:1509-1516; Smedsrod et al 1997, Biochem J 1997;322 ( Pt 2):567-573).
  • the LSEC has a role in the hepatic immune function through the expression of numerous immune antigens (Limmer et ah, Nat Med 2QQ0; ⁇ : ⁇ 3A%- ⁇ 3SA) and by permitting interactions between lymphocytes and hepatocytes that have been shown to occur through the fenestrations (Warren et al. Hepatology 2006;44:l 182-1190).
  • Fenestrations are arranged in sieve plates, average lOOnm in diameter and occupy 6-8% of the sinusoidal surface area. They permit the passage of macromolecules, such as lipoproteins, into the extravascular space for subsequent hepatocellular uptake and metabolism. This function has led to liver sinusoidal endothelial cells being termed "the liver sieve”. Both the number and the size of fenestrations can be regulated by a variety of processes.
  • Capillarization refers to the changes that occur in hepatic cirrhosis consisting of defenestration, basal lamina formation, upregulation of antigens such as von Willebrands factor and CD31 and perisinusoidal collagen deposition (DeLeve et al. Am J Physiol Gastrointest Liver Physiol 2004;287:G757-763; Onori et al J Hepatol 2000;33:555-563; Urashima et al.
  • Pseudocapillarization refers to the changes that occur in old age including defenestration in the absence of changes in light microscopic appearance or stellate cell activation (Le Couteur et al. Hepatology 2001;33:537-543; Le Couteur et al. Anatomical Record 2001 ; ⁇ m press); Warren et al Exp Gerontol 2005;40:807-812; Ito et al Exp Gerontol 2007;(in press)).
  • Loss of fenestrations has been proposed to be a novel mechanism for dyslipidaemia in the elderly and accordingly, modulation of fenestrations might be a novel target for the management of dyslipidaemia (Hilmer et al 2005, supra; Le Couteur et al. Lancet 2002;359:1612-1615).
  • the LSECs are also influenced by toxins such as oxidants (Cogger et al.
  • liver fenestrations In addition to the impact of fenestration abnormalities in the context of liver metabolism, there also exists a potential immunological impact which is linked to the fact that liver fenestrations have been shown to provide a portal through which T cells and hepatocytes can interact, in the absence of T cell activation having first occurred. Accordingly, the liver is an exception to the rule that T cells must be activated by antigen preventing cells in order to cross the endothelial cell barrier. Accordingly, reduction in fenestration number and/or functionality may also lead to altered immune functioning, such as in the context of the induction of hepatic immune tolerance.
  • identifying means such as pharmacological manipulation, to effect modulation of fenestrations is a high priority in terms of developing means of treating conditions which are linked to abnormalities in the structure or number of liver sinusoidal fenestrations.
  • the study of LSECs has been limited by the lack of a suitable cellular model that is fenestrated and performs endocytosis. Intact animal experiments are constrained by the capacity to only perform a single perturbation at a single concentration and timepoint in each animal.
  • LSECs isolated from rat livers have been the major model for studying LSEC biology.
  • liver tumour cell line SK Hep-1 is, in fact, fenestrated, these fenestrations being modulatable by appropriate extracellular stimulation.
  • a mechanism of visualising the fenestrations, other than by electron microscopy has been successfully designed and is exemplified herein in the context of a second generation cell line.
  • This cell line comprises a genetic modification to enable the expression of a detectable marker of fenestration which enables the number, distribution and size of cellular fenestrations to be identified by means other than electron microscopy, specifically, by fluorescence microscopy. This development significantly reduces the need to perform technically demanding microscopy and provides a viable means of performing high throughput screening assays.
  • the term "derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source. Further, as used herein the singular forms of "a”, “and” and “the” include plural referents unless the context clearly dictates otherwise.
  • One aspect of the present invention is directed to a genetically modified mammalian cell, which cell is capable of forming fenestrations, wherein said cell has been genetically modified to express one or more detectable markers of fenestration.
  • a genetically modified mammalian endothelial cell which cell is capable of forming fenestrations, wherein said endothelial cell has been genetically modified to express one or more markers of fenestration.
  • a genetically modified mammalian sinusoidal endothelial cell which cell is capable of forming fenestrations, wherein said sinusoidal endothelial cell has been genetically modified to express one or more detectable markers of fenestration.
  • liver sinusoidal endothelial cell which cell is capable of forming fenestrations, wherein said liver sinusoidal endothelial cell has been genetically modified to express one or more detectable markers of fenestration.
  • SK Hep-1 cell wherein said SK Hep-1 cell has been genetically modified to express one or more detectable markers of fenestration and wherein said markers are selected from:
  • NOS or functional derivative, homologue or variant thereof NOS or functional derivative, homologue or variant thereof; or - serotonin receptor or functional derivative, homologue or variant thereof.
  • the present invention provides a genetically modified SK Hep-1 cell wherein said SK Hep-1 cell has been genetically modified to express detectable caveolin or functional derivative, homologue or variant thereof.
  • the present invention provides a genetically modified SK Hep-1 cell wherein said SK Hep-1 cell has been genetically modified to express detectable actin or functional derivative, homologue or variant thereof.
  • SK Hep-1 cell wherein said SK Hep-1 cell has been genetically modified to express two detectable markers of fenestration, which markers are:
  • a genetically modified mammalian endothelial cell which cell is capable of forming fenestrations, wherein said endothelial cell has been genetically modified to express one or more detectable markers of fenestration and wherein said detectable markers are markers detectable by fluorescent means.
  • a further aspect of the present invention provides a genetically modified SK Hep-1 cell wherein said SK Hep-1 cell has been genetically modified to express one or more detectable markers of fenestration and wherein said markers are detectably by fluorescent means and selected from:
  • caveolin or functional derivative, homologue or variant thereof Ca ATPase or functional derivative, homologue or variant thereof; endothelial or functional derivative, homologue or variant thereof; - dynamin or functional derivative, homologue or variant thereof;
  • Another further aspect of the present invention provides in one embodiment a genetically modified mammalian endothelial cell, which cell is capable of forming fenestrations, said genetic modification comprising the transfection of said cell with a vector, which vector comprises a nucleic acid molecule encoding a GFP-actin fusion protein.
  • a genetically modified mammalian endothelial cell which cell is capable of forming fenestrations, said genetic modification comprising the transfection of said cell with a vector, which vector is pAcGFP-actin.
  • a genetically modified mammalian endothelial cell which cell is capable of forming fenestrations, said genetic modification comprising the transfection of said cell with a vector, which vector comprises a nucleic acid molecule encoding a GFP-caveolin fusion protein.
  • Yet another aspect of the present invention provides a method of analysing mammalian cellular fenestrations, said method comprising exposing the genetically modified cells of the present invention to a stimulus and screening for the detectable marker of fenestration.
  • a method of assessing the effect of a treatment or culture regime on the cellular fenestration of the genetically modified cells as hereinbefore described said method comprising subjecting said cells to said treatment or culture regime and screening for the detectable marker of fenestration.
  • a method of identifying a stimulus which modulates cellular fenestrations comprising contacting the genetically modified cells as hereinbefore described with said stimulus and screening for the detectable marker of fenestration.
  • Still another aspect of the present invention is directed to an agent identified in accordance with the screening method hereinbefore defined when used to modulate endothelial cell fenestration expression, distribution and/or pore diameter.
  • Yet another aspect of the present invention is directed to a method of modulating mammalian endothelial cell fenestrations, said method comprising administering to said mammal an effective amount of an agent identified in accordance with the screening method of the present invention.
  • Figure 1 is an image of A: vascular cast showing extensive sinusoidal network.
  • Figure 2 is an image of (A, B) Transmission em of young and old F344 rat liver. (C, D) Scanning em of young and old liver. (E, F) Immunohistochemistry for von Willebrand factor. In old age, the LSEC becomes thicker, defenestrated and there is upregulation of endothelial antigens not seen in the healthy young liver.
  • Figure 3 is an image of LSECs isolated from rat liver after 1, 6, 20 and 24 hrs shows increase in fenestrations at 6 hrs followed by marked defenestration and loss of cellular viability.
  • Figure 4 is an image of a scanning em of SK Hep-1 cells (A-E) and, for comparison, an LSEC isolated from a rat liver (F).
  • the SK Hep-1 cells perforated with fenestrations of varying size and sometimes arranged in linear groups (B), but rarely in full sieve plates such as seen in isolated LSECs (F).
  • Figure 5 is a graphical representation of the frequency distribution of fenestrations of SK Hep-1 cells.
  • Figure 6 is an image of a scanning em of digested SK Hep-1 cells showing actin cytoskeleton. Sieve-like structures are apparent (A). After incubation of VEGF for 24 hours, there is a marked disruption of the actin cytoskeleton (B).
  • Figure 7 is an image and graphical representation of the effects of VEFG on SK Hep-1 cells.
  • A control.
  • B after incubation with VEGF 40 ng/mL for 24 hrs.
  • Bar l ⁇ M.
  • Figure 9 is a fluorescent image of SK Hep-1 cells transfected stably with GFP-caveolin-1 (A) and actin (B).
  • Figure 10 is a fluorescent image of SK Hep-1 cells after incubation with FITC-FSA. There is uptake of FSA into the cells as indicated by the bright fluorescent particles.
  • Figure 11 is a fluorescent image of SK Hep-1 cells transfected stably with GFP-actin (A) and caveolin-1 (D). The effect of VEGF at 1 hr (B 5 E) and 12 hrs (C 5 F) is shown.
  • Figure 12 is an image of a transmission of immunogold staining for caveolin-1 showing staining of vesicles that are transacted fenestrations.
  • Figure 13 is a schematic of the restriction map of p AcGFPl -Actin.
  • Figure 14 is a schematic of the restriction map of pEGFP-caveolin-1.
  • the present invention is predicated, in part, on the determination that an immortalised liver sinusoidal endothelial cell line is, in fact, fenestrated, these fenestrations being modulatable by extracellular signals. Still further, there has been developed a means for visualising changes to the fenestrations in these cells by genetically modifying these cells to express a detectable marker of fenestration. This determination, and the generation of cells based thereon, has now provided a means of simply and routinely analysing liver cell fenestrations, in particular in the context of changes to their number and/or structure, by means other than highly complex and costly electron microscopy.
  • one aspect of the present invention is directed to a genetically modified mammalian cell, which cell is capable of forming fenestrations, wherein said cell has been genetically modified to express one or more detectable markers of fenestration.
  • a cell "capable of forming fenestrations” should be understood as a reference to a cell which expresses fenestrations constitutively and/or can be induced to express fenestrations upon the receipt of an appropriate stimulus, such as a cytokine signal.
  • the subject cell is one in which the fenestrations, whether constitutive or inducible, are themselves also responsive to stimuli which can modulate their structure and/or expression levels.
  • a fenestration is a pore which is generally found in sinusoidal endothelial cells. Fenestrations are arranged in sieve plates and average lOOnm in diameter. They generally occupy 6-8% of the sinusoidal surface area and, in the context of liver metabolism, permit the passage of macromolecules, such as lipoproteins, into the extravascular space for subsequent hepatocellular uptake and metabolism.
  • the subject cells may have been freshly isolated from an individual (such as an individual who may be the subject of treatment) or they may have been sourced from a non-fresh source, such as from a culture (for example, where cell numbers were expanded) or a frozen stock of cells (for example, an established cell line such as the SK Hep-1 cell line), which had been isolated at some earlier time point either from an individual or from another source.
  • a non-fresh source such as from a culture (for example, where cell numbers were expanded) or a frozen stock of cells (for example, an established cell line such as the SK Hep-1 cell line), which had been isolated at some earlier time point either from an individual or from another source.
  • the subject cells prior to undergoing the genetic manipulation of the present invention, may have undergone some other form of treatment or manipulation, such as but not limited to enrichment or purification, modification of cell cycle status or the formation of a cell line.
  • the subject cell may be a primary cell or a secondary cell.
  • a primary cell is one which has been isolated from an individual.
  • said fenestrated cell is an endothelial cell.
  • fenestrated endothelial cells are generally found in capillaries. They demonstrate pores of a diameter of 50-200 nm whereas the non-fenestrated endothelial cells effect a continuous coverage of the vascular lumen.
  • the fenestrae are open and very wide in sinusoids which also lack a basal lamina (eg. the sinusoids of liver and spleen).
  • Other fenestrated endothelial cells with open fenestrae are present in the kidney.
  • said endothelial cell is a sinusoidal endothelial cell and preferably a liver sinusoidal endothelial cell.
  • the liver is the major metabolic and detoxification organ.
  • the blood delivered to the liver via the portal vein from the gut, spleen and pancreas contains a vast array of nutrients, toxins and hormones.
  • the portal blood circulates between plates of hepatocytes through an intricate network of sinusoids.
  • Liver sinusoidal endothelial cells line the hepatic sinusoids and facilitate this exchange.
  • the fenestrae act as a dynamic filter which filters fluids, solutes and particles which are exchanged between the sinusoidal luman and the space of Disse, allowing only particles smaller than the fenestrations to reach the parenchymal cells or to leave the space of Disse.
  • a genetically modified mammalian endothelial cell which cell is capable of forming fenestrations, wherein said endothelial cell has been genetically modified to express one or more markers of fenestration.
  • Endothelial cell should be understood as a reference to the endothelial cells which line the blood vessels, lymphatics or other serous cavities or sinusoids such as fluid filled cavities.
  • endothelial cells should also be understood as a reference to cells which exhibit one or more of the morphology, phenotype and/or functional activity of endothelial cells and is also a reference to mutants or variants thereof.
  • “Variants” include, but are not limited to, cells exhibiting some but not all of the morphological or phenotypic features or functional activities of endothelial cells at any differentiative stage of development. “Mutants” include, but are not limited to, endothelial cells which have been naturally or non-naturally modified such as cells which are genetically modified. It should also be understood that the endothelial cells of the present invention may be at any differentiative stage of development. Accordingly, the cells may be immature but, provided that they do possess the capability of forming fenestrations, fall within the scope of the present invention.
  • a genetically modified mammalian sinusoidal endothelial cell which cell is capable of forming fenestrations, wherein said sinusoidal endothelial cell has been genetically modified to express one or more detectable markers of fenestration.
  • said sinusoidal endothelial cell is a liver sinusoidal endothelial cell.
  • liver sinusoidal endothelial cell should be understood as a reference to a sinusoidal endothelial cell which is associated with any part of the liver.
  • liver sinusoidal endothelial cell which cell is capable of forming fenestrations, wherein said liver sinusoidal endothelial cell has been genetically modified to express one or more detectable markers of fenestration.
  • said liver sinusoidal endothelial cell is a SK Hep-1 cell, which cell is capable of forming fenestrations, wherein said SK Hep-1 cell has been genetically modified to express one or more detectable markers of fenestration.
  • mammal should be understood to include reference to a mammal such as but not limited to human, primate, livestock (animal (eg. sheep, cow, horse, donkey, pig), companion animal (eg. dog, cat), laboratory test animal (eg. mouse, rabbit, rat, guinea pig, hamster), captive wild animal (eg. fox, deer).
  • animal eg. sheep, cow, horse, donkey, pig
  • companion animal eg. dog, cat
  • laboratory test animal eg. mouse, rabbit, rat, guinea pig, hamster
  • captive wild animal eg. fox, deer
  • SK Hep-1 is an immortal, human cell line derived from the ascitic fluid of a patient with adenocarcinoma of the liver. It has now been determined that these cells are of endothelial origin, despite the location of the tumor from which SK Hep-1 was derived. The cell line does not exhibit properties of hepatocytes. Northern blot analysis of total cellular RNA shows no messenger RNA for the hepatic-specific proteins albumin, alpha-fibrinogen, or gamma-fibrinogen. Rather, endothelial characteristics are seen by transmission electron microscopy.
  • a cell line thought to be of hepatocyte origin is in fact a fenestrated liver sinusoidal endothelial cell
  • the fenestrations are constitutively expressed yet nevertheless respond to cytokine manipulation.
  • This renders this cell line an ideal model of liver sinusoidal fenestration functionality.
  • sinusoidal endothelial fenestrae are dynamic structures whose diameter and number vary in response to a variety of hormones, drugs, toxins, or even to changes in the underlying extracellular matrix. Accordingly, SK Hep-1 cells provide an excellent model for studying fenestration functionality.
  • Reference to a "marker of fenestration” should be understood as a reference to a molecule which is either directly or indirectly associated with a fenestration.
  • a molecule which is "directly” associated with a fenestration should be understood as a reference to a molecule which forms part of the fenestration structure itself while a marker which is "indirectly” associated with a fenestration should be understood as a reference to a molecule which although not necessarily associated with the structure of the fenestration itself, nevertheless forms part of the cellular structure which surrounds or supports the fenestration. Changes in the expression pattern of these molecules are therefore indicative of structural changes to the fenestration itself.
  • said marker is a protein.
  • proteins which form part of the fenestration pore itself include, but are not limited to, caveolin (which is found in the walls of fenestrations), Ca ATPase, endothelin-1 and dynamin.
  • proteins which are indirectly associated with a fenestration include, but are not limited to actin, filamen, nitric oxide synthase (NOS), serotonin receptor and calcium channels.
  • actin forms a cytoskeleton which supports the liver sieve plates and fenestrations while NOS associates with caveolin and filamen acts to link caveolin to actin. Accordingly, changes to the intracellular distribution, concentration or expression of any one or more of these molecules is indicative of the modulation of fenestration formation, whether that be upregulation or downregulation of the induction of fenestration, the maintenance of basal levels of fenestrations, the loss of fenestrations or changes to fenestration diameter or distribution.
  • markers for example caveolin, Ca ATPase, endothelin-1, dynamin, actin, filamen, NOS and serotonin receptor, should be understood as a reference to all forms of these markers and to functional derivatives and homologues thereof. This includes, for example, any isoforms which arise from alternative splicing of the mRNA encoding these molecules or functional mutants or polymorphic variants of these proteins.
  • functional is meant that the subject mutant, homologue, derivative or variant would nevertheless be incorporated into the structure to which the protein of which it is a mutant, homologue, derivative or variant would normally form part.
  • a murine homologue of caveolin or actin would be expected to also incorporate into the cellular fenestration structures with which human caveolin and actin form part, thereby rendering the expression of these murine homologues suitable for use in the context of the genetically modified cells of the present invention.
  • Derivatives of the molecules herein described include functional fragments, parts, portions or variants. Derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the functionality of the resulting product. Deletional variants are characterised by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in a sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins, as detailed above.
  • Derivatives also include fragments having particular regions of the entire protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules.
  • Derivatives of nucleic acid sequences which may be utilised in accordance with the method of the present invention may similarly be derived from single or multiple nucleotide substitutions, deletions and/or additions including fusion with other nucleic acid molecules.
  • Derivatives of nucleic acid sequences also include degenerate variants.
  • a "variant" should be understood to mean a molecule which exhibits at least some of the functional activity of the form of molecule of which it is a variant. A variation may take any form and may be naturally or non-naturally occurring.
  • homologue is meant that the molecule is derived from a species other than that from which the cells of the present invention were derived. This may occur, for example, where it is determined that a species other than that which is being treated produces a form of the subject molecule which exhibits suitable functionality.
  • the marker of fenestration is not necessarily one which is exclusively expressed only in the context (either directly or indirectly) of a cellular fenestration. Rather, it may also be expressed in the context of other cellular structures which are unrelated to fenestrations. However, due to the unique tubular structure and distribution pattern of fenestrations, the skilled person can determine from the expression pattern of the marker, following visualisation of the detection tag, which aspects of its expression pattern are indicative of fenestrations. To this end, depending on the nature of the markers which are selected for use, it may be sufficient to screen with a single marker or else it may be more appropriate to screen with two or more markers, either on the same or different cellular samples.
  • markers selected for use is one which is directly associated with fenestrations, such as caveolin
  • this marker alone could be analysed since even if caveolin is expressed by other structures within the cell, the unique ring-like staining characteristic of a fenestration renders the fenestration-related staining highly distinctive.
  • Markers which are indirectly associated with fenestrations may be equally informative when analysed on their own. For example, markers which are linked to caveolin may demonstrate the distinctive staining pattern which is characteristic of caveolin. Other markers, such as actin, which are widely distributed across the cell are indicative of the presence of fenestrations due to changes which can be found in the confluence of their staining pattern.
  • SK Hep-1 cell wherein said SK Hep-1 cell has been genetically modified to express one or more detectable markers of fenestration and wherein said markers are selected from:
  • NOS or functional derivative, homologue or variant thereof or serotonin receptor or functional derivative, homologue or variant thereof.
  • the present invention provides a genetically modified SK Hep-1 cell wherein said SK Hep-1 cell has been genetically modified to express detectable caveolin or functional derivative, homologue or variant thereof.
  • the present invention provides a genetically modified SK Hep-1 cell wherein said SK Hep-1 cell has been genetically modified to express detectable actin or functional derivative, homologue or variant thereof.
  • Reference to "two or more” should be understood as a reference to two, three, four, five, six or more.
  • said two or more detectable markers of fenestration is two or three.
  • SK Hep-1 cell wherein said SK Hep-1 cell has been genetically modified to express two detectable markers of fenestration, which markers are:
  • caveolin or functional derivative, homologue or variant thereof and actin or functional derivative, homologue or variant thereof.
  • the subject marker is "detectable". By “detectable” is meant that the expression of the subject marker can be visualised.
  • Detection tags and visualisation techniques are well known to those of skill in the art and any suitable technique, such as fluorescence, radiolabelling, chemiluminescence or enzymatic based techniques can be utilised.
  • fluorescence related techniques there exist a wide range of molecules which are suitable for use in the context of the cells of the present invention.
  • naturally fluorescent proteins such as green fluorescent protein (GFP/EGFP)
  • GFP/EGFP green fluorescent protein
  • Other naturally fluorescent proteins which could also be used include, but are not limited to, yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), emerald green fluorescent protein (EMGFP) or red fluorescent protein (RFP).
  • Nucleic acid molecules encoding these proteins are well known in the art and vectors incorporating these sequences are commercially available (eg. Invitrogen).
  • Other technologies which are suitable for use in this context include tetracysteine tag detection wherein a small 6 amino acid tag is expressed as a fusion together with the marker of interest, this peptide being subsequently detectable with a fluorescent reagent which is targeted to the tag. Accordingly, the protein of interest is fluorescent only when the labelling reagent is added.
  • the application of this technology is familiar to those skilled in the art and is, in fact, commercially available (see for example the TC-FIAsH II In-cell Tetracysteine Tag Detection Kit, Invitrogen).
  • Still other detection means suitable for use in this context include protein detection tags to which visualisation means can bind or otherwise interact, such as via immunological or other means which are either directly or indirectly detectable (for example, biotin-avidin based detection).
  • protein molecules such as epitopes are suitable for use in antibody based screening methods and can be easily and routinely expressed together with the marker of interest.
  • a genetically modified mammalian endothelial cell which cell is capable of forming fenestrations, wherein said endothelial cell has been genetically modified to express one or more detectable markers of fenestration and wherein said detectable markers are markers detectable by fluorescent means.
  • said endothelial cell is a sinusoidal endothelial cell, more preferably a liver sinusoidal endothelial cell and still more preferably SK Hep-1.
  • the present invention provides a genetically modified SK Hep-1 cell wherein said SK Hep-1 cell has been genetically modified to express one or more detectable markers of fenestration and wherein said markers are detectably by fluorescent means and selected from:
  • said detectable marker is GFP labelled caveolin.
  • said detectable marker is GFP labelled actin.
  • the cells of the present invention are genetically modified.
  • genetically modified is meant that the subject cell has undergone some form of molecular manipulation relative to that which is observed in the context of a corresponding unmodified cell. Such modifications include, but are not limited to:
  • the cell is rendered transgenic via the introduction of all or part of one or more genes. This clearly occurs in the context of the transfection of a nucleic acid molecule encoding the detectable marker of fenestration.
  • the cell is preferably permanently transfected with cDNA or genomic DNA encoding the detectable marker of fenestration.
  • cells may be generated which transiently express a nucleic acid molecule encoding these molecules. This may be useful in certain circumstances where, for example, one is seeking to generate a population of cells suitable for an application where ongoing expression of the detectable marker is not desired but yet initial characterisation of the cells in terms of their fenestration functionality is desirable.
  • genes relevant to optimising generation of the subject cells include genes encoding other marker proteins suitable for purposes such as identification of successful transformants.
  • Selection markers such as antibiotic resistance genes (for example G418 resistance gene which enables the selection of mammalian cells using the neomycin analogue G418 or puromycin resistance gene), provide a convenient means of selecting for successful transformants while the incorporation of a suicide gene, such as the pMCl -thymidine kinase gene, facilitates the in vivo elimination of the genetically modified cells if they were to be administered in vivo.
  • nucleic acid should be understood as a reference to both deoxyribonucleic acid and ribonucleic acid thereof.
  • the subject nucleic acid molecule may be any suitable form of nucleic acid molecule including, for example, a genomic, cDNA or ribonucleic acid molecule.
  • expression refers to the transcription and translation of DNA or the translation of RNA resulting in the synthesis of a peptide, polypeptide or protein.
  • a DNA construct for example, corresponds to the construct which one may seek to transfect into a cell for subsequent expression while an example of an RNA construct is the RNA molecule transcribed from a DNA construct, which RNA construct merely requires translation to generate the protein of interest.
  • Reference to "expression product” is a reference to the product produced from the transcription and translation of a nucleic acid molecule.
  • protein should be understood to encompass peptides, polypeptides and proteins. It should also be understood that these terms are used interchangeably herein.
  • the protein may be glycosylated or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the protein such as lipids, carbohydrates or other peptides, polypeptides or proteins (such as occurs in preferred embodiments of the present invention where the marker protein is produced as a fusion protein with the detection means).
  • Reference hereinafter to a "protein” includes a protein comprising a sequence of amino acids as well as a protein associated with other molecules such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • genetic material is generally conveniently introduced to cells via the use of an expression construct.
  • a cell type which either naturally or as a result of earlier random or directed genetic manipulation is already characterised by one or more of the genetic modifications of interest (for example, modifications to render the cell immortal or to modify its MHC profile in order to render the cell less immunogenic in the event that it was to be transplanted).
  • Means of introducing the genetic construct to the cell are also well known in the art.
  • transfection methods fall into three broad categories: physical (e.g., electroporation, direct gene transfer and particle bombardment), chemical (lipid-based carriers, or other non-viral vectors) and biological (virus-derived vector and receptor uptake).
  • physical e.g., electroporation, direct gene transfer and particle bombardment
  • chemical lipid-based carriers, or other non-viral vectors
  • biological virus-derived vector and receptor uptake
  • non- viral vectors may be used which include liposomes coated with DNA. Such liposome/DNA complexes may be directly injected intravenously into the patient.
  • vectors or the "naked" DNA of the gene may be directly injected into the desired cells.
  • Chemical methods of transfection may involve a lipid based compound, not necessarily a liposome, to ferry the DNA across the cell membrane.
  • Lipofectins or cytofectins lipid- based positive ions that bind to negatively charged DNA, may be used to cross the cell membrane and provide the DNA into the interior of the cell.
  • Another chemical method may include receptor-based endocytosis, which involves binding a specific ligand to a cell surface receptor and enveloping and transporting it across the cell membrane.
  • Viral vectors such as retrovirus vectors to insert genes into cells.
  • Viral vectors may be selected from the group including, but are not limited to, retroviruses, other RNA viruses such as poliovirus or Sindbis virus, adenovirus, adeno-associated virus, herpes viruses, SV 40, vaccinia and other DNA viruses.
  • Replication-defective murine retroviral vectors are the most widely utilized gene transfer vectors and are preferred.
  • Adenoviral vectors may be delivered bound to an antibody that is in turn bound to collagen coated stents.
  • DNA delivery may be employed and include, but are not limited to, fusogenic lipid vesicles such as liposomes or other vesicles for membrane fusion, lipid particles of DNA incorporating cationic lipid such as lipofectin, polylysine-mediated transfer of DNA, direct injection of DNA, such as microinjection of DNA into germ or somatic cells, pneumatically delivered DNA-coated particles, such as the gold particles used in a "gene gun", inorganic chemical approaches such as calcium phosphate transfection and plasmid DNA incorporated into polymer coated stents.
  • Ligand-mediated transfection may also be employed involving complexing the DNA with specific ligands to form ligand-DNA conjugates, to direct the DNA to a specific cell or tissue.
  • the DNA of the plasmid may or may not integrate into the genome of the cells.
  • Non- integration of the transfected DNA would allow the transfection and expression of gene product proteins in terminally differentiated, non-proliferative tissues for a prolonged period of time without fear of mutational insertions, deletions, or alterations in the cellular or mitochondrial genome.
  • the DNA could be reinjected periodically to maintain the gene product level without mutations occurring in the genomes of the recipient cells.
  • Non- integration of exogenous DNAs may allow for the presence of several different exogenous DNA constructs within one cell with all of the constructs expressing various gene products.
  • said genetic modification is the transfection of a cell capable of forming fenestrations with an expression construct comprising one or more DNA regions comprising a promoter operably linked to a sequence encoding a marker of fenestration and a second DNA region encoding a detection means and, optionally, a third DNA region encoding a selection marker.
  • the subject promoter may be constitutive or inducible. Where the subject construct expresses more than one protein of interest, these may be under the control of separate promoters (for example the detectable marker of fenestration is separately controlled to a selection marker, such as an antibiotic resistance gene), or they may be under the control of a single promoter, such as occurs in the context of a bicistronic vector which makes use of an IRES sequence to facilitate the translation of more than one protein product, in an unfused form, from a single RNA transcript. This latter technology may be particularly useful where one is seeking to co-express two detectable markers of fenestration but wishes to do so in the context of a single transfection event with one vector.
  • a polycistronic vector could be used.
  • the subject construct may additionally be designed to facilitate use of the Cre recombinase mediated splicing inducible gene expression system.
  • nucleic acid "expression construct” should be understood as a reference to a nucleic acid molecule which is transmissible to a cell and designed to undergo transcription. The RNA molecule is then transcribed therefrom.
  • expression constructs are also referred to by a number of alternative terms, which terms are widely utilised interchangeably, including "expression cassette” and "vector".
  • the expression construct of the present invention may be generated by any suitable method including recombinant or synthetic techniques.
  • the subject construct may be constructed from first principles, as would occur where an entirely synthetic approach is utilised, or it may be constructed by appropriately modifying an existing vector. Where one adopts the latter approach, the range of vectors which could be utilised as a starting point are extensive and include, but are not limited to:
  • Plasmids are small independently replicating pieces of cytoplasmic DNA, generally found in prokaryotic cells, which are capable of autonomous replication. Plasmids are commonly used in the context of molecular cloning due to their capacity to be transferred from one organism to another. Without limiting the present invention to any one theory or mode of action, plasmids can remain episomal or they can become incorporated into the genome of a host. Examples of plasmids which one might utilise include the bacterial derived pBR322 and pUC.
  • Bacteriophages are viruses which infect and replicate in bacteria. They generally consist of a core of nucleic acid enclosed within a protein coat (termed the capsid). Depending on the type of phage, the nucleic acid may be either DNA (single or double stranded) or RNA (single stranded) and they may be either linear or circular. Phages may be filamentous, polyhedral or polyhedral and tailed, the tubular tails to which one or more tubular tail fibres are attached. Phages can generally accommodate larger fragments of foreign DNA than, for example, plasmids. Examples of phages include, but are not limited to the E.coli lambda phages, Pl bacteriophage and the T-even phages (e.g. T4).
  • Artificial chromosomes such as yeast artificial chromosomes or bacterial artificial chromosomes.
  • Hybrid vectors such as cosmids, phagemids andphasmids
  • Cosmids are generally derived from plasmids but also comprise cos sites for lambda phage while phagemids represent a chimaeric phage-plasmid vector.
  • Phasmids generally also represent a plasmid-phage chimaera but are defined by virtue of the fact that they contain functional origins of replication of both. Phasmids can therefore be propagated either as a plasmid or a phage in an appropriate host strain.
  • an appropriate vector for modification to the extent that one chooses to do this rather than synthetically generate a construct, will depend on a number of factors including the ultimate use to which the genetically modified cell will be put. For example, where the cell is to be administered in vivo into a human, it may be less desirable to utilise certain types of vectors, such as viral vectors. Further, it is necessary to consider the amount of DNA which is sought to be introduced to the construct. It is generally understood that certain vectors are more readily transfected into certain cell types. For example, the range of cell types which can act as a host for a given plasmid may vary from one plasmid type to another.
  • the size of the inserted DNA can vary depending on factors such as the size of the DNA sequence encoding the protein of interest, the number of proteins which are sought to be expressed, the number of selection markers which are utilised and the incorporation of features such as linearisation polylinker regions and the like.
  • the expression construct which is used in the present invention may be of any form including circular or linear.
  • a "circular" nucleotide sequence should be understood as a reference to the circular nucleotide sequence portion of any nucleotide molecule.
  • the nucleotide sequence may be completely circular, such as a plasmid, or it may be partly circular, such as the circular portion of a nucleotide molecule generated during rolling circle replication (this may be relevant, for example, where a construct is being initially replicated, prior to its introduction to a cell population, by this type of method rather than via a cellular based cloning system).
  • the "circular" nucleotide sequence corresponds to the circular portion of this molecule.
  • linear nucleotide sequence should be understood as a reference to any nucleotide sequence which is in essentially linear form.
  • the linear sequence may be a linear nucleotide molecule or it may be a linear portion of a nucleotide molecule which also comprises a non-linear portion such as a circular portion.
  • An example of a linear nucleotide sequence includes, but is not limited to, a plasmid derived construct which has been linearised in order to facilitate its integration into the chromosomes of a host cell or a construct which has been synthetically generated in linear form. To this end, it should also be understood that the configuration of the construct of the present invention may or may not remain constant.
  • a circular plasmid-derived construct may be transfected into a cell where it remains a stable circular episome which undergoes replication and transcription in this form.
  • the subject construct may be one which is transfected into a cell in circular form but undergoes intracellular linearisation prior to chromosomal integration. This is not necessarily an ideal situation since such linearisation may occur in a random fashion and potentially cleave the construct in a crucial region thereby rendering it ineffective.
  • the vector may be designed to effect its recombination into a specific chromosomal site rather than, for example by site directed homologous recombination, rather than by random insertion.
  • this vector remains episomal subsequently to transfection and is not linearised or integrated into the chromosome.
  • the endogenous marker gene continues to also be expressed.
  • the vector since the vector has been optimised to increase translation efficiency, it can efficiently compete with the endogenous marker for integration into a forming fenestration.
  • the nucleic acid molecules which are utilised in the method of the present invention are derivable from any human or non-human source.
  • Non-human sources contemplated by the present invention include primates, livestock animals (eg. sheep, pigs, cows, goats, horses, donkeys), laboratory test animal (eg. mice, hamsters, rabbits, rats, guinea pigs), domestic companion animal (eg. dogs, cats), birds (eg. chicken, geese, ducks and other poultry birds, game birds, emus, ostriches) captive wild or tamed animals (eg. foxes, kangaroos, dingoes), reptiles, fish, insects, prokaryotic organisms or synthetic nucleic acids.
  • livestock animals eg. sheep, pigs, cows, goats, horses, donkeys
  • laboratory test animal eg. mice, hamsters, rabbits, rats, guinea pigs
  • domestic companion animal eg.
  • the constructs of the present invention may comprise nucleic acid material from more than one source.
  • the construct may originate from a bacterial plasmid, in modifying that plasmid to introduce the features defined herein nucleic acid material from non-bacterial sources may be introduced.
  • sources may include, for example, viral DNA (e.g. IRES DNA), mammalian DNA or synthetic DNA (e.g. to introduce specific restriction endonuclease sites).
  • the cell type in which it is proposed to express the subject construct may be different again in that it does not correspond to the same organism as all or part of the nucleic acid material of the construct.
  • a construct consisting of essentially bacterial and viral derived DNA may nevertheless be expressed in the mammalian stem cells contemplated herein.
  • the present invention is exemplified in terms of the generation of two genetically modified SK Hep-1 cell lines.
  • One cell line has been modified to express a GFP-actin fusion protein while the other has been modified to express a GFP-caveolin fusion protein.
  • this is expressed by the vector pAcGFPl-Actin ( Figure 13) while the GFP-caveolin fusion protein is expressed by the vector pEGFP-caveolin-1.
  • the actin vector encodes GFP from Aequorea coerulescens as a fusion molecule together with the human cytoplasmic b-actin gene (Ponte et al. 1984, Nucleic Acid Res. 12:1687- 1696). Still without limiting the present invention in any way, the vector exemplified herein comprises SV40 polyadenylation signals downstream of the AcGFPl -Actin fusion to direct proper processing of the 3' end of the AcGFPl mRNA.
  • AcGFPl contains silent mutations that create an open reading frame comprised almost entirely of optimized human codons. These changes increase the translational efficiency of the AcGFPl mRNA and consequently the expression of AcGFPl in mammalian and plant cells.
  • the vector backbone also contains an SV40 origin for replication in any mammalian cell line that expresses the SV40 T-antigen.
  • a neomycin resistance cassette (Neo 1 ), consisting of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the herpes simplex virus thymidine kinase (HS V-TK) gene, allows stably transfected eukaryotic cells to be selected using G418.
  • a bacterial promoter upstream of this cassette drives expression of the gene encoding kanamycin resistance in E.coli
  • the pAcGFPl-Actin backbone also provides a pUC origin of replication for propagation in E.coli and an fl origin for single-stranded DNA production.
  • the caveolin encoding vector as exemplified herein corresponds to the actin vector but with the exception that the actin encoding DNA segment is substituted with a caveolin encoding DNA segment.
  • the present invention therefore provides in one embodiment a genetically modified mammalian endothelial cell, which cell is capable of forming fenestrations, said genetic modification comprising the transfection of said cell with a vector, which vector comprises a nucleic acid molecule encoding a GFP-actin fusion protein.
  • said vector is p AcGFPl -actin.
  • a genetically modified mammalian endothelial cell which cell is capable of forming fenestrations, said genetic modification comprising the transfection of said cell with a vector, which vector is pAcGFP -actin.
  • a genetically modified mammalian endothelial cell which cell is capable of forming fenestrations, said genetic modification comprising the transfection of said cell with a vector, which vector comprises a nucleic acid molecule encoding a GFP-caveolin fusion protein.
  • said vector is pEGFP-caveolin-1.
  • said endothelial cell is a sinusoidal cell and even more preferably a liver sinusoidal endothelial cell.
  • said cell is SK Hep-1.
  • said genetically modified cell corresponds to the cells deposited at ECACC on 27 July 2007 under Accession No. 07072602.
  • said genetically modified cell corresponds to the cells deposited at ECACC on 27 July 2007 under Accession No. 07072603.
  • the generation of the cells of the present invention may require the application of a screening and selection step to identify and isolate cells which have successfully incorporated the genetic modification of interest. Identification methods would be well known to the person of skill in the art and include, but are not limited to:
  • Detection of specific proteins may be conveniently effected via fluorescence microscopy, for example.
  • this method can be utilised to identify cell types via either a positive or negative selection step based on the expression of any one or more molecules.
  • RT-PCR Real-time PCR
  • other methods which can be used include hybridization microarray ("RNA chip") or Northern blotting or Southern blotting.
  • RNA chip hybridization microarray
  • Northern blotting Southern blotting.
  • RT-PCR can be used to detect specific RNAs encoding essentially any protein, such as the proteins detailed in point (i) above, or proteins which are secreted or otherwise not conveniently detectable via the methodology detailed in point (i). (Hi) Detection of specific cellular functional activity.
  • fenestrae are dynamic structures, whose diameter and number vary in response to a variety of hormones, drugs, toxins, diseases or even to changes in the underlying extracellular matrix. Structural integrity of the fenestrated sinusoidal liver endothelium, for example, is believed to be essential for the maintenance of a normal exchange of fluids, solutes, particles and metabolites between the hepatocytes and sinusoidal blood. Its alteration can have adverse effects on hepatocytes and liver function in general.
  • the identification and generation of the cells of the present invention has changed the landscape in this regard in that a method of routinely and simply performing high throughput screening has now been facilitated.
  • the cells of the present invention enable very sensitive and informative information to be obtained in relation to the concentration or distribution of cellular fenestrations or else changes to these features subsequently to treatment of the cell with an agent of interest.
  • Once cells have been identified which either exhibit a change to fenestration structure or are at least indicative of a change to fenestration structure (such as either their upregulation or downregulation in number) those specific cells may be further analysed, if necessary, such as in the context of electron microscopy in order to determine if the fenestrations which have been observed correspond to a folly formed pore.
  • yet another aspect of the present invention provides a method of analysing mammalian cellular fenestrations, said method comprising exposing the genetically modified cells of the present invention to a stimulus and screening for the detectable marker of fenestration.
  • the detection tag is a fluorescent protein.
  • fluorescent protein is detectable via fluorescence microscopy, as demonstrated in terms of the exemplified embodiment of the present invention. Fluorescence microscopy, in this context, is a highly informative technique since it provides a topographical analysis of the stimulated cells.
  • a detection tag such as an enzyme substrate (eg. alkaline phosphatase) requires enzymatic treatment in order to effect visualisation.
  • the tag is a molecule which is uniquely recognisable by immunointeractive or other specific means, visualisation is effected by treating said cell with a molecule which binds to the tag and enables visualisation of this complex.
  • the subject immunointeractive or other molecule may itself be appropriately labelled to facilitate the application of fluorescent, enzymatic or other suitable visualisation means.
  • the visualisation of an increase in the intensity/concentration of detection tag subsequently to exposure of the cells of the present invention to a stimulus is indicative of an upregulation in fenestration number due to formation of new cellular fenestrations.
  • the formation of new fenestrations may not be indicative of an overall increase in fenestration number but may be indicative of a redistribution of the localisation of fenestrations, this being characterised by a loss of fenestrations in some regions and formation of new fenestrations in other regions of the cell. Still further, one may alternatively or additionally observe the induction of a change in the diameter of existing fenestrations. In yet another example, there may be observed the loss of fenestrations. It would be appreciated that in order for an analysis of modulation of fenestrations to be most effective, it would be desirable to compare the results obtained post-stimulus with those of a control/unstimulated sample.
  • antagonists of specific fenestration modulators such as VEGF which activates fenestration formation
  • VEGF which activates fenestration formation
  • the identification both of molecules which upregulate and molecules which downregulate fenestration formation, distribution or diameter is of importance.
  • the stimulus may be an agent such as a proteinaceous and non-proteinaceous molecules which may have been derived from a wide variety of sources.
  • the proteinaceous molecules described above may be derived from any suitable source such as natural, recombinant or synthetic sources and includes fusion proteins or molecules which have been identified following, for example, natural product screening.
  • the reference to non-proteinaceous molecules may be, for example, a reference to a nucleic acid molecule (such as antisense nucleic acids which prevent transcription or translation of the genes or mRNA of components, RNA (particularly siRNA), ribosomes, DNAzymes or RNA aptamers) or it may be a molecule derived from natural sources, such as for example natural product screening, or may be a chemically synthesised molecule.
  • a nucleic acid molecule such as antisense nucleic acids which prevent transcription or translation of the genes or mRNA of components, RNA (particularly siRNA), ribosomes, DNAzymes or RNA aptamers
  • the agents which are utilised in accordance with the method of the present invention may take any suitable form.
  • proteinaceous agents may be glycosylated or unglycosylated, phosphorylated or dephosphorylated to various degrees and/or may contain a range of other molecules used, linked, bound or otherwise associated with the proteins such as amino acids, lipid, carbohydrates or other peptides, polypeptides or proteins.
  • the subject non-proteinaceous molecules may also take any suitable form.
  • Both the proteinaceous and non-proteinaceous agents herein described may be linked, bound or otherwise associated with any other proteinaceous or non-proteinaceous molecules.
  • said agent is associated with a molecule which permits its targeting to a localised region.
  • the subject proteinaceous or non-proteinaceous molecule may act either directly or indirectly to modulate the expression of fenestrations.
  • Said molecule acts directly if it associates with a fenestration-related nucleic acid molecule or expression product to modulate expression.
  • Said molecule acts indirectly if it associates with a molecule other than a fenestration-related nucleic acid molecule or expression product which other molecule either directly or indirectly modulates the expression of the fenestration-related nucleic acid molecule or expression product, respectively.
  • the method of the present invention encompasses the regulation of fenestration-related nucleic acid molecule expression or expression product activity via the induction of a cascade of regulatory steps.
  • the subject agents include chemical and functional equivalents which exhibit any one or more of the functional activities of a naturally occurring modulator of fenestration, which functional equivalents may be derived from any source such as being chemically synthesised or identified via screening processes such as natural product screening.
  • chemical or functional equivalents can be designed and/or identified utilising well known methods such as combinatorial chemistry or high throughput screening of recombinant libraries or following natural product screening.
  • libraries containing small organic molecules may be screened, wherein organic molecules having a large number of specific parent group substitutions are used.
  • a general synthetic scheme may follow published methods (eg., Bunin BA, et al. (1994) Proc. Natl. Acad. ScI USA, 91 /4708-4712; DeWitt SH, et al. (1993) Proc. Natl. Acad. ScI USA, 90:6909-6913). Briefly, at each successive synthetic step, one of a plurality of different selected substituents is added to each of a selected subset of tubes in an array, with the selection of tube subsets being such as to generate all possible permutation of the different substituents employed in producing the library.
  • One suitable permutation strategy is outlined in US. Patent No. 5,763,263.
  • oligomeric or small-molecule library compounds capable of interacting specifically with a selected biological agent, such as a biomolecule, a macromolecule complex, or cell, are screened utilising a combinational library device which is easily chosen by the person of skill in the art from the range of well-known methods, such as those described above.
  • a selected biological agent such as a biomolecule, a macromolecule complex, or cell
  • each member of the library is screened for its ability to interact specifically with the selected agent.
  • a biological agent is drawn into compound-containing tubes and allowed to interact with the individual library compound in each tube. The interaction is designed to produce a detectable signal that can be used to monitor the presence of the desired interaction.
  • the biological agent is present in an aqueous solution and further conditions are adapted depending on the desired interaction. Detection may be performed as hereinbefore described.
  • the stimulus is a natural product library such as NP_LLP, which has been generated from over 40,000 biota harvested from natural sources such as plants and marine life.
  • the subject stimulus may also take the form of a change to the extracellular environment, such as a change to the cell culture conditions, pH or temperature.
  • one may seek to observe the effect of subjecting the subject cells to an electric current or sonication.
  • a method of assessing the effect of a treatment or culture regime on the cellular fenestration of the genetically modified cells as hereinbefore described said method comprising subjecting said cells to said treatment or culture regime and screening for the detectable marker of fenestration.
  • the method of the present invention can be used to screen and/or test drugs or other treatment regimes, such as electrical treatments.
  • the treatment to which the cells or tissues of the present invention are subjected is an exposure to a compound.
  • the compound is a drug or a physiological ion.
  • the compound can be a growth factor or differentiation factor. Since drugs used for treating various diseases can unexpectedly result in desired or undesired effects to cellular functionality, it is highly desirable to have available a method which is capable of predicting such side effects on fenestrated endothelial tissue prior to administering the drug.
  • the present invention provides a means of assessing the effect of a treatment regime for any condition on the functionality of the liver sinusoidal endothelial cells.
  • a method of identifying a stimulus which modulates cellular fenestrations comprising contacting the genetically modified cells as hereinbefore described with said stimulus and screening for the detectable marker of fenestration.
  • said stimulus is a proteinaceous or non-proteinaceous molecule such as the molecule of a natural product library.
  • Reference to "modulates" cellular fenestrations should be understood as a reference to upregulating or downregulating fenestration formation, increasing or decreasing fenestration diameter or altering fenestration distribution.
  • Reference to modulating fenestration formation should be understood as a reference to partially or completely inducing or removing a fenestration. To this end, it should be appreciated that to the extent that a fenestration may only have been partially formed or partially downregulated, this may not be conclusively determinable via the visualisation of the detection tag.
  • an increase in the level of a detectable marker of fenestration is indicative of an upregulation in fenestration formation while a decrease in the level of a detectable marker of fenestration is indicative of a decrease in cellular fenestration number.
  • Still another aspect of the present invention is directed to an agent identified in accordance with the screening method hereinbefore defined when used to modulate endothelial cell fenestration expression, distribution and/or pore diameter.
  • Yet another aspect of the present invention is directed to a method of modulating mammalian endothelial cell fenestrations, said method comprising administering to said mammal an effective amount of an agent identified in accordance with the screening method of the present invention.
  • said genetically modified cells are preferably endothelial cells, more preferably sinusoidal endothelial cells, still more preferably liver sinusoidal endothelial cells and most preferably SK Hep-1 cells.
  • said cell is transfected with one or more markers selected from:
  • - caveolin or functional derivative, homologue or variant thereof Ca ATPase or functional derivative, homologue or variant thereof; endothelin-1 or functional derivative, homologue or variant thereof; - dynamin or functional derivative, homologue or variant thereof; actin or functional derivative, homologue or variant thereof; filamen or functional derivative, homologue or variant thereof; NOS or functional derivative, homologue or variant thereof; or serotonin receptor or functional derivative, homologue or variant thereof;
  • said detection tag is a fluorescent protein, epitope or enzymatic substrate.
  • said fluorescent protein is GFP, YFP, CFP, BFP, EmGFR or RFP.
  • said detectable marker of fenestration is GFP labelled caveolin or GFP labelled actin.
  • said genetically modified cell is SK Hep-1 transfected with pAcGFPI-actin or pEGFP-caveolin-1.
  • said genetically modified cell corresponds to the cells deposited at ECACC on 27 July 2007 under Accession No. 07072602 or Accession No. 07072603.
  • Liver sinusoidal endothelial cells are very thin, lack any basal lamina or supportive connective tissue, and are perforated with pores called fenestrations (Figure 1).
  • LSECs occupy a critical and strategic position in the hepatic sinusoid because they facilitate the bidirectional transfer of substrates between blood and hepatocytes.
  • the extreme vascularity of the liver also generates a huge surface area for LSEC interactions with blood cells and particulate substrates for endocytosis (16).
  • Isolated LSECs are only viable for about 24 hours and there is a dramatic change in morphology, particularly of the fenestrations during this period (Figure 3) (Gatmaitan et al. 1993 supra).
  • Reagents included Ml 99 and G418 culture medium (Gibco, Invitrogen, Australia), fetal calf serum, human recombinant vascular endothelial growth factor (VEGF) 165 (Calbiochem (La Jolla, CA), pAcGFPl-actin and EGFP-caveolin-1 ( ⁇ Biosciences, Ryde, Australia), lipofectamine 2000 (Invitrogen, Mt. Waverley, Australia) hexamethyl- disilazane (Sigma, St Louis, MO), antibodies to VEGF (Abeam, Cambridge, UK) and caveolin-1 (Santa Cruz Biotechnology, Santa Cruz, CA).
  • VEGF vascular endothelial growth factor
  • SK Hep-1 cells were obtained from the American Type Tissue Culture Collection (ATCC, Manassas, VA) and were cultured in a humidified 5% CO2 incubator at 37 0 C. Cells were grown in Ml 99 supplemented with 10 % fetal calf serum and antibiotics. Cells were plated in flasks coated with collagen IV. In some experiments VEGF 165 was added at a concentration of 40ng/mL and incubated with the cells for 24 hours. For comparison, LSECs were isolated from male Fischer F344 rats as described previously (Cogger et al. 2004, supra; Cogger et al. 2006, Atherosclerosis 2006;189:273-281) and fixed for electron microscopy 1 hr after isolation.
  • SK Hep 1 cells were plated on collagen coated 12-well dishes. One day prior to transfection, when cells were approximately 75 % confluent, complete culture medium was replaced with M 199 supplemented with 10% fetal calf serum without antibiotics. Transfection of pAcGFPl-actin and Pegfp-caveolin- 1 was carried out using lipofectamine 2000 as per the manufacturers instructions. Stably expressing lines were created by passaging cells into fresh growth medium 24 hours after transfection. G418 supplemented media (750 mg /mL) was added after 24 hours to select for vector expressing cells.
  • Sequence images were exported as single TIFF files. Quantitation of mean fluorescence intensity in selected regions of interest was performed using NIH Image 1.62. Quicktime movies were produced using NIH Image 1.62 or OpenLab 2.0. Scanning electron microscopy (EM)
  • Cells were grown on thermanox coverslips coated with collagen IV. Once cells were 90% confluent, they were fixed with 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer with 1% sucrose. Cells on coverslips were osmicated (1% OsO 4 /0.1 mol/L sodium cacodylate buffer), dehydrated in an ethanol gradient to 100% and incubated for 2 minutes in hexamethyl-disilazane. Coverslips were then mounted on stubs, sputter coated with platinum, and examined using a Jeol 6380 (Tokyo) scanning electron microscope.
  • Jeol 6380 Jeol 6380
  • Formaldehyde-treated serum albumin (FSA) labelled with fluorescein isothiocyanate (FITC) (6mg/ml, 0.1ml) was kindly provided by Professor Bard Smedsr ⁇ d (University of Tromso, Norway). This was added to SK Hep-1 cells and incubated at 37 0 C. After 1 hour cells were washed with PBS and fixed with 4 % paraformaldehyde. Cells were examined using a Zeiss inverted confocal microscope.
  • Cellular VEGF and caveolin-1 was quantified with Western Blots.
  • Cell lysate was mixed with sample buffer containing 10 Mm dithiothreitol, heated to 100°C for 5 minutes, and separated by SDS-PAGE. After transfer onto nitrocellulose membrane (Amersham Biosciences, Australia) the blot was blocked, incubated with primary antibodies to VEGF and caveolin-1, washed and incubated with a rabbit anti-goat IgG secondary antibody conjugated to horseradish peroxidase. Proteins were visualized using chemiluminescence, quantified with a BioDocAnalyze system (Biometra, Gottingen, Germany) and expressed as arbitrary units (AU) normalized to cell protein.
  • BioDocAnalyze system Biometra, Gottingen, Germany
  • Immunogold labelling was performed to localize the distribution of caveolin-1 in SK Hep- 1 cells.
  • Cells were centrifuged, fixed, embedded in 12% gelatin, cut into blocks and infiltrated with 2.3M sucrose. Cryosections were obtained and immunogold labelling was performed on ultra-thin sections collected on carbonized formwar-coated 200 mesh Ni grids. Grids were incubated for 10 minutes with 1% cold fish skin gelatin in PBS and then with primary antibody caveolin-1 over-night at 4 0 C. Grids were incubated with protein-A gold (IOnm) for 15 minutes. Grids were washed as above and contrasted with 1% uranyl acetate in methylcellulose and viewed with a JEOL JEM-1010 transmission electron microscope.
  • SK Hep-1 cells grew rapidly to confluence and were very robust in standard Ml 99 medium.
  • SK Hep-1 cells respond to VEGF
  • SK Hep-1 cells express VEGF and caveolin-1
  • SK Hep-1 cells were found to express both VEGF and caveolin-1 on Western blotting (Figure 9A). Immunogold and transmission em revealed staining for caveolin-1 in transected fenestrations but not elsewhere ( Figure 8).
  • SK Hep-1 cells take up FITC-FSA
  • FIG. 10 shows SK Hep-1 cells containing FITC-FSA after 1 hr incubation. There is uptake of FITC-FSA into the SK Hep-1 cells and this result is consistent with the presence of stabilin receptors and active endocytosis.
  • Figure 11 shows the GFP-actin and GFP-caveolin-1 appearance in control cells, and the response to incubation with VEGF (40 ng/niL. Actin was distributed throughout the cell whereas the caveolin-l had a punctate appearance consistent with fenestrations. Following incubation with VEGF, F-actin constricted to the perinuclear area then gradually redistributed into the cytoplasm over the next 12 hours.
  • VEGF 40 ng/niL. Actin was distributed throughout the cell whereas the caveolin-l had a punctate appearance consistent with fenestrations.
  • F-actin constricted to the perinuclear area then gradually redistributed into the cytoplasm over the next 12 hours.
  • SK Hep-1 cells (with either GFP-caveolin-1 or GFP-actin) are grown in Ml 99 (Gibco) supplemented with 5% Fetal Calf Serum (Gibco), glutamine and antibiotics.
  • Cells are plated at a density of 106 per well (6 well Biocoat plate BD Biosciences) in 2ml of medium and left to adhere for 24 hours prior to experimentation.
  • HCS are performed using the Evotec OperaTM Quadruple Excitation High Sensitivity Confocal Cell Imager in the laboratory of AI Avery. Optimisation of conditions required for screening, such as cell number, culture conditions, screening dose, staining, and imaging conditions is undertaken.
  • the NP_ LLP is tested in 384 well plates at various time points against cells containing the GFP-caveolin 1 construct and retested against both cells transfected with GFP-calveolin 1 and GFP-actin. EC 50 values is determined for all confirmed active samples. A propidium iodide stain is used to identify cytotoxic compounds resulting in necrosis. Image acquisition and cytometric analysis
  • Data for a minimum of 200 cells per well is collected using the Evotec OperaTM according to the optimised exposure protocol.
  • Data stored as image files undergo analysis using AcapellaTM Image Analysis software. Parameters such as number of positive cells, number of fluorescent spots, fluorescence intensity, distribution and size is determined using optimised algorithms for each specific parameter.
  • the bioactive component(s) Since hits identified during NPJLPP screening are chromatography peaks that may contain more than one compound, the bioactive component(s) must be isolated and fully characterized. Initially, the hits are analyzed by MS to determine the MW of the bioactive component(s). The biota samples corresponding to the hit is then subjected to MS-guided fractionation using preparative HPLC systems equipped with fraction collectors and linked to mass spectrometers.
  • LSECS VEGF, CYTOCHALASIN, PHALLOIDIN, LATRUNCULIN, ENDOTHELIN, SEROTONIN, CALCIUM IONOPHORES, CALCIUM CHANNEL
  • SK Hep-1 cells are grown in Ml 99 (Gibco) supplemented with 5% Fetal Calf Serum (Gibco), glutamine and antibiotics.
  • Cells are plated at a density of 106 per well (6 well Biocoat plate BD Biosciences) in 2ml of medium and left to adhere for 24 hours prior to experimentation.
  • Agents reported to influence fenestrations previously are added initially according to the following published conditions: VEGF I 5 10, 100 ng/ml (Esser et al. 1998, Journal of Cell Biology 140:947-59); cytochalasin 1, 10, 50 ⁇ g/ml (Braet et al.
  • the fixative is 2.5% EM Grade glutaraldehyde (ProSciTech) in 0.1M Na-cacodylate buffer (pH 7.4) and 1% sucrose. After fixation for Ih, cells are incubated in 1% tannic acid in 0.15M Na-cacodylate buffer for lhr followed by 1% OsO4 in 0.1M Na-cacodylate buffer, dehydrated with ethanol then hexamethyldisilazane and coated with platinum.
  • LSECs are isolated using collagenase A and elutriation with a 2-step Percoll gradient. Purity is enhanced by selective adherence of Kupffer cells. LSECs are cultivated in 24 multiwell plates on Biocoat cover slips in serum -free culture RPMI- 1640 medium. Fenestrations in isolated LSECs are quantified using scanning electron microscopy. These studies determine the effects of hits established using fluorescence in SK Hep-1 cells on the fenestral porosity in LSECs determined with electron microscopy.
  • Gatmaitan Z, Arias IM Hepatic endothelial cell fenestrae. In: Knook DL, Wisse E, eds. Cells of the Hepatic Sinusoid. Volume 4. Leiden: Kupffer Cell Foundation, 1993; 3-7.
  • TLR9 Toll-like receptor 9 is present in murine liver sinusoidal endothelial cells (LSECs) and mediates the effect of CpG-oligonucleotides. J Hepatol 2006,44:939-946.

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Abstract

La présente invention concerne d'une manière générale une population de cellules génétiquement modifiées pour exprimer des marqueurs de fenestration détectables, ainsi que les utilisations de ces cellules. Les cellules de la présente invention sont utilisables dans une grande diversité d'applications, en particulier dans le contexte de systèmes de criblage in vitro permettant de tester l'efficacité et/ou la toxicité de schémas posologiques thérapeutiques ou prophylactiques potentiels.
PCT/AU2007/001066 2006-07-31 2007-07-31 Cellules génétiquement modifiées pour exprimer des marqueurs de fenestration et leurs utilisations WO2008014547A1 (fr)

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CN112195154A (zh) * 2019-07-08 2021-01-08 江苏汇智生物科技有限公司 一种基因改造表达改造型pla2r受体的细胞及其应用

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US20050064522A1 (en) * 1998-08-11 2005-03-24 Genentech, Inc. EG-VEGF nucleic acids and polypeptides and methods of use
WO2006055488A2 (fr) * 2004-11-15 2006-05-26 (Osi) Eyetech, Inc. Marqueur de fenetres sous-endotheliales

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
US20050064522A1 (en) * 1998-08-11 2005-03-24 Genentech, Inc. EG-VEGF nucleic acids and polypeptides and methods of use
WO2006055488A2 (fr) * 2004-11-15 2006-05-26 (Osi) Eyetech, Inc. Marqueur de fenetres sous-endotheliales

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ESSER ET AL.: "Vascular Endothelial Growth Factor Induces Endothelial Fenestrations In Vitro", JOURNAL OF CELL BIOLOGY, vol. 140, 1998, pages 947 - 959, XP009142423, DOI: doi:10.1083/jcb.140.4.947 *
ROBERTS ET AL.: "Increased Microvascular Permeability and Endothelial Fenestration Induced by Vascular Endothelial Growth Factor", JOURNAL OF CELL SCIENCES, vol. 108, 1995, pages 2369 - 2379, XP002917690 *
STAN ET AL.: "PV1 Is a Key Structural Component for the Formation of the Stomatal and Fenestral Diaphragms", MOLECULAR BIOLOGY OF THE CELL, vol. 15, 2004, pages 3615 - 3630, XP002378015, DOI: doi:10.1091/mbc.E03-08-0593 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112195154A (zh) * 2019-07-08 2021-01-08 江苏汇智生物科技有限公司 一种基因改造表达改造型pla2r受体的细胞及其应用
CN112195154B (zh) * 2019-07-08 2024-03-29 江苏汇智生物科技有限公司 一种基因改造表达改造型pla2r受体的细胞及其应用

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