WO2008014547A1

WO2008014547A1 - Cells genetically modified to express markers of fenestration and uses thereof

Info

Publication number: WO2008014547A1
Application number: PCT/AU2007/001066
Authority: WO
Inventors: David Le Couteur; Victoria Cogger
Original assignee: David Le Couteur; Victoria Cogger
Priority date: 2006-07-31
Filing date: 2007-07-31
Publication date: 2008-02-07

Abstract

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.

Description

CELLS GENETICALLY MODIFIED TO EXPRESS MARKERS OF FENESTRATION AND USES THEREOF

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Liver sinusoidal endothelial cells (LSECs) 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. First, 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. Next, 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). Connective tissue macromolecules are almost exclusively cleared by LSECs and a broad range of other substrates is also endocytosed (Smedsrod et al. 1994, supra; Smedsrod Comp Hepatol 2004;3 Suppl 1:S22; Seternes et al. Proc Natl Acad Sci USA 2002;99:7594-7597; Martin-Armas et al. J Hepatol 2006;44:939-946). Finally, the LSEC has a role in the hepatic immune function through the expression of numerous immune antigens (Limmer et ah, Nat Med 2QQ0;β:\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.

Concordant with the important physiological role of the LSEC and its fenestrations has come recognition of changes in LSECs in various pathological conditions. 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. Alcohol & Alcoholism 1993;28:77-84; Schaffner and Popper, Gastroenterology 1963;44:239-242). 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. Journal ofHepatology 2004;41 :370-376) and acetaminophen (McCuskey, CHn Hemorheol Microcirc 2006;34:5-10) which tend to cause gap formation in LSECs and hepatitis which is associated with defenestration (Warren et al J Hepatol 2007; 46:239-246).

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.

Accordingly, 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. However the yield typically only permits 6-12 wells per liver and is highly dependent on the methodology with some methods failing to generate well fenestrated cells (DeLeve et al Am J Physiol Gastrointest Liver Physiol 2006;291 :G1187-1189). Isolated LSECs are only viable for about 24 hours and there is a dramatic change in morphology, particularly of the fenestrations during this period (Gatmaitan et al. In:

Knook DL, Wisse E, eds. Cells of the Hepatic Sinusoid. Volume 4. Leiden: Kupffer Cell Foundation, 1993; 3-7; Martinez et al. (submitted) 2007). To overcome these problems a method to isolate LSECs from pig livers has been developed but has obvious problems related to availability for most laboratories (Gerlach et al. J Surg Res 2001;100:39-45). An immortalized LSEC cell line, Ml has been reported but is not fenestrated unless co- cultured with other cells and treated with actin disruptors (Saito et al. Comp Hepatol 2004;3 Suppl 1 :S28). The identification of a fenestrated immortal LSEC cell line is thus highly desirable and would transform the study of LSEC and fenestration biology.

In work leading up to the invention, it has been determined that the liver tumour cell line SK Hep-1 is, in fact, fenestrated, these fenestrations being modulatable by appropriate extracellular stimulation. Still further, 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.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

As used herein, 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.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs .

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.

In another aspect there is provided 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.

In yet another aspect there is provided 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.

In still another aspect there is provided a genetically modified mammalian 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.

In a further aspect of the present invention there is provided 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 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.

In another aspect 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.

In another aspect 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.

In yet another aspect of the present invention there is provided a genetically modified 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. In still yet another aspect of the present invention there is provided 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;

■ - 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.

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.

In another aspect there is provided 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.

In another aspect there is provided 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.

In still another aspect, there is provided 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.

In yet still another aspect there is provided a method of identifying a stimulus which modulates cellular fenestrations, said method 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. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an image of A: vascular cast showing extensive sinusoidal network. B: TEM showing fenestrations (f) perforating the endothelium. C: SEM showing fenestrations grouped into liver sieve plates.

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). Some SK Hep-1 cells had long cytoplasmic extensions (A₅E). Bar = lμM.

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. C: there is an increase in porosity of the fenestrations (P=0.002). Bar = lμM. Figure 8 is an image of Western blots for caveolin-1 and actin in SK Hep-1 cells (A) and immunogold staining for caveolin-1, showing staining in transacted fenestrations (—>). Bar = 200 nm.

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 (B₅E) and 12 hrs (C₅F) 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.

DETAILED DESCRIPTION OF THE INVENTION

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.

Accordingly, 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.

Reference to 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. In one embodiment 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. Without limiting the present invention to any one theory or mode of action 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. It should also be understood that 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. Accordingly, the subject cell may be a primary cell or a secondary cell. A primary cell is one which has been isolated from an individual. A secondary cell is one which, following its isolation, has undergone some form of in vitro manipulation prior to the genetic manipulation of the present invention.

In one embodiment, said fenestrated cell is an endothelial cell. Without limiting the present invention to any one theory or mode of action, 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. In another embodiment, said endothelial cell is a sinusoidal endothelial cell and preferably a liver sinusoidal endothelial cell. As detailed hereinbefore, 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. Accordingly, 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.

According to this embodiment there is provided 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. Reference to "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. The phrase "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.

In yet another embodiment there is provided 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.

Preferably, said sinusoidal endothelial cell is a liver sinusoidal endothelial cell.

Reference to a "liver" sinusoidal endothelial cell should be understood as a reference to a sinusoidal endothelial cell which is associated with any part of the liver.

There is therefore provided, according to this embodiment, a genetically modified mammalian 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.

Preferably, 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.

Reference to "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). Preferably the mammal is a human or primate. Most preferably the mammal is a human.

Without limiting the present invention in any way, 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. These features include numerous pinocytotic vesicles, electron dense granules consistent with Weibel- Palade bodies, and abundant intermediate filaments, identified immunocytochemically as vimentin. Cultures grown on plastic dishes grow in bundles of polygonal to spindle- shaped cells. Proteins characteristic for endothelial cells have been identified by immunocytochemistry. Addition of basement membrane material (Matrigel) or type I collagen to the cultures induces these cells to organise into a tubular network.

As detailed hereinbefore, in addition to the unexpected determination that a cell line thought to be of hepatocyte origin is in fact a fenestrated liver sinusoidal endothelial cell, it has still further been determined that the fenestrations are constitutively expressed yet nevertheless respond to cytokine manipulation. This renders this cell line an ideal model of liver sinusoidal fenestration functionality. As would be known to the person of skill in the art, 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. However, a limitation in this regard has been that the visualisation of endothelial fenestrations, for example in order to assess changes to their number and/or structure, has only been possible via electron microscopy. Nevertheless a visualisation technique has now been developed based on genetically modifying these cells to express a detectable marker of fenestration. This has enabled a range of features of the cellular fenestrations, such as their number, distribution and diameter, to be assessed significantly more easily by means which are based on screening for the detectable marker.

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. The person of skill in the art would be familiar with the well known structural biology of fenestrations and could therefore determine molecules appropriate for use as markers herein. Preferably, said marker is a protein. Examples of 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. Examples of 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. In terms of the indirect markers, 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. Reference to said "marker", 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. By "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. For example, 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. By "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.

It should be understood by the person of skill in the art that 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. For instance, where the markers selected for use is one which is directly associated with fenestrations, such as caveolin, it is feasible that 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. This is therefore indicative of changes to fenestration patterns and may form the basis of justifying further analyses, such as analysis with additional markers or electron microscopy. Alternatively, visualisation of more than one marker may be pursued, either as double staining or via single staining of two separate cellular populations which have each been genetically modified in the context of a single specific marker. For example, one may seek to analyse both actin and caveolin since these results would provide clear visualisation of the co- localisation of the sieve plate structure with the fenestration. It would be within the skill of the person in the art to determine an appropriate marker or combination of markers which are suitable as the focus of the genetic modification herein described.

In one embodiment of the present invention there is provided 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 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.

In another embodiment 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.

In another embodiment 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.

As detailed earlier, to the extent that it is desired to screen for fenestrations, one may use a cellular population which expresses a single detectable marker of fenestration and, to this end, may choose to screen across more than one cellular population, where each cellular population expresses a different detectable marker of fenestration. Alternatively, one may screen a single cellular population which has been genetically modified to express two or more detectable markers of fenestration. Reference to "two or more" should be understood as a reference to two, three, four, five, six or more. Preferably, said two or more detectable markers of fenestration is two or three.

Accordingly, in yet another embodiment of the present invention there is provided a genetically modified 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.

In the context of the cells of the present invention, 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. In terms of 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. For example, naturally fluorescent proteins, such as green fluorescent protein (GFP/EGFP), are suitable for expression together with the marker of the present invention. 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. Again, 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). For example, 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.

Accordingly, in yet another aspect of the present invention there is provided 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.

Preferably, said endothelial cell is a sinusoidal endothelial cell, more preferably a liver sinusoidal endothelial cell and still more preferably SK Hep-1.

Accordingly, 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; 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. In one embodiment, said detectable marker is GFP labelled caveolin.

In another embodiment, said detectable marker is GFP labelled actin.

As detailed hereinbefore, the cells of the present invention are genetically modified. By "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:

(i) The introduction of homologous or heterologous nucleic acid material to the cell.

For example, 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. To this end, the cell is preferably permanently transfected with cDNA or genomic DNA encoding the detectable marker of fenestration. However, 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.

In addition to the modification of the cells of the present invention to produce proteins which are directly relevant to fenestration analysis, other genes relevant to optimising generation of the subject cells, and which may also be introduced, 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.

(ii) The modulation of expression of a gene, for example by inducing upregulation of expression of a gene which would otherwise not be expressed, or the mutation of endogenous DNA, for example to downregulate or render non-functional an unwanted gene, such as the endogenous marker gene.

Reference to a "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. To this end, the term "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.

The term "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.

It would be appreciated by the person of skill in the art that the mechanism by which these genetic modifications are introduced may take any suitable form which would be well known and understood by those of skill in the art. For example, genetic material is generally conveniently introduced to cells via the use of an expression construct. Alternatively, one may seek to use, as the starting cellular population, 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 (ie. transfection) are also well known in the art.

Briefly, 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). For example, non- viral vectors may be used which include liposomes coated with DNA. Such liposome/DNA complexes may be directly injected intravenously into the patient. Additionally, 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.

Many transfection methodologies employ 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.

Mechanical methods of 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.

Most preferably, 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. To the extent that one may seek to co-express three or more detectable markers of fenestration, 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.

Reference to a 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. In general, 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. To this end, 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:

(i) Plasmids

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.

(ii) Bacteriophage

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).

(iii) Baculovirus

These are any of a group of DNA viruses which multiply only in invertebrates and are generally classified in the family Baculoviridae. Their genome consists of double-stranded circular DNA.

(iv) Artificial Chromosomes

Artificial chromosomes such as yeast artificial chromosomes or bacterial artificial chromosomes.

(v) 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.

(vi) Commercially available vectors which are themselves entirely synthetically generated or are modified versions of naturally occurring vectors, such as the pIRESpuro3 bicistronic vector.

It would be understood by the person of skill in the art that the selection of 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. In still yet another example, the larger the DNA insert which is required to be inserted, the more limited the choice of vector from which the expression construct of the present invention is generated. To this end, 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. In this context, a "circular" nucleotide sequence should be understood as a reference to the circular nucleotide sequence portion of any nucleotide molecule. For example, 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). In this context, the "circular" nucleotide sequence corresponds to the circular portion of this molecule. A ^* "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. For example, 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. However, in another example, 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. In the alternative, 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. In the context of the vector exemplified herein, 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. However, 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.

It should be understood that the constructs of the present invention may comprise nucleic acid material from more than one source. For example, whereas 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. These sources may include, for example, viral DNA (e.g. IRES DNA), mammalian DNA or synthetic DNA (e.g. to introduce specific restriction endonuclease sites). Still further, 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. For example, a construct consisting of essentially bacterial and viral derived DNA may nevertheless be expressed in the mammalian stem cells contemplated herein.

Without limiting the present invention to any one theory or mode of action, 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. With respect to the GFP-actin 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 (Neo¹), 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.

Preferably, said vector is p AcGFPl -actin.

Accordingly, in another embodiment there is provided 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.

In another embodiment there is provided 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.

Preferably, said vector is pEGFP-caveolin-1.

Still more preferably, said endothelial cell is a sinusoidal cell and even more preferably a liver sinusoidal endothelial cell. Most preferably, said cell is SK Hep-1. Most preferably, said genetically modified cell corresponds to the cells deposited at ECACC on 27 July 2007 under Accession No. 07072602.

In another preferred embodiment, said genetically modified cell corresponds to the cells deposited at ECACC on 27 July 2007 under Accession No. 07072603.

As would be appreciated by the person of skill in the art, 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:

(i) Detection of specific cellular proteins.

Detection of specific proteins, such as a GFP -marker fusion protein may be conveniently effected via fluorescence microscopy, for example. In this regard, 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.

(H) Detection of specific cellular RNA or DNA.

This method is preferably effected using RT-PCR or real-time (qRT-PCR). Alternatively, other methods, which can be used include hybridization microarray ("RNA chip") or Northern blotting or 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.

Although the analysis of a cell population in terms of its functioning is generally regarded as a less convenient method than the screening methods of points (i)-(ϋ), in some instances this may not be the case. For example, to the extent that one is seeking to establish the existence of a modulatable fenestration, analysis of transfected cells after stimulation with VEGF may be pursued.

(iv) Other means of screening for the stable integration and maintenance of the modification (for example in the context of cell line generation and therefore long term cellular culturing) may be performed and include screening for the expression of a selection marker, which provides a most convenient means for establishing the integration of a genetic modification.

It should be understood that in terms of characterising the population of cells generated in the context of the present invention, any one or more of the techniques detailed above may be utilised.

The development of the cells of the present invention has now facilitated the development of in vitro based screening systems for testing or otherwise analysing molecules in terms of their functional effect on fenestration formation, maintenance, downregulation, contraction and dilation. As discussed hereinbefore, 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. In the past twenty years, numerous publications have appeared which discuss the role of these dynamic structures under various physiological and pathological situations. (Braet & Wisse 2002, Comparative Hepatology 1 :1). Their role and involvement in processes such as lipoprotein metabolism, hypoxia, endotoxic shock, virus infection, cirrhosis, fibrosis and liver cancer is well known.

In light of the crucial role of fenestrations in this regard, it is of great importance to identify drugs or other molecules which could modulate the number or diameter of fenestrations in order to treat a wide variety of disease conditions associated with fenestration-related liver function. To date, however, the only mechanism of achieving this was to treat freshly harvested fenestrated endothelial cells with a molecule of interest and to thereafter perform electron microscopy in order to determine whether there has occurred any structural change to the fenestrations of the subject cell. As previously mentioned, fenestrations are too small to be visualised by light microscopy. This has been a cumbersome and costly method of conducting fenestration related research and has essentially prohibited the type of large scale high throughput screening which is required in a drug discovery program.

The identification and generation of the cells of the present invention, however, has changed the landscape in this regard in that a method of routinely and simply performing high throughput screening has now been facilitated. Specifically, 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. To this end, depending on the particular detectable marker with which the cells of the invention are transfected and, further, how many different markers are assessed in the context of a given test, it may or may not be necessary to perform additional analyses such as electron microscopy or functional tests. For example, where one marker but not another exhibits change (such as actin by not caveolin or vice versa), the fact that the expression pattern of a least one marker has changed is indicative of the induction of a change to the fenestrations, hi this case, further analyses to conclusively determine this may be justified. However, where two or more direct and highly specific markers of fenestration demonstrate altered staining patterns which clearly show a consistent change to the pattern of fenestration rings, no further testing may elect to be sought due to the strongly indicative nature of these results, although electron microscopy would confirm that the fenestration pores which have been identified via topographical screening are, in fact, fully formed. Still further, where the subject visualisation screen has indicated a change in fenestration pore diameter, further electron microscopy screening may enable highly accurate measurement of the precise degree of change to that diameter. In addition to electron microscopy analysis in terms of additional analyses, one may elect to proceed with in vitro or annual model functional analysis of the molecules of interest.

Accordingly, 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.

Reference to "screening for the detectable marker of fenestration" should be understood as a reference to screening for the detection tag associated with the marker of fenestration. The type of screening step which is performed will depend on the nature of the detection tag which has been used. Still further, a technique which provides topographical analysis may be particularly useful in the context of some screening scenarios. In one embodiment, the detection tag is a fluorescent protein. Such a tag 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. This enables one to assess not just the overall upregulation or downregulation of fenestration numbers but, further, the cellular distribution of the fenestrations and changes to their diameter. To the extent that topographical analysis is not required, it may be sufficient to analyse relative fluorescence intensity via a technique such as FACS. It would also be appreciated that to the extent that the detection tag is not an autofiuorescent protein, it may be necessary to treat the cells prior to visualisation in order to enable visualisation/detection of the tag. For example, to the extent that a tetracysteine tag detection system is used, a green or red fluorescent reagent is required to be used in order to visualise, fluorescently, the localisation of the tag. In yet another example, a detection tag such as an enzyme substrate (eg. alkaline phosphatase) requires enzymatic treatment in order to effect visualisation. To the extent that 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. In terms of visualising such a complex, the subject immunointeractive or other molecule may itself be appropriately labelled to facilitate the application of fluorescent, enzymatic or other suitable visualisation means.

In terms of screening for the detection tag, and as detailed hereinbefore, since the method of the present invention is predicated on the incorporation into newly formed fenestrations of the detectable marker of fenestration, 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. In another example, 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. However, in some situations it may be sufficient merely to analyse the test sample in isolation. In yet another example, antagonists of specific fenestration modulators, such as VEGF which activates fenestration formation, may be screened for by conducting assays based on competitive inhibition. In terms of the application of the screening method of this aspect of the present invention, the identification both of molecules which upregulate and molecules which downregulate fenestration formation, distribution or diameter is of importance. For example, although in the context of aging one may be seeking molecules which either upregulate or at least increase the diameter of existing fenestrations, in order to improve liver functionality (either at the metabolic or immunological level), for patients with hepatic cancer, it may be desirable to reduce fenestration number of diameter.

Reference to a "stimulus" should be understood as a reference to any type of stimulus to which one may seek to expose a fenestrated cell. 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.

The agents which are utilised in accordance with the method of the present invention may take any suitable form. For example, 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. Similarly, 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. For example, in one embodiment of the present invention, 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. Accordingly, 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. For example 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.

For example, 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.

There is currently widespread interest in using combinational libraries of random organic molecules to search for biologically active compounds (see for example U.S. Patent No. 5,763,263). Ligands discovered by screening libraries of this type may be useful in mimicking or blocking natural ligands or interfering with the naturally occurring ligands of a biological target. These ligands may be used in combination libraries formed by various solid-phase or solution-phase synthetic methods (see for example U.S. Patent No. 5,763,263 and references cited therein). By use of techniques, such as that disclosed in U.S. Patent No. 5,753,187, millions of new chemical and/or biological compounds may be routinely screened in less than a few weeks. Of the large number of compounds identified, only those exhibiting appropriate biological activity are further analysed.

With respect to high throughput library screening methods, 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. In such a method, each member of the library is screened for its ability to interact specifically with the selected agent. In practising the method, 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. Preferably, 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.

In one embodiment, 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. However, 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. In yet another example, one may seek to observe the effect of subjecting the subject cells to an electric current or sonication.

Hence, the method of the present invention can be used to screen and/or test drugs or other treatment regimes, such as electrical treatments. Preferably, the treatment to which the cells or tissues of the present invention are subjected is an exposure to a compound. Preferably, the compound is a drug or a physiological ion. Alternatively, 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. In a preferred embodiment, 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.

Preferably, 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. In this case, if it is evident that some form of change has occurred, one may elect to either functionally test the fenestration related properties of the cell or else one may employ electron microscopy to further analyse the fenestrations of the cell, such as to determine whether a newly formed fenestration has actually formed a complete pore. Similarly, detailed analysis of alteration to pore diameter may be followed up with electron microscopy in order to more accurately determine the extent of this change.

Accordingly, in an embodiment of the present invention, 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.

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.

In accordance with these aspects 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.

In yet another embodiment, 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;

and said detection tag is a fluorescent protein, epitope or enzymatic substrate.

More preferably, said fluorescent protein is GFP, YFP, CFP, BFP, EmGFR or RFP.

Yet more preferably, said detectable marker of fenestration is GFP labelled caveolin or GFP labelled actin.

Still more preferably said genetically modified cell is SK Hep-1 transfected with pAcGFPI-actin or pEGFP-caveolin-1.

Most preferably, said genetically modified cell corresponds to the cells deposited at ECACC on 27 July 2007 under Accession No. 07072602 or Accession No. 07072603.

The present invention is further described by reference to the following non-limiting examples.

EXAMPLE 1 THE STRUCTURE AND FUNCTION OF THE LSEC

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). These features underpin many important physiological roles; such as: filtering lipoproteins via fenestrations, endocytosis and aging.

The presence of dramatic age-related changes in the morphology of the LSEC. The presence of dramatic age-related changes in the morphology of the LSEC has been reported in rats and subsequently similar changes in old mice, the non-human primate papio hamadryas and humans has been found (Le Couteur et al. 2001 supra; 33:537-543; Cogger et al. Experimental Gerontology 2003;38:l 101-1107; McLean et al. Journal of Pathology 2003;200:l 12-117; Warren et al. 2005, supra) (Figure 2). Specifically there is a 30-50% increase in the thickness and 30-50% reduction in the porosity of the LSEC in old age. The reduction of porosity is mediated by both a reduction in fenestral frequency and diameter, and is associated with a loss of sieve plate structure. This is called

"pseudocapillarization". In addition to these ultrastructural changes, there was an increase in basal lamina formation and extravascular collagen deposition. There were also variable increases in the immunohistochemical expression of various vascular antigens not usually seen in young healthy livers such as von Willebrands factor, laminin and collagens (especially collagen IV). Perisinusoidal staining with trichrome Masson, Sirius red and reticulin stains were more prominent in old livers. It was found that caloric restriction is associated with reversal of these ageing changes thus indicating it is a fundamental ageing change and preventable (Jamieson et al. Experimental Gerontology 2007 (in press)).

This age-related reduction in fenestrations impedes the passage of lipoproteins from the sinusoid to the hepatocyte for receptor mediated uptake and subsequent hepatic metabolism. The impulse response technique in perfused livers of rats aged 6 and 24 months was used. The volume of distribution of lipoproteins (diameter 56 nm) was 102 ± 14% of the extracellular volume in young rats, indicating that these lipoproteins cross the LSEC unimpeded. However in the old rats the lipoproteins were restricted to the vascular space (92 ± 7% of the extracellular volume, P<0.02) proving that the defenestrated LSEC in old age prevents the passage of lipoproteins (Hilmer 2005, supra).

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).

EXAMPLE 2

IDENTIFICATION OF SK HEP-I CELLS AS FENESTRATED LIVER SINUSOIDAL ENDOTHELIAL CELLS

Experimental procedures

Reagents

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 (βπ 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).

Cell Culture

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 37⁰C. 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.

Transfection with AcGFP 1-Actin and Pegfp-caveolin 1

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.

Confocal Microscopy

Cells plated in coverslip chambers were maintained at 37°C and covered with mineral oil. Quantitative live cell image analysis was performed using a Zeiss LSM 510 META (Carl Zeiss, Thornwood, NY) inverted confocal microscope with a 63 x oil immersion objective, NA 1.32 or 1.40. Live cells were held at 37⁰C by an ASI 400 air stream incubator (Nevtek, Burnsville, VA). Images were captured at 900 second intervals over 14 hours.

Image Analysis

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% OsO₄/0.1 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.

In order to visualise the cytoskeleton, scanning em was also performed on digests of SK Hep-1 cells. To digest cell membranes, cells were incubated for 1 hour at room temperature in 100 mmol/L Pipes buffer (pH 6.9) containing 1 mmol/L EGTA, 4% polyethylene glycol 6000 and 0.1% triton X. Cells were then washed, fixed and prepared as above.

Endocytosis of FITC-FSA

Formaldehyde-treated serum albumin (FSA) labelled with fluorescein isothiocyanate (FITC) (6mg/ml, 0.1ml) was kindly provided by Professor Bard Smedsrød (University of Tromso, Norway). This was added to SK Hep-1 cells and incubated at 37⁰C. 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

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.

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 4⁰C. 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.

Statistics

All cell studies were performed at least in triplicate. Results are presented as mean ± standard deviation. Comparisons between two groups were performed using the Students T test and considered significant when P < 0.05.

Results

Cell culture characteristics

SK Hep-1 cells grew rapidly to confluence and were very robust in standard Ml 99 medium.

SK Hep-1 cells are fenestrated

On scanning em, SK Hep-1 cells were fenestrated (Figure 4). The average diameter of the fenestrations was 55+28 and the overall porosity was 2.0+1.4% (n= 453 fenestrations). The frequency distribution of the diameter of the fenestrations is shown in Figure 5. The fenestrations were scattered over the cells and were not clustered in typical liver sieve plates that are seen in native LSECs. However there was a tendency for the fenestrations to be clustered into parallel groups of linearly orientated fenestrations that are reminiscent of sieve plates. Long filaments were seen extending from the cytoplasm in many cells (Figure 4E). (For comparison, electron microscopy is shown from LSECs isolated from a rat liver where fenestrations are larger than those seen in SK Hep-1 cells (Figure 4F). The actin cytoskeleton formed a pattern seen in LSECs with circular structures that presumptively support these sieve plates (Figure 6).

SK Hep-1 cells respond to VEGF

VEGF increased the porosity and diameter of SK Hep-1 cells (Figure 7). After incubation with VEGF, the porosity of the SK Hep-1 cells increased 4.8+2.6% = 0.002 and the diameter of the fenestrations increased to 104+59 nm (P<0.001, n=921 fenestrations). The change in the cytoskeleton is shown in Figure 3B where there is marked rare faction of the cytoskeletal filaments.

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

Figure 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. SKHep-1 cells transfected with GFP-actin and GFP-caveolin-1

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 caused an initial decrease in caveolin-l intensity with contraction to the perinuclear region over the first hour and then the number of dots increased over the next 12 hours from 560+222 to 1605+963 dots per cell (n=5 cells in each group, P<0.05), presumptively consistent with an increased number of fenestrations.

EXAMPLE 3

HIGH CONTENT SCREENING OF THE SK HEP-I CONSTRUCT AGAINST THE NP LIBRARY TO DETECT NOVEL AGENTS THAT MODULATE

FENESTRATIONS

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 Opera™ 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. EC₅₀ 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 Opera™ according to the optimised exposure protocol. Data stored as image files undergo analysis using Acapella™ 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.

Isolation and structure elucidation of active compounds

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.

Once the bioactive natural products are purified their chemical structures are determined using ID (¹H, ¹³C, DEPT) and 2D NMR (gCOSY, TOCSY, gHSQC, gHMBC, ROESY), IR, UV, CD and MS data.

EXAMPLE 4

CHARACTERISATION OF THE EFFECTS OF SMALL MOLECULE LEAD COMPOUNDS AND TOOLS ON SK HEP-I CELL FENESTRATIONS

COMPARED TO AGENTS KNOWN TO INFLUENCE FENESTRATIONS IN

LSECS (VEGF, CYTOCHALASIN, PHALLOIDIN, LATRUNCULIN, ENDOTHELIN, SEROTONIN, CALCIUM IONOPHORES, CALCIUM CHANNEL

BLOCKERS, INSULIN)

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 I₅ 10, 100 ng/ml (Esser et al. 1998, Journal of Cell Biology 140:947-59); cytochalasin 1, 10, 50 μg/ml (Braet et al. 1996, Hepatology 24:627-35); latrunculin 0.01, 0.1, 1 μg/ml (Braet et al. 1996, supra); endothelin-1 10^"9, lO^"8, 10^'7 M (Kamegaya et al. 2002, Hepatology Research 22:89-101); serotonin 0.01, 0.1, 1 μM (Gatmaitan et al. 1996, American Journal of Pathology 148:2027-41), calcium ionophore A23187 10^"7, 10^"6, 10-⁵ M (Gatmaitan et al. 1996, supra), verapamil 10^"7, 10^'6, 10^'5 M (Gatmaitan et al 1996, supra); insulin 0.2, 2, 20 μg/1 (insulin upregulates VEGF in isolated LSECs) (Qiao et al. 2005, World Journal of Gastroenterology 2005; 11:5978-83). Active compounds identified are tested in dose response (InM - lOOμM) to evaluate their ability to modulate fenestrations. Additional concentrations are investigated depending upon the responses to these concentrations. Changes in fenestral diameter have been recorded in isolated LSECs over minutes and hours. For electron microscopy, 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.

Samples are examined using a Joel JSM 6380 scanning electron microscope and analysis of fenestral diameter, frequency and number of fenestrations per cell are made using the Image J image analysis program (NIH) and SigmaScanPro. Fluorescence is captured with Confocal Z Stacks over 14 hours with images taken at least every 15 mins. Change in fluorescence is determined quantitatively (total fluorescence) and qualitatively (changes in patterns of cellular staining). These experiments are used to establish the responsiveness of fenestrations in SK Hep-1 cells and to confirm the changes with electron microscopy. Furthermore, in order to establish the effects on native LSECs, novel chemicals (hits) that are identified using HTS are assessed for their effects on LSECs isolated from rats. 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.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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Claims

CLAIMS:

1. 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.

2. The genetically modified cell of claim 1 wherein said cell is an endothelial cell.

3. The genetically modified cell of claim 2 wherein said endothelial cell is a sinusoidal endothelial cell.

4. The genetically modified cell of claim 3 wherein said sinusoidal endothelial cell is a liver sinusoidal endothelial cell.

5. The genetically modified cell of claim 4 wherein said liver sinusoidal endothelial cell is a SK Hep-1 cell.

6. The genetically modified cell of any one of claims 1 to 5 wherein said markers are 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.

7. The genetically modified cell of claim 6 wherein said cell has been modified to express one detectable marker of fenestration and said marker is caveolin or functional derivative, homologue or variant thereof.

8. The genetically modified cell of claim 6 wherein said cell has been modified to express one detectable marker of fenestration and said marker is actin or functional derivative, homologue or variant thereof.

9. The genetically modified cell of claim 6 wherein said cell has been modified to express one detectable marker of fenestration and said marker is Ca ATPase or functional derivative, homologue or variant thereof.

10. The genetically modified cell of claim 6 wherein said cell has been modified to express one detectable marker of fenestration and said marker is endothelin-1 or functional derivative, homologue or variant thereof.

11. The genetically modified cell of claim 6 wherein said cell has been modified to express one detectable marker of fenestration and said marker is dynamin or functional derivative, homologue or variant thereof.

12. The genetically modified cell of claim 6 wherein said cell has been modified to express one detectable marker of fenestration and said marker is filamen or functional derivative, homologue or variant thereof.

13. The genetically modified cell of claim 6 wherein said cell has been modified to express one detectable marker of fenestration and said marker is NOS or functional derivative, homologue or variant thereof.

14. The genetically modified cell of claim 6 wherein said cell has been modified to express one detectable marker of fenestration and said marker is serotonin receptor or functional derivative, homologue or variant thereof.

15. The genetically modified cell of claim 6 wherein said cell has been modified to express two detectable markers of fenestration.

16. The genetically modified cell of claim 6 wherein said cell has been modified to express three detectable markers of fenestration.

17. The genetically modified cell of claim 6 wherein said cell has been modified to express four detectable markers of fenestration.

18. The genetically modified cell of any one of claims 7 to 17 wherein said cell is a SK Hep-1 cell.

19. The genetically modified cell of any one of claims 6 to 18 wherein said detectable marker is a fluorescent marker, an enzyme substrate or a tag with which a visualisation means can interact.

20. The genetically modified cell of claim 19 wherein said fluorescent marker is selected from green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, emerald green fluorescent protein or red fluorescent protein.

21. The genetically modified cell of claim 19 wherein said tag with which a visualisation means can interact is a tetracysteine tag and wherein said visualisation means is a fluorescent reagent targeted to said tag.

22. The genetically modified cell of claim 19 wherein said tag is an antigen or epitope and wherein said visualisation means is a detectable antibody or functional fragment thereof.

23. The genetically modified cell of claim 22 wherein said antibody is either directly or indirectly detectable by fluorescent, radiological or enzymatic means.

24. The genetically modified cell of claim 19 wherein said tag is an enzymatic substrate.

25. The genetically modified cell of claim 24 wherein said enzymatic substrate is alkaline phosphatase.

26. The genetically modified SK Hep-1 cell of claim 5 wherein said detectable marker of fenestration is fluorescently labelled caveolin or functional derivative, homologue or variant thereof.

27. The genetically modified SK Hep-1 cell of claim 5 wherein said detectable marker of fenestration is fluorescently labelled actin or functional derivative, homologue or variant thereof.

28. The genetically modified SK Hep-1 cell of claim 5 wherein said detectable marker of fenestration is fluorescently labelled Ca ATPase or functional derivative, homologue or variant thereof.

29. The genetically modified SK Hep-1 cell of claim 5 wherein said detectable marker of fenestration is fluorescently labelled endothelin-1 or functional derivative, homologue or variant thereof.

30. The genetically modified SK Hep-1 cell of claim 5 wherein said detectable marker of fenestration is fluorescently labelled dynamin or functional derivative, homologue or variant thereof.

31. The genetically modified SK Hep-1 cell of claim 5 wherein said detectable marker of fenestration is fluorescently labelled filamen or functional derivative, homologue or variant thereof.

32. The genetically modified SK Hep-1 cell of claim 5 wherein said detectable marker of fenestration is fluorescently labelled NOS or functional derivative, homologue or variant thereof.

33. The genetically modified SK Hep-1 cell of claim 5 wherein said detectable marker of fenestration is fluorescently labelled serotonin receptor or functional derivative, homologue or variant thereof.

34. The genetically modified cell according to claim 26 wherein said fluorescent label is green fluorescent protein.

35. The genetically modified cell according to claim 27 wherein said fluorescent label is green fluorescent protein.

36. The genetically modified cell according to any one of claims 28 to 33 wherein said fluorescent label is green fluorescent protein.

37. The genetically modified cell according to claim 34 wherein said genetic modification comprises the transfection of said cell with a vector, which vector encodes a nucleic acid molecule encoding a GFP-caveolin fusion protein.

38. The genetically modified cell according to claim 37 wherein said vector is pEGFP- caveolin-1.

39. The genetically modified cell according to claim 38 wherein said cell corresponds to the cells deposited at ECACC on 27 July 2007 under Accession No. 07072602.

40. The genetically modified cell according to claim 35 wherein said genetic modification comprises the transfection of said cell with a vector, which vector encodes a nucleic acid molecule encoding a GFP-actin fusion protein.

41. The genetically modified cell of claim 40 wherein said vector is pAcGFP-actin.

42. The genetically modified cell according to claim 41 wherein said cell corresponds to the cells deposited at ECACC on 27 July 2007 under Accession No. 07072603.

43. A method of analysing mammalian cellular fenestrations, said method comprising exposing the genetically modified cells of any one of claims 1 to 42 to a stimulus and screening for the detectable marker of fenestration.

44. A method of assessing the effect of a treatment or culture regime on the cellular fenestration of the genetically modified cells of any one of claims 1 to 42 said method comprising subjecting said cells to said treatment or culture regime and screening for the detectable marker of fenestration.

45. The method according to claim 43 or 44 wherein said analysis or assessment is directed to fenestration expression, distribution and/or pore diameter.

46. A method of identifying a stimulus which modulates cellular fenestrations, said method comprising contacting the genetically modified cells according to any one of claims 1 to 42 with said stimulus and screening for the detectable marker of fenestration.

47. The method according to claim 46 wherein modulation of cellular fenestration is upregulation or downregulation of fenestration expression, distribution and/or pore diameter.

48. The method according to any one of claims 43 to 47 wherein said cells are the cells of claims 34, 35 or 37 to 42.

49. The method according to claim 48 wherein said screening method is fluorescence microscopy.

50. The method according to claim 48 wherein said screening method is FACS.

51. The method according to claim 49 or 50 wherein said screening step is optionally followed by electron microscopic analysis of said cells.

52. The method according to claim 49, 50 or 51 wherein said screening step is optionally followed by in vitro or animal model functional analysis of said cells.

53. The method according to any one of claims 43 to 52 wherein said stimulus is a cytokine, hormone, cell surface receptor ligand, proteinaceous or non-proteinaceous molecule derived from a natural product library, nucleic acid molecule, drug or chemically synthesised molecule.

54. The method according to claim 53 wherein said natural product library is NP-LLP.

55. The method according to any one of claims 43 to 52 wherein two or more genetically modified cell lines are screened and wherein each cell line expresses a different detectable marker of fenestration.

56. An agent identified in accordance with the screening method of any one of claims 43-54 when used to modulate endothelial cell fenestration expression, distribution and/or pore diameter.

57. 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 any one of claims 43-54.