WO2000052479A2 - Diagnostics and therapeutics for drusen associated ocular disorders - Google Patents
Diagnostics and therapeutics for drusen associated ocular disorders Download PDFInfo
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- WO2000052479A2 WO2000052479A2 PCT/US2000/005858 US0005858W WO0052479A2 WO 2000052479 A2 WO2000052479 A2 WO 2000052479A2 US 0005858 W US0005858 W US 0005858W WO 0052479 A2 WO0052479 A2 WO 0052479A2
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Definitions
- Macular degeneration is a clinical term that is used to describe a variety of diseases that are all characterized by a progressive loss of central vision associated with abnormalities of Bruch's membrane, the neural retina and the retinal pigment epithelium. These disorders include very common conditions that affect older patients (age-related macular degeneration or AMD) as well as rarer, earlier-onset dystrophies that in some cases can be detected in the first decade of life (Best F. Z, Augenheilkd., 13:199-212, 1905; Sorsby, A., et al., Rr J. Opthalmol. 33:67-97, 1949; Stargardt, K., Albrecht Von Graefes Arch Klin Exp Opthalmol.
- Macular degeneration diseases include, for example, age- related macular degeneration, North Carolina macular dystrophy, Sorsby's fundus dystrophy, Stargardt's disease, pattern dystrophy, Best disease, malattia leventinese, Doyne's honeycomb choroiditis, dominant drusen and radial drusen.
- age-related macular degeneration North Carolina macular dystrophy, Sorsby's fundus dystrophy, Stargardt's disease, pattern dystrophy, Best disease, malattia leventinese, Doyne's honeycomb choroiditis, dominant drusen and radial drusen.
- Ip21-ql3 for recessive Stargardt's disease or fundus flavi maculatus
- Age-related macular degeneration the most prevalent macular degeneration is associated with progressive diminution of visual acuity in the central portion of the visual field, changes in color vision, and abnormal dark adaptation and sensitivity
- Brown & Lovie-Kitchin 1983; Brown, et al., 1986; Sunness, et al., 1985; Sunness, et al., 1988; Sunness, et al., 1989; Eisner, et al., 1987; Massof, et al, 1989; Chen, et al., 1992).
- AMD AMD is the leading cause of legal blindness in North America and Western Europe (Hyman, 1992) and has become a significant health problem as the percentage of individuals above the age of 50 increases.
- the incidence of AMD was estimated to be 9.2% for persons over the age of 40 (Klein, et al., 1995).
- the Framingham Eye Study found the overall incidence of AMD to be 8.8%, with a 27.9%) incidence in the 75-85 year old population (Kahn, et al., 1977; Leibowitz, et al., 1980).
- 18.5% of those over age 85 were estimated to be afflicted with AMD (O'Shea, 1996). Variations in estimated incidence are likely a result of the use of different criteria for a diagnosis of AMD in different studies, or they may result from different risk factors among the various populations studied.
- neovascular "membranes” derived from the choroidal vasculature invade Bruch's membrane, leak, and often cause detachments of the RPE and/or the neural retina (Elman and Fine, 1989). This event can occur over a short period of time and can lead to rapid and permanent loss of central vision. If one eye is affected, there is a high degree of probability that the second eye will develop a choroidal neovascular membrane within five years of the initial event (Macular Photocoagulation Study, 1977).
- neovascular AMD Important clinical signs of neovascular AMD include gray-green neovascular membranes, dome-shaped RPE detachments, and disciform scars (caused by proliferation of fibroblasts and retinal glial cells) which are best visualized by their hyperfluorescence on fluorescein angiography (Elman and Fine, 1989). Killingsworth et al. (1990) suggested that macrophages may participate in the breakdown of Bruch's membrane in the neovascular stage of AMD and in drusen regression, and show one electron micrograph depicting structures resembling drusen cores.
- AMD has a genetic component, based upon the examination of the rates of AMD in different racial groups and the degree of familial aggregation of AMD (Hyman, et al., 1983). For example, Caucasians appear to be at greater risk than individuals of Hispanic origin (Cruickshanks, et al., 1997). In addition, a black population on Barbados had a lower incidence of advanced AMD than the local Caucasian population (Schachat, et al., 1995).
- AMD is not a single, genetic disease, since different diseases with distinct chromosomal loci share morphologic differences (Holz, et al., 1995a; Mansergh et al., 1995; and (2) that drusen may develop as a result of a biological pathway induced by a variety of different insults, genetic or otherwise. Determining whether AMD is a genetic or an acquired disorder is problematic, since AMD may actually be several diseases, and thus defy simple categorization; indeed, both genetic and environmental factors appear to play some role in its development.
- Environmental conditions may modulate the rate at which an individual develops AMD or the severity of the disease.
- Light exposure has been proposed as a possible risk factor, since AMD most severely affects the macula, where light exposure is high. (Young, 1988; Taylor, et al., 1990; Schalch, 1992).
- the amount of time spent outdoors is associated with increased risk of choroidal neovascularization in men, and wearing hats and/or sunglasses is associated with a decreased incidence of soft drusen (Cruickshanks, et al., 1993).
- Accidental exposure to microwave irradiation has also been shown to be associated with the development of numerous drusen (Lim, et al., 1993).
- Cataract removal and light iris pigmentation has also been reported as a risk factor in some studies (Sandberg, et al., 1994). This suggests that: 1) eyes prone to cataracts may be more likely to develop AMD; 2) the surgical stress of cataract removal may result in increased risk of AMD, due to inflammation or other surgically-induced factors; or 3) cataracts prevent excessive light exposure from falling on the macula, and are in some way prophylactic for AMD. While it is possible that dark iris pigmentation may protect the macula from light damage, it is difficult to distinguish between iris pigmentation alone and other, cosegregating genetic factors which may be actual risk factors. Dietary factors may also influence an individual's risk of developing AMD.
- Drusen appear to affect vision prior to the loss of visual acuity; changes in color contrast sensitivity (Frennesson, et al., 1995; Holz, et al., 1995b; Midena, et al., 1994; Stangos, et al., 1995; Tolentino, et al, 1994), macular recovery function, central visual field sensitivity, and spatiotemporal contrast sensitivity (Midena, et al., 1997) have been reported.
- Drusen accumulate between the RPE basal lamina and the inner collagenous layer of Bruch's membrane. They cause a lateral stretching of the RPE monolayer and physical displacement of the RPE from its immediate vascular supply, the choriocapillaris. This displacement creates a physical barrier that may impede normal metabolite and waste diffusion between the choriocapillaris and the retina. It is likely that wastes may be concentrated near the RPE and that the diffusion of oxygen, glucose, and other nutritive or regulatory serum-associated molecules required to maintain the health of the retina and RPE are inhibited. It has also been suggested that drusen perturb photoreceptor cell function by placing pressure on rods and cones (Rones, 1937) and/or by distorting photoreceptor cell alignment (Kincaid, 1992).
- drusen phenotypes are hard and soft (see, for example, Eagle, 1984; Lewis, et al., 1986; Yanoff and Fine, 1992; Newsome, et al., 1987; Mimoun, et al., 1990; van der Schaft, et al., 1992; Spraul and Grossniklaus, 1997), although numerous drusen phenotypes exist (Mullins & Hageman, 1999, Mol. Vision).
- Hard drusen are typically defined as small distinct deposits comprised of homogeneous eosinophilic material. Histologically, they are round or hemispherical, without sloped borders.
- Soft drusen are larger and have sloped, indistinct borders. Unlike hard drusen, soft drusen are not usually homogeneous, and typically contain inclusions and spherical profiles. An eye with many large/soft drusen is at a significantly higher risk of developing complications of AMD than is an eye with no drusen or a few, small drusen.
- the term "diffuse drusen,” or “basal linear deposit,” is used to describe the amorphous material which forms a layer between the inner collagenous layer of Bruch's membrane and the RPE. This material can appear similar to soft drusen histologically, with the exception that it is not mounded.
- drusen composition is scant. Wolter and Falls (1962) observed that drusen stain with oil red O, indicating the presence of neutral lipids in at least some drusen. Pauleikhoff, et al. (1992) used lipid-based histochemical staining approaches to show that different phenotypes of drusen contain either phospholipids or neutral lipids. These "hydrophilic" drusen were also bound by an anti- fibronectin antibody. Pauleikhoff et al. (1992) concluded that phospholipid-containing, but not neutral lipid-containing, drusen were anti-fibronectin antibody-reactive.
- drusen are comprised of sialomucins (glycoproteins with O- glycosidically-linked oligosaccharides) and cerebrosides and/or gangliosides.
- sialomucins glycoproteins with O- glycosidically-linked oligosaccharides
- cerebrosides and/or gangliosides glycoproteins with O- glycosidically-linked oligosaccharides
- Newsome et al. (1987) described labeling of soft drusen with antibodies directed against fibronectin, and to hard and soft drusen with antibodies directed against IgG and IgM.
- drusen result from secretion of abnormal material derived from RPE or photoreceptors ("deposition theories” ⁇ Muller, 1856; Ishibashi, et al., 1986; Young, 1987); transformation of degenerating RPE cells into drusen ("transformation theories"-Donders, 1854; Rones, 1937; Fine, 1981; El Baba, et al., 1986) or some combination of these pathways.
- Drusen-associated membranous profiles were inferred to be derived from the RPE, due to their localization between the RPE basal lamina and the inner collagenous zone of Bruch's membrane. While a number of investigators cite ultrastructural evidence for the derivation of drusen from RPE, the presence of melanin, lipofuscin or other RPE-derived organelles in drusen has not been reported.
- the invention Based on the elucidation of the role of dendritic cells in drusen biogenesis and a greater understanding of the pathology of drusen associated ocular disorders, the invention features novel diagnostics, therapeutics, treatment modalities and drug screening assays for drusen associated ocular disorders.
- the invention provides methods for diagnosing a subject for the presence of or predisposition for developing a drusen-associated ocular disorders.
- the method comprises detecting the presence, activity or level of at least one "drusen associated marker" (i.e. a phenotypic or genotypic marker that is involved with the development of drusen and ultimately the etiology of a drusen-associated ocular disorder).
- drusen associated marker i.e. a phenotypic or genotypic marker that is involved with the development of drusen and ultimately the etiology of a drusen-associated ocular disorder.
- choroidal neovascularization and/or choroidal fibrosis e.g. spiral collagens, elastin fibrils and microfilaments.
- genes expressed by dysfunctional or dying RPE cells include: HLA-DR, CD68, vitronectin, apolipoprotein E, clusterin and S-100.
- Genes expressed by chorodial and RPE cell in AMD include heat shock protein 70, death protein, proteasome,
- Markers involved in immune mediated events associated with drusen formation include: autoantibodies (e.g. directed against drusen, RPE and/or retina components), leukocytes, dendritic cells, myo fibroblasts, type VI collagen, and a cadre of chemokines and cytokines.
- Molecules associated with drusen include: immunoglobulins, amyloid A, amyloid P component, HLA- DR, fibrinogen, Factor X, prothrombin, complements 3, 5, 9, and 5b-9, C- reactive protein (CRP) apolipoprotein A, apolipoprotein E, antichymotrypsin, ⁇ 2 microglobulin, thrombospondin, and vitronectin.
- Markers of drusen associated dendritic cells include: CD la, CD4, CD14, CD68, CD83, CD86, and CD45, S100, PECAM, MMP14, ubiquitin, and FGF.
- Important dendritic cell-associated accessory molecules that participate in T cell recognition include ICAM-1, LFA1, LFA3, and B7, IL-1, IL-6, IL-12, TNF-alpha, GM-CSF and heat shock proteins. Markers associated with dendritic cell expression include: colony stimulating factor, TNF ⁇ , and H-1. Markers associated with dendritic cell proliferation include: GM-CSF, IL-4, 11-3, SCF, FLT-3 and TNF ⁇ . Markers associated with dendritic cell differentiation include IL-10, M-CSF, IL-6 and IL-4. In a preferred embodiment, a genetic fingerprint of the subject is analyzed to determine whether the subject has or is predisposed to developing a drusen associated ocular disorder.
- the invention provides therapeutic compositions and methods for treating or preventing the development of a drusen-associated ocular disease, comprising providing to the subject an effective amount of an agent which inhibits DC migration, proliferation or differentiation, prevents RPE cell dysfunction and death, prevents choroidal fibrosis, or otherwise inhibits drusen formation or enhances drusen resolution.
- the agent is selected from the group consisting of cytokines, chemokines and agonists and antagonists thereof.
- Preferred agents for reducing or inhibiting DC migration include the DCRMs granulocyte macrophage colony stimulating factor (GM-CSF), tumor necrosis factor a (TNFa) or interleukin-1 (IL-1) and agonists thereof.
- GM-CSF granulocyte macrophage colony stimulating factor
- TNFa tumor necrosis factor a
- IL-1 interleukin-1
- the agent or method reduces or inhibits dendritic cell migration into the subRPE space.
- the agent or method provide a means for inhibiting the protrusion of a cellular process, such as a dendritic cell process, into the subRPE space.
- Preferred agents for reducing or inhibiting DC proliferation include DCRMs that are antagonists for GM-CSF, IL-4, IL-3, SCF, FLT-3 or TNFa.
- Preferred agents for reducing or inhibiting DC differentiation include the DCRMs IL-10, macrophage colony stimulating factor (M-CSF), IL-6 and IL-4 and agonists thereof.
- DCRMs that are antagonists for LPS, TNFa, IL-1, IL-4, IL-13 or GM-CSF.
- Preferred agents that prevent or inhibit choroidal fibrosis include anti-angiogenic factors, collagenases and elastases.
- the invention provides therapeutic compositions and methods for treating or preventing drusen-associated disease, comprising providing to the subject an effective amount of an agent which reduces or inhibits the gene expression or activity of one or more drusen-associated molecules (DRAM).
- DRAM drusen-associated molecules
- the DRAM is an amyloid A protein, amyloid P component, antichymotrypsin, apolipoprotein E, b2 microglobulin, complement 3, complement C5, complement C5b-9 terminal complexes, factor X, fibrinogen, immunoglobulins (kappa and lambda), prothrombin, thrombospondin or vitronectin.
- DRAM gene regulation or activity is reduced or inhibited by one or more of a specific antisense nucleic acid, a ribozyme, a peptide, an antibody, or an enzyme.
- the DRAM antibody is conjugated to a reactive group.
- the reactive group is a photoreactive dye or a toxin.
- the invention features in vitro and in vivo assays for identifying therapeutics for drusen associated ocular disorders.
- the Figure is a schematic representation of the retina and choroid, as seen in (A) histological section, and (B) retinal neurons shown diagrammatically.
- A amacrine cells; B, bipolar cells; BM, Bruch's membrane; C, cone cells; CC, choriocapillaris; ELM, external limiting membrane; G, ganglion cells; GCL, ganglion cell layer; H, horizontal cells; ILM, inner limiting membrane; INL, internal nuclear layer; IPM, interphotoreceptor matrix; IS, inner segments of rods and cones; IPL, internal plexiform layer; NFL, nerve fiber layer; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segments of rods and cones; PE, pigment epithelium; PRL, photoreceptor layer; PT, photorecptor cell terminals; R, rod cells; ST, stroma vascularis of choroid.
- agonist is meant to refer to an agent that enhances or upregulates (e.g., potentiates or supplements) the production or activity of a gene product.
- An agonist can also be a compound which increases the interaction of a gene product, molecule or cell with another gene product, molecule or cell, e.g., of a gene product with another homologous or heterologous gene product, or of a gene product with its receptor.
- a preferred agonist is a compound which enhances or increases binding or activation of a transcription factor to an upstream region of a gene and thereby activates the gene.
- Any agent that activates gene expression, e.g., by increasing RNA or protein synthesis or decreasing RNA or protein turnover, or gene product activity may be an agonist whether the agent acts directly on the gene or gene product or acts indirectly, e.g., upstream in the gene regulation pathway.
- Agonists may be RNAs, peptides, antibodies and small molecules, or a combination thereof.
- animal model includes transgenic animals, naturally occurring animals with genetic mutations and non-transgenic animals that have been treated with one or more agents, or combinations thereof (e.g., a skid mouse), any of which may serve as experimental models for a disease, e.g., macular degeneration.
- a transgenic mouse may be a mouse in which a gene is knocked out or in which a gene is overexpressed.
- antagonist as used herein is meant to refer to an agent that downregulates (e.g., suppresses or inhibits) the production or activity of a gene product.
- Such an antagonist can be an agent which inhibits or decreases the interaction between a gene product, molecule or cell and another gene product, molecule or cell.
- a preferred antagonist is a compound which inhibits or decreases binding or activation of a transcription factor to an upstream region of a gene and thereby blocks activation of the gene.
- Any agent that inhibits gene expression or gene product activity may be an antagonist whether the agent acts directly on the gene or gene product or acts indirectly, e.g., upstream in the gene regulation pathway.
- An antagonist can also be a compound that downregulates expression of a gene or which reduces the amount of gene product present, e.g., by decreasing RNA or protein synthesis or increasing RNA or protein turnover.
- Antagonists may be RNAs, peptides, antibodies and small molecules, or a combination thereof.
- sociate or “interact” as used herein is meant to include detectable relationships or associations (e.g., biochemical interactions) between molecules, such as interaction between protein-protein, protein-nucleic acid, nucleic acid-nucleic acid, protein- carbohydrate, carbohydrate-carbohydrate, protein-lipid, lipid-lipid, etc., and protein-small molecule or nucleic acid-small molecule in nature.
- Bruch's Membrane is a trilaminar extracellular matrix complex that lies between the retinal RPE and the primary capillary bed of the choroid, the choriocapillaris.
- Bruch's membrane is comprised of two collagen layers, referred to as the inner and outer collagenous layers, that flank a central domain comprised largely of elastin.
- the strategic location of Bruch's membrane between the retina and its primary source of nutrition, the choroidal vasculature is essential for normal retinal fimction (Marshall et al., 1998; Guymer and Bird, 1998).
- Type VI is associated specifically with the elastic lamina, types IV and V with the basal laminae of the choriocapillaris and RPE, and types I and III with the inner and outer collagenous layers.
- Type I, III, IV and V has also been confirmed biochemically.
- choroid refers to the highly vascularized tissue lying between the sclera and retinal pigment epithelium of the eye. This tissue is comprised of numerous pericytes, melanocytes, fibroblasts, myofibroblasts and transitional leukocytes. "Bruch's membrane, a trilamellar extracellular matrix comprised of inner and outer collagenous layers and an elastic lamina, is a component of the choroid. It is positioned between the basal lamina of the RPE and the choriocapillaris. The remaining extracellular matrix of the choroid is comprised of a variety of extracellular matrix constituents that are loosely organized.
- dendritic cell refers to hematopoietic cells characterized by their unusual dendritic morphology, their potent antigen-presenting capability and their lack of lineage-specific markers such as CD3, CD 19, CD 16, CD 14, which distinguishes them respectively from T cells, B cells, NK cells, and monocytes.
- lineage-specific markers such as CD3, CD 19, CD 16, CD 14, which distinguishes them respectively from T cells, B cells, NK cells, and monocytes.
- Myeloid-committed precursors which give rise to granulocytes and monocytes can also differentiate into Langerhans cells of the skin and myeloid related dendritic cells in the secondary lymphoid tissue.
- lymphoid-derived dendritic cells See Lotze, M.T. and Thomson, A.W. (Eds.) (1999) "Dendritic Cells", Academic Press, San Diego, CA, for a number of reviews on dendritic cells, the teachings of which are incorporated herein by reference).
- dendritic cell precursor or “DC precursor” as used herein refers to cell types from which a dendritic cell is derived upon differentiation and maturation.
- a dendritic cell precursor may be a bone marrow stem cell, a lymphiod cell lineage-committed cell or a myeloid cell lineage-committed cell from which a dendritic cell may develop after exposure to certain DCRMs.
- DC precursors of the myeloid lineage can be induced to differentiate into DCs by treatment with GM-CSF.
- dendritic cell process refers to a cellular portion of a dendritic cell which projects or extends away from the center of the dendritic cell.
- drusen as used herein encompasses a number of phenotypes, all of which develop, between the inner collageous layer of Bruch's membrane and the RPE basal lamina.
- Hard drusen are small distinct deposits comprised of homogeneous eosinophilic material and are usually round or hemispherical, without sloped borders.
- Soft drusen are larger, usually not homogeneous, and typically contain inclusions and spherical profiles. Some drusen may be calcified.
- the term "diffuse drusen,” or “basal linear deposit,” is used to describe amorphous material which forms a layer between the inner collagenous layer of Bruch's membrane and the retinal pigment epithelium (RPE). This material can appear similar to soft drusen histologically, with the exception that it is not mounded.
- drusen-associated marker refers to a phenotype or genotype that is involved or associated with the development of drusen formation and ultimately the development of a drusen associated ocular disease or disorder.
- phenotypic markers include: RPE dysf ncation and/or death, immune mediated events, dendritic cell activation, migration, differentiation and extrusion of the DC process into the sub RPE space (e.g. by detecting the presence or level of a dendritic cell marker such as CD68, CD la and SI 00), the presence of geographic atrophy or disciform scars, the presence of choroidal neovascularization and/or choroidal fibrosis, especially in the macula.
- genotypic markers include mutant genes and/or a distinct pattern of differential gene expression (Drusen Development Pathway"), including genes that are upregulated or downregulated in drusen forming ocular tissue associated with drusen biogenesis.
- genes expressed by dysfunctional and or dying RPE cells include: HLA-DR, CD68, vitronectin, apolipoprotein E, clusterin and S-100.
- Markers involved in immune mediated events associated with drusen formation include: autoantibodies (e.g.
- Molecules associated with drusen include: immunoglobulins, amyloid A, amyloid P component, HLA-DR, fibrinogen, Factor X, prothrombin, complements 3, 5, 9, and 5b-9, C- reactive protein (CRP) apolipoprotein A, apolipoprotein E, antichymotrypsin, ⁇ 2 microglobulin, thrombospondin, and vitronectin.
- CRP C- reactive protein
- Markers of drusen associated dendritic cells include: CDla, CD4, CD14, CD31 (PECAM-1), CD45, CD64/1 (FcR), CD68, CD83, CD86 and HLA-DR, particular preferred dendritic cell markers include CDla, CD14, CD45, CD68, CD83 and HLA-DR.
- Important dendritic cell-associated accessory molecules that participate in T cell recognition include ICAM-1, LFA1, LFA3, and B7, IL-1, IL-6, IL-12, TNF-alpha, GM-CSF and heat shock proteins.
- Markers associated with dendritic cell expression include: colony stimulating factor, TNF ⁇ , and II- 1. Markers associated with dendritic cell proliferation include: GM-CSF, IL-4, 11-3, SCF, FLT-3 and
- Markers associated with dendritic cell differentiation include IL-10, M-CSF, IL-6 and IL-4.
- Markers of fibrosis include: a decrease in BIG H3, increase in ⁇ l- integrin, increase in various growth factors (e.g. fibroblast growth factors (FGF), chemokines and cytokines, increase in collagen (e.g. collagen 6 a2 and collagen 6 a3) or procollagen e.g. I and III and peptides thereof, increase in elastin or elastin peptides, and increase in FSP-1 and an increase in human metalloelastase (HME).
- FGF fibroblast growth factors
- chemokines and cytokines increase in collagen
- procollagen e.g. I and III and peptides thereof
- elastin or elastin peptides increase in FSP-1 and an increase in human metalloelastase (HME).
- Molecules that are known or suspected to participate in systemic fibrosis and are therefore potential candidates for choroidal fibrosis include, but are not limited to: BIGh3, calpain, cathepsin D, collagens (I, III, IV, VI, VII), CTGF, desmosine, elastin, emilin, endothelin, bFGF, fibrillins 1-2, fibroblasst specific proteins (FSP-1), fibronectin, fibrosin, fibulins 1-5, ficolin, GM-CSF, 4-hydroxy-nonenal, HLA antigens, HME, IFG-1, IFN- ⁇ , IL-2, 11-4, IL-6, IL-8, IL-10, integrins ⁇ l ⁇ l and ⁇ 2 ⁇ l, laminins Cl-3, laminin receptors, LOXL, LTBP 1-4, MCP-1, MFAP 1-4, MMPs, oncostatin M, osteopontin, P
- drusen-associated ocular disorder refers to any disease or disorder which involves drusen formation.
- the accumulation of drusen creates a physical barrier that appears to impede normal metabolite and waste diffusion between the choriocapillaris and the retina.
- the diffusion of oxygen, glucose, and other nutritive or regulatory serum-associated molecules required to maintain the health of the retina and RPE are inhibited.
- drusen-associated molecule refers to any protein, carbohydrate, glycoconjugate (e.g., glycoprotein or glycolipid), other lipid, nucleic acid or other molecule which is found in association with, or interacting with, a drusen deposit.
- DRAMS may include cellular fractions or organelles that are not normally found deposited in, or in association with, a tissue unless it is affected by drusen or which is not present in drusen- affected and normal tissue in equivalent amounts.
- ECM extracellular matrix
- collagens e.g., the collagens, proteoglycans, non-collagenous glycoproteins and elastins that surround cells and provide structural and functional support for cells as well as maintain various functions of cells, such as cell adhesion, proliferation, differentiation and protein synthesis.
- proteoglycans e.g., the collagens, proteoglycans, non-collagenous glycoproteins and elastins that surround cells and provide structural and functional support for cells as well as maintain various functions of cells, such as cell adhesion, proliferation, differentiation and protein synthesis.
- elastins e.g., the collagens, proteoglycans, non-collagenous glycoproteins and elastins that surround cells and provide structural and functional support for cells as well as maintain various functions of cells, such as cell adhesion, proliferation, differentiation and protein synthesis.
- a skilled artisan will appreciate that the precise composition and physical properties of ECM, as well as its function, vary between various cell types, between
- Fibrosis refers to a disease process, typically observed in chronic diseases, characterized by progressive accumulation and/or deposition of extracellular matrix proteins (e.g. collagens) and activation, differentiation and/or transformation of various interstitial cell types (e.g. fibroblasts).
- extracellular matrix proteins e.g. collagens
- activation, differentiation and/or transformation of various interstitial cell types e.g. fibroblasts.
- inhibitor means to prevent or prohibit and is intended to include total inhibition, partial inhibition, reduction or decrease.
- macular degeneration refers to any of a number of conditions in which the retinal macula degenerates or becomes dysfunctional, e.g., as a consequence of decreased growth of cells of the macula, increased death or rearrangement of the cells of the macula (e.g., RPE cells), loss of normal biological function, or a combination of these events. Macular degeneration results in the loss of integrity of the histoarchitecture of the cells of the normal macula and/or the loss of function of the cells of the macula. The term also encompasses extramacular changes that occur prior to, or following dysfunction and/or degeneration of the macula.
- any condition which alters or damages the integrity or function of the macula may be considered to fall within the definition of macular degeneration.
- Other examples of diseases in which cellular degeneration has been implicated include retinal detachment, chorioretinal degenerations, retinal degenerations, photoreceptor degenerations, RPE degenerations, mucopolysaccharidoses, rod-cone dystrophies, cone-rod dystrophies and cone degenerations.
- modulation means modulation or alteration, modulate “, or “alter” are used interchangeably herein to refer to both upregulation (i.e., activation or stimulation (e.g., by agonizing or potentiating) and downregulation (i.e., inhibition or suppression (e.g., by antagonizing, decreasing or inhibiting)) of an activity.
- the activity that is modulated may be gene expression or may be the growth, proliferation, migration or differentiation of dendritic cells.
- “Modulates” or “alters” is intended to describe both the upregulation or downregulation of a process, since, as is well known to a skilled artisan, a process which is upregulated by a certain stimulant may be inhibited by an antagonist to that stimulant.
- a process that is downregulated by a certain stimulant may be inhibited by an antagonist to that stimulant.
- an antagonist to that stimulant.
- the identification of an agent that induces a cellular response modulates or alters cellular behavior in an inductive manner and it is inherently understood that the response may be modulated in an inhibitory manner by an inhibitor of that agent (e.g., by an antibody or antisense RNA, as is well understood and described in the art).
- nucleic acid refers to polynucleotides or oligonucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
- DNA deoxyribonucleic acid
- RNA ribonucleic acid
- the term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.
- polymorphism refers to the coexistence of more than one form of a gene or portion (e.g., allelic variant) thereof.
- a portion of a gene of which there are at least two different forms, i.e., two different nucleotide sequences, is referred to as a "polymorphic region of a gene".
- a polymorphic region can be a single nucleotide, the identity of which differs in different alleles.
- a polymorphic region can also be several nucleotides long.
- a "polymorphic gene” refers to a gene having at least one polymorphic region.
- protein protein
- polypeptide and peptide are used interchangeably herein when referring to a gene product comprising amino acids.
- recombinant protein refers to a polypeptide of the present invention which is produced by recombinant DNA techniques, wherein generally DNA encoding a polypeptide is inserted into a suitable expression vector which is in rum used to transform a host cell to produce the heterologous protein.
- recombinant nucleic acid refers to a nucleic acid or DNA of the present invention which is produced by recombinant DNA techniques, wherein generally DNA encoding a polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein.
- phrase "derived from”, with respect to a recombinant gene is meant to include within the meaning of "recombinant protein” those proteins having an amino acid sequence of a native polypeptide, or an amino acid sequence similar thereto which is generated by mutations including substitutions and deletions (including truncation) of a naturally occurring form of the polypeptide.
- RPE Retinal Pigment Epithelium
- the RPE derives developmentally from, and is indeed contiguous with, the same neuroectodermal layer as the neural retina.
- the RPE possesses numerous large pigment granules (melanosomes) which participate in the prevention of light scattering.
- the RPE plays a critical role in the maintenance of photoreceptor cell viability and function by the phagocytosis and removal of photoreceptor outer segment disks, the processing and secretion of various molecules necessary for photoreceptor function and viability (such as vitamin A derivatives and growth factors), the regulation of macromolecular traffic between the retina and choroid, and the mediation of retinal adhesion.
- Small molecule as used herein, is meant to refer to a composition which has a molecular weight of less than about 5 kD and most preferably less than about 4 kD.
- Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids (e.g., glycolipids and pig-tail lipids) or other organic (carbon containing) or inorganic molecules.
- lipids e.g., glycolipids and pig-tail lipids
- a "therapeutic” as used herein refers to an agonist or antagonist of the bioactivity of a drusen associated marker.
- Preferred therapeutics reduce or inhibit RPE cell death, factors involved in the inflammatory response, factors involved in fibroblast proliferation and migration resulting in fibrosis and/or dendritic cell activation, migration or differentiation into drusen.
- modulators of fibrosis include, but are not limited to: L-tryptophan dimer, superoxide, nitric oxide, corticosteroid, retinoid, halofuinone, Tranilast, CTGF, interferons, relaxin, TGF ⁇ 3, HGF, prolyl hydroxylase, C-proteinase, lysyl oxidase, and antisense oligonucleotides.
- agents that have shown some efficacy in treating or preventing aortic diseases include agents that have shown some efficacy in treating or preventing aortic diseases (e.g. AAA), including: antiinflammatory agents (e.g. anti CD-I 8 antibody), protease inhibitors, inhibitors of elastolytic MMPs (e.g. the hydroxamate based RS312908, batimastat, antibiotics (e.g. doxycycline), tetracycline), inhibitors of prostaglandin synthesis and beta-blockers (e.g. propanalol).
- antiinflammatory agents e.g. anti CD-I 8 antibody
- protease inhibitors e.g. the hydroxamate based RS312908, batimastat
- antibiotics e.g. doxycycline
- tetracycline tetracycline
- beta-blockers e.g. propanalol
- transcriptional regulatory sequence is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked.
- transfection means the introduction of a nucleic acid, e.g., via an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.
- Transformation refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA.
- transgene means a nucleic acid sequence (encoding, e.g., one of the polypeptides of the invention, or an antisense transcript thereto) which has been introduced into a cell.
- a transgene could be partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or can be homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout or may result in over expression).
- a transgene can also be present in a cell in the form of an episome.
- a transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as 5' UTR sequences, 3' UTR sequences, or introns, that may be necessary for optimal expression of a selected nucleic acid.
- a "transgenic animal” refers to any animal, preferably a non-human mammal, bird or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
- the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
- the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachro mosomally replicating DNA.
- the transgene causes cells to fail to express a specific normal gene product, to express a recombinant form of one or more DRAM polypeptides, e.g., either agonistic or antagonistic forms, or molecules that regulate the biosynthesis, accumulation or resorption of DRAMs or dendritic cells.
- Transgenic knockouts may, for example, be produced which cause alterations in dendritic cell behavior (e.g., cell growth, proliferation, migration, differentiation or gene expression).
- dendritic cell behavior e.g., cell growth, proliferation, migration, differentiation or gene expression.
- transgenic animals in which the recombinant DCRM or DRAM gene is silent are also contemplated, as for example, the FLP or CRE recombinase dependent constructs.
- transgenic animal also includes those recombinant animals in which gene disruption is caused by human intervention, including both recombination and antisense techniques.
- treating is intended to encompass curing as well as ameliorating at least one symptom of the condition or disease.
- vector refers to a nucleic acid molecule, which is capable of transporting another nucleic acid to which it has been linked.
- One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
- Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
- Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors.”
- expression system refers to an expression vector under conditions whereby an mRNA may be transcribed and/or an mRNA may be translated into protein.
- the expression system may be an in vitro expression system, which is commercially available or readily made according to art known techniques, or may be an in vivo expression system, such as a eukaryotic or prokaryotic cell containing the expression vector.
- expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
- plasmid and vector are used interchangeably as a plasmid is the most commonly used form of vector.
- the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
- wild-type allele refers to an allele of a gene which, when present in two copies in a subject results in a wild-type phenotype. There can be several different wild- type alleles of a specific gene, since certain nucleotide changes in a gene may not affect the phenotype of a subject having two copies of the gene with the nucleotide changes.
- the invention is based, at least in part, on the elucidation of the etiology of AMD and other drusen-associated ocular disorders, essentially as described below.
- dendritic cells in inflammatory lesions are well-recognized. It is clear that dendritic cells must be recruited, activated, and migrate to, sites of inflammation, rather than passively migrating to these sites. Dendritic cells are typically recruited to sites of tissue damage by various chemoattractants, heat shock proteins, DNA fragments, and others. Choroidal dendritic cell processes are associated with the smallest of drusen, and are often observed in the sub-RPE space in association with whole, or portions of, RPE cells that have been shunted into Bruch's membrane, prior to the time that drusen, per se, are detectable.
- choroidal dendritic cells are activated and recruited by locally damaged and/or sublethally injured RPE cells.
- This idea is consistent with recent data showing that dendritic cells, and thus the innate immune system, can be activated by microenvironmental tissue damage. In this state, these cells extend a cellular process through Bruch's membrane in order to gain access to the site of tissue damage.
- choroidal dendritic cells may thus serve as sentinel receptors with the capacity to respond to local cell injury, and ultimately provide for the overall integration of immune-mediated processes that determine the outcome of the overall response.
- the injured RPE itself may serve as a source of soluble cytokines or other stimulatory factors that initiate dendritic cell recruitment and activation.
- the data presented herein clearly supports accelerated RPE cell death in eyes derived from donors with AMD, as compared to age-matched controls. Based on available information from other systems, and upon previous suggestions pertaining to the etiology of AMD, RPE cell death might occur by several mechanisms, including ischemia, necrosis, gene-mediated injury, Bruch's membrane-induced dysfunction, oxidative injury from light or systemic factors (e.g. smoking-generated compounds), lipofuscin accumulation, or autoimmune phenomena, to list a few. Based on data disclosed herein, it is likely that RPE cell death would most likely have to be due to necrosis, rather than to apoptosis, since cells undergoing apoptotic cell death are not known to be capable of recruiting dendritic cells.
- cytokines such as IL-1, IL-6, IL-12, TNF-alpha, and GM-CSF
- heat shock proteins altered expression of cell surface proteins and DNA in the presence of free radicals.
- HLA-DR CD68
- vitronectin S-100
- clusterin apolipoprotein E
- dendritic cells might then contribute to the chronicity (induced chronic inflammatory lesions) of AMD by any number of mechanisms, including immune complex formation, complement activation, and/or in situ activation of choroidal T-cells, other phagocytic cells, and matrix proteolysis.
- immune complex formation including immune complex formation, complement activation, and/or in situ activation of choroidal T-cells, other phagocytic cells, and matrix proteolysis.
- the presence of numerous immune-associated constituents in drusen, including immunoglobulins, complement proteins, and some acute phase proteins, could be explained by such an event.
- This type of self- limiting control is typically accomplished in other systems via turnover of dendritic cells; the influx of new dendritic cell precursors and the concomitant reduction in the influx of mature dendritic cells into the lymph nodes is typically sufficient to shift the balance back to tolerance.
- natural killer cells recognize mature dendritic cells as targets, providing a negative feedback effect on antigen presentation, forcing the system into tolerance.
- cyclical events of RPE cell death may occur over a period of many years that do not allow the system to return to tolerance. In one example, this might occur as a result of genetic preprogramming, as in the case of a RPE gene mutation.
- dendritic cells may be activated by local tissue injury might also initiate an autoimmune response to retinal and/or RPE antigens that are uncovered during tissue damage.
- the availability and amount of RPE debris/antigen will most likely determine which ensuing pathway is involved.
- Such autoimmune responses have been documented as a consequence of ischemia or injury to the heart.
- the inventors have recently identified autoantibodies in the sera of individuals with AMD that are directed against retinal and RPE proteins of 35kDa and 53kDa. This might occur as a consequence of aberrant delayed-type hypersensitivity responses, perhaps explaining the presence of serum autoantibodies in at least some AMD patients. It is also conceivable that the groundwork for this autoimmune process is primed earlier in life by necrosis of RPE cells.
- DRAMs are circulating plasma proteins
- ligands associated with Bruch's membrane in the aging eye.
- These ligands could be basement membrane components, plasma membrane receptors, secretory products derived from RPE or choroidal cells, or byproducts of cellular autolysis.
- a number of drusen-associated molecules including apolipoprotein E, vitronectin, fibrinogen, C reactive protein, and transthyretin, have been synthesized by the RPE and/or retina. Although unexpected, these data support the concept that some DRAMs may be synthesized and secreted locally.
- This model also predicts an imbalance in extracellular matrix synthesis, degradation, and/or turnover, which would thereby lead to events such as choroidal neovascularization, a hallmark characteristic of some forms of AMD, cellular proliferation, cellular differentiation, and interstitial fibrosis.
- fibrosis and fibrogenesis is a common complication of tissue injury, independent of the initial site of said injury.
- the recruitment of immune cells, and their activation and/or modulation by resident cells represents a key step in the cascade of events that ultimately lead to fibrosis. More recent studies suggest that distinct functional fibroblast phenotypes may play a central role in early fibrosis, including the initial recruitment of immune cells.
- choroidal fibrosis The inventors have documented "choroidal fibrosis" in a subset of donor eyes. There is a significant correlation between choroidal fibrosis and age. Furthermore, preliminary data suggest that there is a strong correlation between choroidal fibrosis, drusen, AMD, aortic aneurysms, aortic stenosis, and possibly COPD. These fibrotic choroids are characterized ultrastructurally by massive accumulations of newly synthesized collagen and elastin fibrils, as well as filamentous collagens and microfilaments, that fill the normally loosely packed choroidal stromas.
- the major collagen fibrils average 0.042-0.063 ⁇ m in diameter as compared to the fibrillar collagen in the sclera, which averages 0.211-0.253 ⁇ m in diameter. Furthermore, the collagen fibrils in these donors exhibit a classic spiraled mo ⁇ hology in longitudinal and cross sections. It is thought that spiraled collagen results from disaggregation of fibrils and/or to inco ⁇ oration of uncleaved procollagen molecules.
- This collagen phenotype is observed in a few heritable connective tissue diseases (Ehler's-Danlos; PXE; dermatoparaxis), as well as in other nongenetic conditions (collagenofibrotic glomerulopathy, scleroderma, atherosclerosis, amyloid, emphysema, atheromatous plaques).
- the deposition of a distinct banded material is also present in donor eyes exhibiting choroidal fibrosis.
- Clear indications of active elastin synthesis including dilated RER, pockets of microfilaments, and elastin exhibiting the mo ⁇ hological characteristics of newly synthesized protein) are also observed along attenuated fibroblast cell processes and interspersed amongst the collagen fibrils. Genes that are and are not differentially expressed in choroidal fibrosis are shown in Table 1 , below.
- RPE retinal pigment epithelial cell
- fragments of RPE cells (identified on the basis of mo ⁇ hologically detectable lipofuscin and pigment granules), can be detected within drusen at both the light and electron microscopic levels of resolution, demonstrating that they contribute to drusen volume and formation.
- RPE cells associated with the smallest of drusen are often characterized by focal expression of molecules not normally associated with these cells. These molecules include HLA-DR, CD68, vitronectin, apolipoprotein E, and perhaps clusterin and S-100. Although it is highly unusual for non- immunocompetent cells to express HLA-DR, this protein is typically expressed by cells early in immune reactions. Indeed, its expression by RPE cells may be a marker of RPE cell dysfunction and is likely to be involved in recognition of dysfunction and or damaged RPE by other cells. Alternatively, the expression of HLA-DR might be a secondary phenomenon related to the presence of dendritic cells.
- drusen or other abnormal changes in the extracellular environment that is Bruch's membrane
- An accumulation of drusen could cause local interference with the exchange of metabolites and waste products between the choriocapillaris and an otherwise normal RPE, leading to RPE dysfunction and death.
- drusen may be a consequence of aberrant RPE gene expression, although the precise biological events that ultimately lead to RPE dysfunction are equally unclear.
- Suggestions range from gene mutations to oxidative insults to lipofuscin accumulation, to programmed cell death.
- Penfold, P.L., et al Modulation of major histocompatibility complex class II expression in retinas with age-related macular degeneration. Investigative Ophthalmology & Visual Science, 1997. 38(10): p. 2125-33.) and other leukocytes (Penfold, P., M. Killingsworth, and S. Sarks, Senile macular degeneration: the involvement of immunocompetent cells. Graefe's Archives for Clinical and Experimental Ophthalmology, 1985. 223:p.69-76);Killingsworth, M., J. Sarks, and S. Sarks, Macrophages related to Bruch's membrane in age-related macular degeneration. Eye, 1990. 4: p.
- type VI collagen a putative component of the basal laminar deposits that are prevalent in eyes of donors with AMD, increases in association with inflammatory processes leading to fibrotic remodeling in diseases such as lung fibrosis, scleroderma, and eosinophilic myalgia syndrome.
- Exhaustive immunohistochemical analyses of drusen composition have revealed a distinct a ⁇ ay of molecules (including immunoglobulins, amyloid A, amyloid P component, C5 and C5b-9 terminal complexes, HLA-DR, fibrinogen, Factor X, and prothrombin) that are common to all phenotypes of hard and soft drusen.
- additional studies have documented that a number of these constituents (many of which have been thought to be synthesized primarily in the liver) are synthesized locally by RPE, retinal, and/or choroidal cells.
- CRP complement reactive protein
- HLA-DR apolipoprotein A
- apolipoprotein E amyloid A
- vitronectin a number of these drusen-associated constituents (DRAMs) are participants in humoral and cellular immune processes.
- DRAMs drusen-associated constituents
- C5b-9 complex is associated with specific immune processes, often involving cell death.
- Dendritic cells are found in primary lymphoid organs and most non-lymphoid tissues and organs (Ibrahim, M., B. Chain, and D. Katz, The injured cell: the role of the dendritic cell system as a sentinel receptor pathway. Immunology Today, 1995. 16: p. 181-6; Matyszak, M. and V. Perry, The potential role of dendritic cells in immune-mediated inflammatory diseases in the central nervous system. Neuroscience, 1996. 74: p. 599-608; Matyszak, M. and V. Perry, Dendritic cells in inflammatory responses in the CNS, in Dendritic cells in fundamental and clinical immunology, Ricciardi-Castagnoli, Editor.
- dendritic cells reside within non-lymphoid tissues. Dendritic cells are powerful antigen-presenting cells that contribute to the pathogenesis of immune-mediated responses in a number of ways, including the primary activation of T lymphocytes, various secondary responses, and the induction of autoimmune responses. Antigen presentation is important in the induction of conventional immune responses, as well as in the induction and maintenance of tolerance. It has been proposed that dendritic cells may provide an essential link between the innate and adaptive immune systems, actively participating in determining the outcome of the immune response. For example, data from recent investigations suggest that dendritic cells, and hence the innate immune system, can be activated by local, microenvironmental tissue damage. In this role, dendritic cells provide a sentinel receptor system that responds to local tissue injury and provides an integrative mechanism that determines the outcome of the immune response.
- dendritic cells After acquiring an antigen, dendritic cells typically (but not always) migrate out of the tissue, into the blood, through the afferent lymphatics, and into the T cell-rich regions of the local lymphoid organs.
- Important dendritic cell-associated accessory molecules that participate in T cell recognition include ICAM-1, LFA1, LFA3, and B7, whereas T cell counter receptors include LFA1, CD2, and CD28. Binding of the B7 ligand to its counter receptor CD28 is especially important in stimulating the synthesis and secretion of IL-2 by T cells.
- Drusen cores are observed in all drusen phenotypes and are present in both macular and extramacular drusen. They may be more prevalent in drusen possessing a height-width ratio of less than 0.5.
- drusen may represent an ocular manifestation of amyloidosis, elastosis, dense deposit disease, and/or atherosclerosis. Although modulated by different genes and/or environmental influences, all these diseases give rise to similar, yet distinguishable, pathological phenotypes by triggering a similar set of biological responses that include inflammation, coagulation, and activation of the immune system.
- the invention provides a valuable recognition of these similarities but also provides a method for diagnosing and treating drusen specifically, as compared to other age- related diseases which manifest themselves in deposits or plaques.
- the invention provides a method for diagnosing, or determining a predisposition to developing a drusen associated disease by detecting one or more markers which are associated with drusen development.
- markers include: RPE dysfuncation and/or death, immune mediated events, dendritic cell activation, migration and differentiation, extrusion of the dendritic cell process into the sub RPE space (e.g. by detecting the presence or level of a dendritic cell marker such as CD68, CDla and SI 00), the presence of geographic atrophy or disciform scars, the presence of choroidal neovascularization and/or choroidal fibrosis, especially in the macula.
- genotypic markers include mutant genes and/or a distinct pattern of differential gene expression (Drusen Development Pathway"), including genes that are upregulated or downregulated in drusen forming ocular tissue associated with drusen biogenesis.
- genes expressed by dysfunctional and/or dying RPE cells include: HLA-DR, CD68, vitronectin, apolipoprotein E, clusterin and S-100.
- Markers involved in immune mediated events associated with drusen formation include: autoantibodies (e.g.
- Molecules associated with drusen include: immunoglobulins, amyloid A, amyloid P component, HLA-DR, fibrinogen, Factor X, prothrombin, complements 3, 5, 9, and 5b-9, c reactive protein (CRP) apolipoprotein A, apolipoprotein E, antichymotrypsin, ⁇ 2 microglobulin, thrombospondin, and vitronectin.
- CRP reactive protein
- Markers of drusen associated dendritic cells include: CDla, CD4, CD14, CD68, CD83, CD86, and CD45, PECAM, MMP14, ubiquitin, and FGF.
- Important dendritic cell-associated accessory molecules that participate in T cell recognition include ICAM-1, LFA1, LFA3, and B7, IL-1, IL-6, IL-12, TNF-alpha, GM-CSF and heat shock proteins.
- Markers associated with dendritic cell expression include: colony stimulating factor, TNF ⁇ , and II- 1.
- Markers associated with dendritic cell proliferation include: GM-CSF, IL-4, 11-3, SCF, FLT-3 and TNF ⁇ .
- Markers associated with dendritic cell differentiation include IL-10, M-CSF, IL-6 and IL-4.
- Markers of fibrosis include: a decrease in BIG H3, increase in ⁇ l- integrin, increase in collagen (e.g. collagen 6 ⁇ 2 and collagen 6 ⁇ 3), increase in elastin, and an increase in human metallo elastase (HME).
- Some drusen-associated markers may be detected by one or more ophthalmological procedures, such as fundus fluorescein angiography (FFA), fundus ophthalmoscopy or photography (FP), electroretinogram (ERG), electrooculogram (EOG), visual fields, scanning laser ophthalmoscopy (SLO), visual acuity measurements, dark adaptation measurements or other standard method.
- FFA fundus fluorescein angiography
- FP fundus ophthalmoscopy or photography
- EEG electroretinogram
- EOG electrooculogram
- visual fields such as scanning laser ophthalmoscopy (SLO), visual acuity measurements, dark adaptation measurements or other standard method.
- SLO scanning laser ophthalmoscopy
- drusen-associated markers can be detected on the molecular level, e.g. by detecting the identity, level and/or activity of the gene, mRNA transcript or encoded protein.
- drusen may be detected by determining the presence of any of the following: amyloid A protein, amyloid P component, antichymotrypsin, apolipoprotein E, ⁇ 2 microglobulin, complement 3, complement C5, complement C5b-9 terminal complexes, factor X, fibrinogen, immunoglobulins (kappa and lambda), prothrombin and thrombospondin.
- the drusen-associated marker is a molecule whose production is altered in a drusen-associated molecular pathological process.
- one pathological process associated with drusen biogenesis is cell death and/or dysfunction of the retinal pigment epithelium (RPE).
- RPE retinal pigment epithelium
- a number of molecular markers have been associated with such dysfunctional RPE cells including: HLA-DR, CD68, vitronectin, apolipoprotein E, clusterin and S-100.
- HLA-DR expression is particularly unique for non-immunocompetent cells (although it is frequently expressed by cells early in an immune reaction).
- Still other molecular markers associated with dysfunctional choroid and RPE cells of AMD-affected eyes include gene products associated with cell death such as: death protein, heat shock protein 70, proteasome, Cu/Zn superoxide dismutase, cathepsins, and death adaptor protein RAIDD.
- drusen biogenesis is facilitated by dendritic cells and various immune-mediated events such as the production of autoantibodies in the sera of AMD patients.
- These autoantibodies are directed against drusen, the RPE and other retinal components.
- the invention provides for diagnostic assays designed to detect the presence and antigen specificity of such autoantibodies by methods known in the art, including standard immunohistochemical and Western blot techniques.
- immune system-associated molecules including Ig mu, lambda, J, and kappa chains and various cytokines are up-regulated in the RPE/choroid in conjunction with the formation of drusen. Accordingly, these immune-associated molecules provide another target for protein-based (e.g.
- drusen-associated molecular markers are those found in conjunction with subpopulation of choroidal cells that possess cellular processes which breach Bruch's membrane and terminate as bulbous, vesicle-filled "cores" within the centers of drusen.
- Specific marker molecules associated with these dendritic cells include: HLA-DR, CDla, CD4, CD14, CD68, CD83, CD86 and CD45.
- Other molecular markers appear to be associated with drusen-associated dendritic cell cores include: PECAM, MMP14, ubiquitin, FGF and HLA.
- the drusen-associated marker may be a cytokine which facilitates the development of drusen via a receptor-ligand interaction between a dendritic cell precursor and an injured tissue.
- cytokines include: IL-1, IL-6, IL-12, TNF-alpha, and GM-CSF.
- Other molecules involved in drusen development include heat shock proteins, DNA fragments, elastolytic peptides, angiogenic agents and factors up regulated, such as ⁇ integrin, collagen 6 ⁇ 2, collagen 6 ⁇ 3, elastin, HME, or down regulated (e.g. BIGH3) in fibrosis.
- a variety of means are currently available for detecting aberrant levels or activities of genes and gene products. For example, many methods are available for detecting specific alleles at human polymo ⁇ hic loci. The preferred method for detecting a specific polymo ⁇ hic allele will depend, in part, upon the molecular nature of the polymo ⁇ hism. For example, the various allelic forms of the polymo ⁇ hic locus may differ by a single base-pair of the DNA. Such single nucleotide polymo ⁇ hisms (or SNPs) are major contributors to genetic variation, comprising some 80% of all known polymo ⁇ hisms, and their density in the human genome is estimated to be on average 1 per 1 ,000 base pairs.
- SNPs are most frequently biallelic- occurring in only two different forms (although up to four different forms of an SNP, corresponding to the four different nucleotide bases occurring in DNA, are theoretically possible). Nevertheless, SNPs are mutationally more stable than other polymo ⁇ hisms, making them suitable for association studies in which linkage disequilibrium between markers and an unknown variant is used to map disease-causing mutations. In addition, because SNPs typically have only two alleles, they can be genotyped by a simple plus/minus assay rather than a length measurement, making them more amenable to automation.
- a variety of methods are available for detecting the presence of a particular single nucleotide polymo ⁇ hic allele in an individual. Advancements in this field have provided accurate, easy, and inexpensive large-scale SNP genotyping. Most recently, for example, several new techniques have been described including dynamic allele-specific hybridization (DASH), microplate a ⁇ ay diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMan system as well as various DNA "chip” technologies such as the Affymetrix SNP chips. These methods require amplification of the target genetic region, typically by PCR.
- DASH dynamic allele-specific hybridization
- MADGE microplate a ⁇ ay diagonal gel electrophoresis
- pyrosequencing oligonucleotide-specific ligation
- TaqMan system as well as various DNA "chip” technologies such as the Affymetrix SNP chips.
- a primer complementary to the allelic sequence immediately 3' to the polymo ⁇ hic site is permitted to hybridize to a target molecule obtained from a particular animal or human. If the polymo ⁇ hic site on the target molecule contains a nucleotide that is complementary to the particular exonuclease-resistant nucleotide derivative present, then that derivative will be inco ⁇ orated onto the end of the hybridized primer. Such inco ⁇ oration renders the primer resistant to exonuclease, and thereby permits its detection.
- a solution-based method is used for determining the identity of the nucleotide of a polymo ⁇ hic site.
- Patent 2,650,840; PCT Appln. No. WO91/02087) As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employed that is complementary to allelic sequences immediately 3' to a polymo ⁇ hic site. The method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymo ⁇ hic site will become inco ⁇ orated onto the terminus of the primer.
- GBA TM Genetic Bit Analysis
- Goelet, P. et al. PCT Appln. No. 92/157112.
- the method of Goelet, P. et al. uses mixtures of labeled terminators and a primer that is complementary to the sequence 3' to a polymo ⁇ hic site.
- the labeled terminator that is inco ⁇ orated is thus determined by, and complementary to, the nucleotide present in the polymo ⁇ hic site of the target molecule being evaluated.
- the method of Goelet, P. et al. is preferably a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase.
- RNA is initially isolated from available tissue and reverse-transcribed, and the segment of interest is amplified by PCR. The products of reverse transcription PCR are then used as a template for nested PCR amplification with a primer that contains an RNA polymerase promoter and a sequence for initiating eukaryotic translation.
- the unique motifs inco ⁇ orated into the primer permit sequential in vitro transcription and translation of the PCR products.
- the appearance of truncated polypeptides signals the presence of a mutation that causes premature termination of translation.
- DNA as opposed to RNA is used as a PCR template when the target region of interest is derived from a single exon.
- the DNA sample is obtained from a bodily fluid, e.g, blood, obtained by known techniques (e.g. venipuncture) or saliva.
- nucleic acid tests can be performed on dry samples (e.g. hair or skin).
- Diagnostic procedures may also be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary.
- Nucleic acid reagents may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, G.J., 1992, PCR in situ hybridization: protocols and applications, Raven Press, NY).
- profiles may also be assessed in such detection schemes.
- Finge ⁇ rint profiles may be generated, for example, by utilizing a differential display procedure, Northern analysis and/or RT-PCR.
- a prefe ⁇ ed detection method is allele specific hybridization using probes overlapping a region of at least one allele of a drusen associated marker, which has at least about 5, 10, 20, 25, or 30 nucleotides around the mutation or polymo ⁇ hic region.
- a solid phase support e.g., a "chip" (which can hold up to about 250,000 oligonucleotides). Oligonucleotides can be bound to a solid support by a variety of processes, including lithography. Mutation detection analysis using these chips comprising oligonucleotides, also termed "DNA probe a ⁇ ays" is described e.g., in Cronin et al. (1996) Human Mutation 7:244.
- a chip comprises all the allelic variants of at least one polymo ⁇ hic region of a gene. The solid phase support is then contacted with a test nucleic acid and hybridization to the specific probes is detected. Accordingly, the identity of numerous allelic variants of one or more genes can be identified in a simple hybridization experiment.
- Amplification techniques are known to those of skill in the art and include, but are not limited to cloning, polymerase chain reaction (PCR), polymerase chain reaction of specific alleles (ASA), ligase chain reaction (LCR), nested polymerase chain reaction, self sustained sequence replication (Guatelli, J.C. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874- 1878), transcriptional amplification system (Kwoh, D.Y. et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), and Q- Beta Replicase (Lizardi, PM.
- Amplification products may be assayed in a variety of ways, including size analysis, restriction digestion followed by size analysis, detecting specific tagged oligonucleotide primers in the reaction products, allele-specific oligonucleotide (ASO) hybridization, allele specific 5' exonuclease detection, sequencing, hybridization, and the like.
- PCR based detection means can include multiplex amplification of a plurality of markers simultaneously. For example, it is well known in the art to select PCR primers to generate PCR products that do not overlap in size and can be analyzed simultaneously.
- hybridization based detection means allow the differential detection of multiple PCR products in a sample.
- Other techniques are known in the art to allow multiplex analyses of a plurality of markers.
- the method includes the steps of (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, (iii) contacting the nucleic acid sample with one or more primers which specifically hybridize 5' and 3' to at least one allele of a drusen-associated marker under conditions such that hybridization and amplification of the allele occurs, and (iv) detecting the amplification product.
- nucleic acid e.g., genomic, mRNA or both
- abe ⁇ ant levels or activities of drusen-asssociated markers are identified by alterations in restriction enzyme cleavage patterns.
- sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis.
- any of a variety of sequencing reactions known in the art can be used to directly sequence the allele.
- Exemplary sequencing reactions include those based on techniques developed by Maxim and Gilbert ((1977) Proc. Natl Acad Sci USA 74:560) or Sanger (Sanger et al (1977) Proc. Nat. Acad. Sci USA 74:5463).
- any of a variety of automated sequencing procedures may be utilized when performing the subject assays (see, for example Biotechniques (1995) 19:448), including sequencing by mass spectrometry (see, for example PCT publication WO 94/16101; Cohen et al. (1996) Adv Chromatogr 36:127-162; and Griffin et al.
- the occu ⁇ ence of only one, two or three of the nucleic acid bases need be determined in the sequencing reaction. For instance, A-track or the like, e.g., where only one nucleic acid is detected, can be carried out.
- protection from cleavage agents can be used to detect mismatched bases in RNA/RNA or RNA/DNA or DNA DNA heteroduplexes (Myers, et al. (1985) Science 230:1242).
- cleavage agents such as a nuclease, hydroxylamine or osmium tetraoxide and with piperidine
- cleavage agents such as a nuclease, hydroxylamine or osmium tetraoxide and with piperidine
- RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digest the mismatched regions.
- either DNA DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al (1988) Proc. Natl Acad Sci USA 85:4397; and Saleeba et al (1992) Methods Enzymol. 217:286-295.
- the control DNA or RNA can be labeled for detection.
- the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes).
- DNA mismatch repair enzymes
- the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
- an appropriate probe is hybridized to a cDNA or other DNA product from a test cell(s).
- the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent No. 5,459,039.
- alterations in electrophoretic mobility will be used to identify aberrant levels or activities of drusen-associated markers.
- SSCP single strand conformation polymo ⁇ hism
- Single-stranded DNA fragments of sample and control locus alleles are denatured and allowed to renature.
- the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
- the DNA fragments may be labeled or detected with labeled probes.
- the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
- the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
- the movement of alleles in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495).
- DGGE denaturing gradient gel electrophoresis
- DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
- a temperature gradient is used in place of a denaturing agent gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
- oligonucleotide primers may be prepared in which the known mutation or nucleotide difference (e.g., in allelic variants) is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al (1989) Proc. Natl Acad. Sci USA 86:6230).
- Such allele specific oligonucleotide hybridization techniques may be used to test one mutation or polymo ⁇ hic region per reaction when oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations or polymo ⁇ hic regions when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
- Oligonucleotides used as primers for specific amplification may carry the mutation or polymo ⁇ hic region of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238.
- amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
- identification of an allelic variant is carried out using an oligonucleotide ligation assay (OLA), as described, e.g., in U.S. Pat. No. 4,998,617 and in Landegren, U. et al. ((1988) Science 241:1077-1080).
- OLA oligonucleotide ligation assay
- the OLA protocol uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target.
- One of the oligonucleotides is linked to a separation marker, e.g,. biotinylated, and the other is detectably labeled.
- oligonucleotides will hybridize such that their termini abut, and create a ligation substrate. Ligation then permits the labeled oligonucleotide to be recovered using avidin, or another biotin ligand.
- Nickerson, D. A. et al. have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson, D. A. et al. (1990) Proc. Natl. Acad. Sci. USA 87:8923-27). In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.
- U.S. Patent No. 5,593,826 discloses an OLA using an oligonucleotide having 3'-amino group and a 5'-phosphorylated oligonucleotide to form a conjugate having a phosphoramidate linkage.
- OLA OLA combined with PCR permits typing of two alleles in a ⁇ ingle micro liter well. By marking each of the allele-specific primers with a unique hapten, i.e.
- each OLA reaction can be detected by using hapten specific antibodies that are labeled with different enzyme reporters, alkaline phosphatase or horseradish peroxidase.
- This system permits the detection of the two alleles using a high throughput format that leads to the production of two different colors.
- kits for detecting a predisposition for developing a drusen-associated ocular disorder may contain one or more oligonucleotides, including 5' and 3' oligonucleotides that hybridize 5' and 3' to at least one drusen-associated marker.
- PCR amplification oligonucleotides should hybridize between 25 and 2500 base pairs apart, preferably between about 100 and about 500 bases apart, in order to produce a PCR product of convenient size for subsequent analysis.
- oligonucleotides may be any of a variety of natural and/or synthetic compositions such as synthetic oligonucleotides, restriction fragments, cDNAs, synthetic peptide nucleic acids (PNAs), and the like.
- the assay kit and method may also employ labeled oligonucleotides to allow ease of identification in the assays. Examples of labels which may be employed include radio-labels, enzymes, fluorescent compounds, streptavidin, avidin, biotin, magnetic moieties, metal binding moieties, antigen or antibody moieties, and the like.
- the kit may, optionally, also include DNA sampling means.
- DNA sampling means are well known to one of skill in the art and can include, but not be limited to substrates, such as filter papers, and the like; DNA purification reagents such as NucleonTM kits, lysis buffers, proteinase solutions and the like; PCR reagents, such as lOx reaction buffers, thermostable polymerase, dNTPs, and the like; and allele detection means such as restriction enzyme, allele specific oligonucleotides, degenerate oligonucleotide primers for nested PCR from dried blood.
- Information obtained using the diagnostic assays described herein may be useful for diagnosing or confirming that a symptomatic subject (e.g. a subject symptomatic for AMD), has a genetic defect (e.g. in an AMD-associated gene or in a gene that regulates the expression of a drusen-associated marker gene), which causes or contributes to the particular disease or disorder.
- a symptomatic subject e.g. a subject symptomatic for AMD
- a genetic defect e.g. in an AMD-associated gene or in a gene that regulates the expression of a drusen-associated marker gene
- the information can be used prognostically.
- a doctor can recommend a regimen (e.g. diet or exercise) or therapeutic protocol, useful for preventing or prolonging onset of the particular disease or condition in the individual.
- an individual's genetic profile or the genetic profile of a disease or condition, to which genetic alterations cause or contribute can enable a doctor to 1) more effectively prescribe a drug that will address the molecular basis of the disease or condition; and 2) better determine the appropriate dosage of a particular drug.
- the expression level of drusen-associated molecular marker proteins can be measured in many patients at various stages of the disease to generate a transcriptional or expression profile of the disease. Expression patterns of individual patients can then be compared to the expression profile of the disease to determine the appropriate drug and dose to administer to the patient.
- the ability to target populations expected to show the highest clinical benefit, based on the genetic profile, can enable: 1) the repositioning of marketed drugs with disappointing market results; 2) the rescue of drug candidates whose clinical development has been discontinued as a result of safety or efficacy limitations, which are patient subgroup- specific; and 3) an accelerated and less costly development for drug candidates and more optimal drug labeling (e.g. since the use of a drusen-associated molecular markers can be useful for optimizing effective dose).
- Cell-free assays can be used to identify compounds which are capable of interacting with a drusen-associated marker or binding partners thereto, to thereby modify their activity and/or interaction.
- a compound can, e.g., modify the structure of a drusen- associated marker or binding partner thereto and thereby effect its activity.
- one exemplary screening assay of the present invention includes the steps of contacting a drusen-associated marker or functional fragment thereof or a binding partner thereto with a test compound or library of test compounds and detecting the presence or absence of complex formation.
- the molecule can be labeled with a specific marker and the test compound or library of test compounds labeled with a different marker.
- Interaction of a test compound with a drusen-associated marker, fragment thereof or a binding partner thereto can then be detected by determining the level of the two labels after an incubation step and a washing step.
- the presence of two labels after the washing step is indicative of an interaction.
- An interaction between molecules can also be identified by using real-time BIA (Biomolecular Interaction Analysis, Pharmacia Biosensor AB) which detects surface plasmon resonance (SPR), an optical phenomenon. Detection depends on changes in the mass concentration of macromolecules at the biospecific interface, and does not require any labeling of interactants.
- a library of test compounds can be immobilized on a sensor surface, e.g., which forms one wall of a micro-flow cell. A solution containing the drusen-associated marker, functional fragment thereof or binding partner thereto is then flown continuously over the sensor surface. A change in the resonance angle as shown on a signal recording, indicates that an interaction has occu ⁇ ed. This technique is further described, e.g., in BIAtechnology Handbook by Pharmacia.
- Another exemplary screening assay of the present invention includes the steps of (a) forming a reaction mixture including: (i) a drusen-associated marker, (ii) a binding partner, and (iii) a test compound; and (b) detecting interaction of the drusen-associated marker and binding partner.
- the drusen-associated marker and binding partner can be produced recombinantly, purified from a source, e.g., plasma, or chemically synthesized, as described herein.
- the compounds of this assay can be contacted simultaneously.
- a drusen-associated marker can first be contacted with a test compound for an appropriate amount of time, following which the binding partner is added to the reaction mixture.
- the efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound.
- a control assay can also be performed to provide a baseline for comparison.
- Complex formation between a drusen-associated marker and a binding partner may be detected by a variety of techniques. Modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled drusen-associated markers or binding partners, by immunoassay, or by chromatographic detection.
- detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled drusen-associated markers or binding partners
- Binding of drusen-associated marker to a binding partner can be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes.
- a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix.
- glutathione-S-transferase (GST) fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
- drusen-associated marker gene product binding partner e.g. an 35s-labeled drusen-associated marker gene product binding partner
- test compound glutathione derivatized microtitre plates
- the drusen-associated marker gene product binding partner e.g. an 35s-labeled drusen-associated marker gene product binding partner
- the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g. beads placed in scintillant), or in the supernatant after the complexes are subsequently dissociated.
- the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of drusen-associated marker gene product protein or associated binding partner found in the bead fraction quantitated from the gel using standard electrophoretic techniques such as described in the appended examples.
- a drusen-associated marker or its cognate binding partner can be immobilized utilizing conjugation of biotin and streptavidin.
- biotinylated drusen-associated marker molecules can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
- antibodies reactive with drusen-associated marker can be derivatized to the wells of the plate, and the drusen associated marker trapped in the wells by antibody conjugation.
- preparations of a drusen-associated marker, a binding partner and a test compound are incubated in the presenting wells of the plate, and the amount of complex trapped in the well can be quantitated.
- Exemplary methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the drusen-associated marker or binding partner, or which are reactive with the drusen-associated marker and compete with the binding partner; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the binding partner, either intrinsic or extrinsic activity.
- the enzyme can be chemically conjugated or provided as a fusion protein with the drusen-associated marker or binding partner.
- the drusen-associated marker or binding partner can be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of polypeptide trapped in the complex can be assessed with a chromogenic substrate of the enzyme, e.g. 3,3'-diamino-benzadine terahydrochloride or 4- chloro-1-napthol.
- a fusion protein comprising the polypeptide and glutathione-S- transferase can be provided, and complex formation quantitated by detecting the GST activity using l-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).
- the protein to be detected in the complex can be "epitope tagged" in the form of a fusion protein which includes, in addition to the drusen- associated marker, a second polypeptide for which antibodies are readily available (e.g. from commercial sources).
- the GST fusion proteins described above can also be used for quantification of binding using antibodies against the GST moiety.
- Other useful epitope tags include myc-epitopes (e.g., see Ellison et al.
- Cell-free assays can also be used to identify compounds which modulate an activity of an drusen-associated marker. Accordingly, in one embodiment, a drusen-associated marker is contacted with a test compound and the catalytic activity of the drusen-associated marker is monitored. In one embodiment, the ability of a drusen-associated marker to bind a target molecule is determined. The binding affinity of a drusen-associated marker to a target molecule can be determined according to methods known in the art.
- Determination of the enzymatic activity of a drusen-associated marker can be performed with the aid of the substrate furanacryloyl-L-phenylalanyl-glycyl-glycine (FAPGG) under conditions described in Holmquist et al. (1979) Anal. Biochem. 95:540 and in U.S. Patent No. 5,259,045.
- FPGG furanacryloyl-L-phenylalanyl-glycyl-glycine
- drusen-associated markers facilitate the generation of cell-based assays, e.g., for identifying small molecule agonists or antagonists.
- a cell expressing a drusen-associated marker on the outer surface of its cellular membrane is incubated in the presence of a test compound alone or in the presence of a test compound and a drusen- associated marker and the interaction between the test compound and the drusen-associated marker or between the drusen-associated marker and the drusen-associated marker binding partner is detected, e.g., by using a microphysiometer (McConnell et al. (1992) Science 257:1906).
- This assay system thus provides a means of identifying molecular antagonists which, for example, function by interfering with drusen-associated marker - ligand (e.g. receptor) interactions, as well as molecular agonist which, for example, function by activating a drusen-associated marker.
- drusen-associated marker - ligand e.g. receptor
- Cell based assays can also be used to identify compounds which modulate expression of a drusen-associated marker gene, modulate translation of a drusen-associated marker mRNA, or which modulate the stability of a drusen-associated marker mRNA or protein. Accordingly, in one embodiment, a cell which is capable of expressing a drusen- associated marker, e.g., a retinal epithelial cell, is incubated with a test compound and the amount of drusen-associated marker produced in the cell medium is measured and compared to that produced from a cell which has not been contacted with the test compound.
- a drusen-associated marker e.g., a retinal epithelial cell
- the specificity of the compound vis a vis a drusen-associated marker can be confirmed by various control analysis, e.g., measuring the expression of one or more control genes.
- Compounds which can be tested include small molecules, proteins, and nucleic acids.
- this assay can be used to determine the efficacy of antisense or ribozymes to drusen-associated marker genes.
- the effect of a test compound on transcription of a drusen-associated marker gene is determined by transfection experiments using a reporter gene operatively linked to at least a portion of the promoter of a drusen-associated marker gene.
- a promoter region of a gene can be isolated, e.g., from a genomic library according to methods known in the art.
- the reporter gene can be any gene encoding a protein which is readily quantifiable, e.g, the luciferase or CAT gene. Such reporter gene are well known in the art.
- This invention further pertains to novel agents identified by the above- described screening assays and uses thereof for treatments as described herein.
- the invention further provides for animal models, including transgenic animals, which can be used for a variety of purposes, e.g., to identify genetic loci involved in the common etiology of drusen associated diseases, and, further, to create animal models for the treatment of drusen associated diseases.
- the transgenic animals can contain a transgene, such as reporter gene, under the control of a drusen-associated marker gene promoter or fragment thereof. These animals are useful, e.g., for identifying drugs that modulate production of the drusen-associated molecular marker, such as by modulating Factor X, HLA-DR, IL-6 or elastin gene expression.
- a target gene promoter can be isolated, e.g., by screening of a genomic library with an appropriate cDNA fragment and characterized according to methods known in the art.
- the transgenic animal containing a reporter gene is used to screen a class of bioactive molecules for their ability to modulate expression of a drusen-associated molecular marker such as a DRAM.
- a drusen-associated molecular marker such as a DRAM.
- non-human animals within the scope of the invention include those in which the expression of the endogenous target gene has been mutated or "knocked out".
- a "knock out" animal is one carrying a homozygous or heterozygous deletion of a particular gene or genes. These animals could be useful to determine whether the absence of the target will result in a specific phenotype, in particular whether these mice have or are likely to develop a drusen associated disease.
- these animals are useful in screens for drugs which alleviate or attenuate the disease condition resulting from the mutation of drusen associated markers.
- These animals are also useful for determining the effect of a specific amino acid difference, or allelic variation, in a target gene. That is, the target knock out animals can be crossed with transgenic animals expressing, e.g., a mutated form or allelic variant of the target gene containing a drusen associated marker, thereby resulting in an animal which expresses only the mutated protein and not the wild-type target gene product.
- Knock out mice are generated by homologous integration of a "knock out" construct into a mouse embryonic stem cell chromosome which encodes the gene to be knocked out.
- gene targeting which is a method of using homologous recombination to modify an animal's genome, can be used to introduce changes into cultured embryonic stem cells. By targeting a specific gene of interest in ES cells, these changes can be introduced into the germlines of animals to generate chimeras.
- the gene targeting procedure is accomplished by introducing into tissue culture cells a DNA targeting construct that includes a segment homologous to a target locus, and which also includes an intended sequence modification to the genomic sequence (e.g., insertion, deletion, point mutation). The treated cells are then screened for accurate targeting to identify and isolate those which have been properly targeted.
- a DNA targeting construct that includes a segment homologous to a target locus, and which also includes an intended sequence modification to the genomic sequence (e.g., insertion, deletion, point mutation).
- the treated cells are then screened for accurate targeting to identify and isolate those which have been properly targeted.
- Gene targeting in embryonic stem cells is in fact a scheme contemplated by the present invention as a means for disrupting a target gene function through the use of a targeting transgene construct designed to undergo homologous recombination with one or more target genomic sequences.
- the targeting construct can be a ⁇ anged so that, upon recombination with an element of a target gene, a positive selection marker is inserted into (or replaces) coding sequences of the gene.
- the inserted sequence functionally disrupts the target gene, while also providing a positive selection trait.
- Exemplary targeting constructs are described in more detail below.
- the embryonic stem cells (ES cells ) used to produce the knockout animals will be of the same species as the knockout animal to be generated.
- mouse embryonic stem cells will usually be used for generation of knockout mice.
- Embryonic stem cells are generated and maintained using methods well known to the skilled artisan such as those described by Doetschman et al. (1985) J. Embryol Exp. Mol Biol. 87:27-45). Any line of ES cells can be used, however, the line chosen is typically selected for the ability of the cells to integrate into and become part of the germ line of a developing embryo so as to create germ line transmission of the knockout construct. Thus, any ES cell line that is believed to have this capability is suitable for use herein.
- ES cell line Another mouse strain that is typically used for production of ES cells, is the 129J strain.
- Another ES cell line is murine cell line D3 (American Type Culture Collection, catalog no. CKL 1934)
- WW6 cell line Still another prefe ⁇ ed ES cell line.
- the cells are cultured and prepared for knockout construct insertion using methods well known to the skilled artisan, such as those set forth by Robertson in: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. IRL Press, Washington, D.C. [1987]); by Bradley et al. (1986) Current Topics in Devel Biol. 20:357-371); and by Hogan et al. (Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [1986].
- a knock out construct refers to a uniquely configured fragment of nucleic acid which is introduced into a stem cell line and allowed to recombine with the genome at the chromosomal locus of the gene of interest to be mutated.
- a given knock out construct is specific for a given gene to be targeted for disruption. Nonetheless, many common elements exist among these constructs and these elements are well known in the art.
- a typical knock out construct contains nucleic acid fragments of not less than about 0.5 kb nor more than about 10.0 kb from both the 5' and the 3' ends of the genomic locus which encodes the gene to be mutated.
- nucleic acid which encodes a positive selectable marker, such as the neomycin resistance gene (neo R ).
- the resulting nucleic acid fragment consisting of a nucleic acid from the extreme 5' end of the genomic locus linked to a nucleic acid encoding a positive selectable marker which is in turn linked to a nucleic acid from the extreme 3' end of the genomic locus of interest, omits most of the coding sequence for the gene of interest to be knocked out.
- the resulting construct recombines homologously with the chromosome at this locus, it results in the loss of the omitted coding sequence, otherwise known as the structural gene, from the genomic locus.
- a stem cell in which such a rare homologous recombination event has taken place can be selected for by virtue of the stable integration into the genome of the nucleic acid of the gene encoding the positive selectable marker and subsequent selection for cells expressing this marker gene in the presence of an appropriate drug (neomycin in this example).
- a "knock-in" construct refers to the same basic arrangement of a nucleic acid encoding a 5' genomic locus fragment linked to nucleic acid encoding a positive selectable marker which in turn is linked to a nucleic acid encoding a 3' genomic locus fragment, but which differs in that none of the coding sequence is omitted and thus the 5' and the 3' genomic fragments used were initially contiguous before being disrupted by the introduction of the nucleic acid encoding the positive selectable marker gene.
- This "knock-in"type of construct is thus very useful for the construction of mutant transgenic animals when only a limited region of the genomic locus of the gene to be mutated, such as a single exon, is available for cloning and genetic manipulation.
- the "knock-in” construct can be used to specifically eliminate a single functional domain of the targetted gene, resulting in a transgenic animal which expresses a polypeptide of the targetted gene which is defective in one function, while retaining the function of other domains of the encoded polypeptide.
- This type of "knock-in” mutant frequently has the characteristic of a so-called “dominant negative” mutant because, especially in the case of proteins which homomultimerize, it can specifically block the action of (or "poison") the polypeptide product of the wild-type gene from which it was derived.
- a marker gene is integrated at the genomic locus of interest such that expression of the marker gene comes under the control of the transcriptional regulatory elements of the targeted gene.
- a marker gene is one that encodes an enzyme whose activity can be detected (e.g., b-galactosidase), the enzyme substrate can be added to the cells under suitable conditions, and the enzymatic activity can be analyzed.
- an enzyme whose activity can be detected (e.g., b-galactosidase)
- the enzyme substrate can be added to the cells under suitable conditions, and the enzymatic activity can be analyzed.
- One skilled in the art will be familiar with other useful markers and the means for detecting their presence in a given cell. All
- homologous recombination of the above described "knock out” and “knock in” constructs is very rare and frequently such a construct inserts nonhomologously into a random region of the genome where it has no effect on the gene which has been targeted for deletion, and where it can potentially recombine so as to disrupt another gene which was otherwise not intended to be altered.
- Such nonhomologous recombination events can be selected against by modifying the abovementioned knock out and knock in constructs so that they are flanked by negative selectable markers at either end (particularly through the use of two allelic variants of the thymidine kinase gene, the polypeptide product of which can be selected against in expressing cell lines in an appropriate tissue culture medium well known in the art - i.e. one containing a drug such as 5- bromodeoxyuridine).
- a prefe ⁇ ed embodiment of such a knock out or knock in construct of the invention consist of a nucleic acid encoding a negative selectable marker linked to a nucleic acid encoding a 5' end of a genomic locus linked to a nucleic acid of a positive selectable marker which in turn is linked to a nucleic acid encoding a 3' end of the same genomic locus which in turn is linked to a second nucleic acid encoding a negative selectable marker
- Nonhomologous recombination between the resulting knock out construct and the genome will usually result in the stable integration of one or both of these negative selectable marker genes and hence cells which have undergone nonhomologous recombination can be selected against by growth in the appropriate selective media (e.g.
- Each knockout construct to be inserted into the cell must first be in the linear form. Therefore, if the knockout construct has been inserted into a vector (described infra), linearization is accomplished by digesting the DNA with a suitable restriction endonuclease selected to cut only within the vector sequence and not within the knockout construct sequence.
- the knockout construct is added to the ES cells under appropriate conditions for the insertion method chosen, as is known to the skilled artisan. For example, if the ES cells are to be electroporated, the ES cells and knockout construct DNA are exposed to an electric pulse using an electroporation machine and following the manufacturer's guidelines for use. After electroporation, the ES cells are typically allowed to recover under suitable incubation conditions. The cells are then screened for the presence of the knock out construct as explained above. Where more than one construct is to be introduced into the ES cell, each knockout construct can be introduced simultaneously or one at a time.
- the cells can be inserted into an embryo. Insertion may be accomplished in a variety of ways known to the skilled artisan, however a prefe ⁇ ed method is by microinjection. For microinjection, about 10-30 cells are collected into a micropipet and injected into embryos that are at the proper stage of development to permit integration of the foreign ES cell containing the knockout construct into the developing embryo. For instance, the transformed ES cells can be microinjected into blastocytes. The suitable stage of development for the embryo used for insertion of ES cells is very species dependent, however for mice it is about 3.5 days. The embryos are obtained by perfusing the uterus of pregnant females. Suitable methods for accomplishing this are known to the skilled artisan, and are set forth by, e.g., Bradley et al. (supra).
- prefe ⁇ ed embryos are male.
- the prefe ⁇ ed embryos also have genes coding for a coat color that is different from the coat color encoded by the ES cell genes.
- the offspring can be screened easily for the presence of the knockout construct by looking for mosaic coat color (indicating that the ES cell was inco ⁇ orated into the developing embryo).
- the embryo selected will carry genes for black or brown fur.
- the embryo may be implanted into the uterus of a pseudopregnant foster mother for gestation. While any foster mother may be used, the foster mother is typically selected for her ability to breed and reproduce well, and for her ability to care for the young. Such foster mothers are typically prepared by mating with vasectomized males of the same species.
- the stage of the pseudopregnant foster mother is important for successful implantation, and it is species dependent. For mice, this stage is about 2-3 days pseudopregnant.
- Offspring that are born to the foster mother may be screened initially for mosaic coat color where the coat color selection strategy (as described above, and in the appended examples) has been employed.
- DNA from tail tissue of the offspring may be screened for the presence of the knockout construct using Southern blots and/or PCR as described above. Offspring that appear to be mosaics may then be crossed to each other, if they are believed to carry the knockout construct in their germ line, in order to generate homozygous knockout animals.
- Homozygotes may be identified by Southern blotting of equivalent amounts of genomic DNA from mice that are the product of this cross, as well as mice that are known heterozygotes and wild type mice.
- Northern blots can be used to probe the mRNA for the presence or absence of transcripts encoding either the gene knocked out, the marker gene, or both.
- Western blots can be used to assess the level of expression of the Target gene knocked out in various tissues of the offspring by probing the Western blot with an antibody against the particular target protein, or an antibody against the marker gene product, where this gene is expressed.
- in situ analysis such as fixing the cells and labeling with antibody
- FACS fluorescence activated cell sorting
- knock-out or disruption transgenic animals are also generally known. See, for example, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
- Recombinase dependent knockouts can also be generated, e.g. by homologous recombination to insert target sequences, such that tissue specific and/or temporal control of inactivation of a target-gene can be controlled by recombinase sequences (described infra).
- Animals containing more than one knockout construct and/or more than one transgene expression construct are prepared in any of several ways.
- the prefe ⁇ ed manner of preparation is to generate a series of mammals, each containing one of the desired transgenic phenotypes.
- Such animals are bred together through a series of crosses, backcrosses and selections, to ultimately generate a single animal containing all desired knockout constructs and/or expression constructs, where the animal is otherwise congenic (genetically identical) to the wild type except for the presence of the knockout construct(s) and or transgene(s) .
- a target transgene can encode the wild-type form of the protein, or can encode homologs thereof, including both agonists and antagonists, as well as antisense constructs.
- the expression of the transgene is restricted to specific subsets of cells, tissues or developmental stages utilizing, for example, cis-acting sequences that control expression in the desired pattern.
- mosaic expression of a target protein can be essential for many forms of lineage analysis and can additionally provide a means to assess the effects of, for example, lack of target expression which might grossly alter development in small patches of tissue within an otherwise normal embryo.
- tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the transgene in certain spatial patterns.
- temporal patterns of expression can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences.
- target sequence refers to a nucleotide sequence that is genetically recombined by a recombinase.
- the target sequence is flanked by recombinase recognition sequences and is generally either excised or inverted in cells expressing recombinase activity.
- Recombinase catalyzed recombination events can be designed such that recombination of the target sequence results in either the activation or repression of expression of one of the subject target proteins.
- excision of a target sequence which interferes with the expression of a recombinant target gene can be designed to activate expression of that gene.
- This interference with expression of the protein can result from a variety of mechanisms, such as spatial separation of the target gene from the promoter element or an internal stop codon.
- the transgene can be made wherein the coding sequence of the gene is flanked by recombinase recognition sequences and is initially transfected into cells in a 3' to 5' orientation with respect to the promoter element.
- inversion of the target sequence will reorient the subject gene by placing the 5' end of the coding sequence in an orientation with respect to the promoter element which allow for promoter driven transcriptional activation.
- transgenic animals of the present invention all include within a plurality of their cells a transgene of the present invention, which transgene alters the phenotype of the "host cell” with respect to regulation of cell growth, death and/or differentiation. Since it is possible to produce transgenic organisms of the invention utilizing one or more of the transgene constructs described herein, a general description will be given of the production of transgenic organisms by referring generally to exogenous genetic material. This general description can be adapted by those skilled in the art in order to inco ⁇ orate specific transgene sequences into organisms utilizing the methods and materials described below.
- crelloxP recombinase system of bacteriophage PI (Lakso et al. (1992) PNAS 89:6232-6236; Orban et al. (1992) PNAS 89:6861-6865) or the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; PCT publication WO 92/15694) can be used to generate in vivo site-specific genetic recombination systems. Cre recombinase catalyzes the site-specific recombination of an intervening target sequence located between loxP sequences.
- loxP sequences are 34 base pair nucleotide repeat sequences to which the Cre recombinase binds and are required for Cre recombinase mediated genetic recombination.
- the orientation of loxP sequences determines whether the intervening target sequence is excised or inverted when Cre recombinase is present (Abremski et al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing the excision of the target sequence when the loxP sequences are oriented as direct repeats and catalyzes inversion of the target sequence when loxP sequences are oriented as inverted repeats.
- genetic recombination of the target sequence is dependent on expression of the Cre recombinase.
- Expression of the recombinase can be regulated by promoter elements which are subject to regulatory control, e.g., tissue-specific, developmental stage-specific, inducible or repressible by externally added agents. This regulated control will result in genetic recombination of the target sequence only in cells where recombinase expression is mediated by the promoter element.
- the activation expression of a recombinant target protein can be regulated via control of recombinase expression.
- crelloxP recombinase system to regulate expression of a recombinant target protein requires the construction of a transgenic animal containing transgenes encoding both the Cre recombinase and the subject protein. Animals containing both the Cre recombinase and a recombinant target gene can be provided through the construction of "double" transgenic animals. A convenient method for providing such animals is to mate two transgenic animals each containing a transgene, e.g., a target gene and recombinase gene.
- transgenic animals containing a target transgene in a recombinase-mediated expressible format derives from the likelihood that the subject protein, whether agonistic or antagonistic, can be deleterious upon expression in the transgenic animal.
- a founder population in which the subject transgene is silent in all tissues, can be propagated and maintained. Individuals of this founder population can be crossed with animals expressing the recombinase in, for example, one or more tissues and/or a desired temporal pattern.
- conditional transgenes can be provided using prokaryotic promoter sequences which require prokaryotic proteins to be simultaneous expressed in order to facilitate expression of the target transgene.
- Exemplary promoters and the co ⁇ esponding trans-activating prokaryotic proteins are given in U.S. Patent No. 4,833,080.
- expression of the conditional transgenes can be induced by gene therapy-like methods wherein a gene encoding the trans-activating protein, e.g.
- a recombinase or a prokaryotic protein is delivered to the tissue and caused to be expressed, such as in a cell- type specific manner.
- a target transgene could remain silent into adulthood until "turned on” by the introduction of the trans-activator.
- the "transgenic non-human animals" of the invention are produced by introducing transgenes into the germline of the non-human animal.
- Embryonal target cells at various developmental stages can be used to introduce transgenes. Different methods are used depending on the stage of development of the embryonal target cell.
- the specific line(s) of any animal used to practice this invention are selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness.
- the haplotype is a significant factor. For example, when transgenic mice are to be produced, strains such as C57BL/6 or FVB lines are often used (Jackson Laboratory, Bar Harbor, ME).
- Prefe ⁇ ed strains are those with H-2 ⁇ , H-2 ⁇ or H-2°l haplotypes such as C57BL/6 or DBA/1.
- the line(s) used to practice this invention may themselves be transgenics, and/or may be knockouts (i.e., obtained from animals which have one or more genes partially or completely suppressed) .
- the transgene construct is introduced into a single stage embryo. The zygote is the best target for micro-injection. In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of l-2pl of DNA solution.
- zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be inco ⁇ orated into the host gene before the first cleavage (Brinster et al. (1985) PNAS 82:4438-4442). As a consequence, all cells of the transgenic animal will carry the inco ⁇ orated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene.
- the nucleotide sequence comprising the transgene is introduced into the female or male pronucleus as described below.
- the male pronucleus is prefe ⁇ ed. It is most prefe ⁇ ed that the exogenous genetic material be added to the male DNA complement of the zygote prior to its being processed by the ovum nucleus or the zygote female pronucleus.
- the exogenous genetic material be added to the male complement of DNA or any other complement of DNA prior to its being affected by the female pronucleus.
- the exogenous genetic material is added to the early male pronucleus, as soon as possible after the formation of the male pronucleus, which is when the male and female pronuclei are well separated and both are located close to the cell membrane.
- the exogenous genetic material could be added to the nucleus of the sperm after it has been induced to undergo decondensation.sperm containing the exogenous genetic material can then be added to the ovum or the decondensed sperm could be added to the ovum with the transgene constructs being added as soon as possible thereafter.
- Introduction of the transgene nucleotide sequence into the embryo may be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection.
- the embryo may be incubated in vitro for varying amounts of time, or reimplanted into the su ⁇ ogate host, or both. In vitro incubation to maturity is within the scope of this invention.
- a zygote is essentially the formation of a diploid cell which is capable of developing into a complete organism.
- the zygote will be comprised of an egg containing a nucleus formed, either naturally or artificially, by the fusion of two haploid nuclei from a gamete or gametes.
- the gamete nuclei must be ones which are naturally compatible, i.e., ones which result in a viable zygote capable of undergoing differentiation and developing into a functioning organism.
- a euploid zygote is prefe ⁇ ed. If an aneuploid zygote is obtained, then the number of chromosomes should not vary by more than one with respect to the euploid number of the organism from which either gamete originated.
- the biological limit of the number and variety of DNA sequences will vary depending upon the particular zygote and functions of the exogenous genetic material and will be readily apparent to one skilled in the art, because the genetic material, including the exogenous genetic material, of the resulting zygote must be biologically capable of initiating and maintaining the differentiation and development of the zygote into a functional organism.
- the number of copies of the transgene constructs which are added to the zygote is dependent upon the total amount of exogenous genetic material added and will be the amount which enables the genetic transformation to occur. Theoretically only one copy is required; however, generally, numerous copies are utilized, for example, 1 ,000-20,000 copies of the transgene construct, in order to insure that one copy is functional. As regards the present invention, there will often be an advantage to having more than one functioning copy of each of the inserted exogenous DNA sequences to enhance the phenotypic expression of the exogenous DNA sequences.
- exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection. Microinjection of cells and cellular structures is known and is used in the art.
- Reimplantation is accomplished using standard methods. Usually, the su ⁇ ogate host is anesthetized, and the embryos are inserted into the oviduct. The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of off spring the species naturally produces.
- Transgenic offspring of the su ⁇ ogate host may be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product.
- DNA is prepared from tail tissue and analyzed by Southern analysis or PCR for the transgene.
- the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis.
- Alternative or additional methods for evaluating the presence of the transgene include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like. Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
- suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like.
- Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
- Progeny of the transgenic animals may be obtained by mating the transgenic animal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic animal.
- the partner may or may not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a different transgene, or both.
- the partner may be a parental line.
- in vitro fertilization is used, the fertilized embryo may be implanted into a su ⁇ ogate host or incubated in vitro, or both. Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.
- the transgenic animals produced in accordance with the present invention will include exogenous genetic material.
- the exogenous genetic material will, in certain embodiments, be a DNA sequence which results in the production of a Target protein (either agonistic or antagonistic), and antisense transcript, or a Target mutant.
- the sequence will be attached to a transcriptional control element, e.g., a promoter, which preferably allows the expression of the transgene product in a specific type of cell.
- Retroviral infection can also be used to introduce transgene into a non-human animal.
- the developing non-human embryo can be cultured in vitro to the blastocyst stage.
- the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) PNAS 73 : 1260- 1264).
- Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986).
- the viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al.
- the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring.
- transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982) supra).
- ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature 322:445-448).
- Transgenes can be efficiently introduced into the ES cells by D ⁇ A transfection or by retrovirus-mediated transduction.
- Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.
- Jaenisch, R. (1988) Science 240:1468-1474 For review see Jaenisch, R. (1988) Science 240:1468-1474.
- the invention provides compositions and methods for treating or preventing the development of drusen associated ocular disorders.
- Appropriate therapeutics can include any molecule or compound that slows or prevents any of the processes involved in drusen biogenesis, including dendritic cell activation and recruitment, immune mediated events, choroidal fibrosis and neovascularization, extracellular matrix disequilibrium, etc.
- an appropriate therapeutic may be an anti-inflammatory agent, such as an antagonist of T ⁇ F- ⁇ , IL-1, GM- CSF, IL-4 or IL-13.
- the therapeutic may also be IL-10, M-CSF, IL-6 and IL-4 or an agonist thereof.
- the agent is selected from the group consisting of cytokines, chemokines and agonists and antagonists thereof.
- useful therapeutics include agents that inhibit inflammation.
- the macular degeneration therapeutic is an inhibitor of the expression of one or more DRAMs, such as, for example, amyloid A protein, amyloid P component, ⁇ l -antichymotrypsin, apolipoprotein E, ⁇ 2 microglobulin, complement 3, complement C5, complement C5b-9 terminal complexes, factor X, fibrinogen, immunoglobulins (kappa and lambda), prothrombin, thrombospondin or vitronectin.
- the invention provides method for treating a drusen associated disease by modulating the production of DRAMs, e.g., inhibiting or antagonizing their gene expression or activity.
- amyloid P and ⁇ i -antichymotrypsin an inhibitor of serine proteases
- amyloid P is also found in non-amyloid deposits associated with atherosclerosis (Niculescu, et al., 1987), keratin intermediate filament aggregates (Hintner, et al., 1988), and dense deposits associated with glomerulonephropathy (Yang, et al., 1992). It associates with elastic fibers and may function as an protease inhibitor in vivo (Li and McAdam, 1984; Vachino, et al., 1988).
- Inhibiting of drusen formation or facilitating drusen clearance or resolution may be accomplished by a number of regimes, such as (1) inhibition of RNA synthesis for one or more DRAMs, (2) enhancement of RNA turnover or degradation of one or more DRAMs, (3) inhibition of translation of RNA for one or more DRAMs into protein, (4) inhibition of protein processing or transport of one or more DRAMs; (5) inhibition of drusen formation by blocking particular protein binding sites on one or more factors which participate in inter- and intramolecular binding necessary for the association of DRAMs which results in a drusen deposit; (6) digestion or perturbation of protein deposits (e.g., using enzymes); (7) targeting and destroying DRAMs in situ (e.g., using enzyme-antibody techniques).
- regimes such as (1) inhibition of RNA synthesis for one or more DRAMs, (2) enhancement of RNA turnover or degradation of one or more DRAMs, (3) inhibition of translation of RNA for one or more DRAMs into protein, (4) inhibition of protein processing or transport of one or
- DRAMs may be targeted by using photoreactive laser therapy, for example, or other means for targeting and destroying a protein in situ which are well known in the art.
- Such means may include antibodies conjugated to a reactive group such as a protease or chemical substance which, when activated, cleaves or denatures the individual components or interferes with the interaction of two or more components.
- therapeutics for drusen-associated diseases include agents which alter the gene expression of factors that regulate the expression of one or more DRAMs and all other drusen biogenesis associated proteins.
- agents may be "antagonists" which inhibit, either directly or indirectly, DRAM biosynthesis.
- the agent may specifically inhibit the transcription or translation of a DRAM, for example.
- it may be preferable to upregulate either directly or indirectly a gene or genes which will increase the synthesis of a naturally occurring therapeutic agent. For example, the increased gene expression of a proteolytic enzyme that degrades one or more DRAMS or a cytokine or drug that modulates immune responses may be desired.
- the invention is therefore also useful for monitoring the efficacy of a drusen therapeutic or preventative treatment, the absence of drusen core formation, the disappearance of drusen or of a drusen core providing evidence of efficacy of the therapeutic or treatment.
- the therapeutics of the invention relate to antisense therapy.
- antisense therapy refers to administration or in situ generation of oligonucleotide molecules or their derivatives which specifically hybridize (e.g., bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding one or more DRAMs so as to inhibit expression of that protein, e.g., by inhibiting transcription and/or translation.
- binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.
- antisense therapy refers to the range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences.
- an antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a DRAM protein.
- the antisense construct can be an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences of a DRAM gene.
- Such oligonucleotide probes are preferably modified oligonucleotides which are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, and are therefore stable in vivo.
- nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Patent Nos. 5,176,996, 5,264,564 and 5,256,775).
- Approaches to constructing oligomers useful in antisense therapy are well known in the art.
- antisense DNA oligodeoxyribonucleotides derived from the translation initiation site, e.g., between the -10 and +10 regions of the drusen-associated component nucleotide sequence of interest, are prefe ⁇ ed.
- Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to a DRAM mRNA, or their agonists or antagonists.
- the antisense oligonucleotides bind to the subject mRNA transcripts and prevent translation or promote degradation of the transcript. Absolute complementarity, although prefe ⁇ ed, is not required.
- a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
- the ability to hybridize depends on both the degree of complementarity and the length of the antisense nucleic acid.
- compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients.
- the compounds and their physiologically acceptable salts and solvates may be formulated for administration by, for example, eye drops, injection, inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
- the compounds of the invention can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical
- a prefe ⁇ ed method of administration is an eye drop.
- injection is prefe ⁇ ed, including intramuscular, intravenous, intraperitoneal, and subcutaneous.
- the compounds of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
- the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
- Other prefe ⁇ ed methods of administration include choroidal injection, transscleral injection or placing a scleral patch, and selective arterial catheterization.
- prefe ⁇ ed deliveries are intraocular, including transretinal, subconjunctival bulbar, scleral pocket and scleral cutdown injections.
- the agent can be alternatively administered intravascularly, such as intravenously (IV) or intraarterially.
- choroidal injection and scleral patching are similar.
- the clinician uses a local approach to the eye after initiation of appropriate anesthesia, including painkillers and ophthalmoplegics.
- a needle containing the therapeutic compound is directed into the patient's choroid or sclera and inserted under sterile conditions.
- the needle When the needle is properly positioned the compound is injected into either or both of the choroid or sclera.
- the clinician may choose a sustained release or longer acting formulation.
- the procedure may need repetition only every several months or several years, depending on the patient's tolerance of the treatment and response.
- the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate).
- binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
- fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
- lubricants e.g., magnesium stearate, talc or silica
- disintegrants e.g., potato star
- Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
- Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
- suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
- emulsifying agents e.g., lecithin or acacia
- non-aqueous vehicles e.g., ationd oil, oily esters,
- the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
- the therapeutic may be administered alone or in combination with other molecules known to have a beneficial effect on retinal attachment or damaged retinal tissue, including molecules capable of tissue repair and regeneration and/or inhibiting inflammation.
- useful cofactors include basic fibroblast growth factor (bFGF), LaVail et al. (1998), / «ve5t. Ophthalmol. Vis. Sci. 39:592-602, ciliary neurotrophic factor (CNTF), LaVail et al. (1998), Invest. Ophthalmol. Vis. Sci. 39:592-602, axokine (a mutein of CNTF), LaVail et al. (1998), Invest. Ophthalmol.
- bFGF basic fibroblast growth factor
- CNTF ciliary neurotrophic factor
- CNTF LaVail et al.
- axokine a mutein of CNTF
- prostaglandin E2 La Vail et al. (1998), Invest. Ophthalmol. Vis. Sci. 39:581-591, 30kD survival factor, taurine, and vitamin A.
- Other useful cofactors include symptom-alleviating cofactors, including antiseptics, antibiotics, antiviral and anti fungal agents and analgesics and anesthetics.
- a therapeutic also may be associated with means for targeting the therapeutics to a desired tissue.
- an antibody or other binding protein that interacts specifically with a surface molecule on the desired target tissue cells also may be used.
- Such targeting molecules further may be covalently associated to a therapeutic, e.g., by chemical crosslinking, or by using standard genetic engineering means to create, for example, an acid labile bond such as an Asp-Pro linkage.
- Useful targeting molecules may be designed, for example, using the simple chain binding site technology disclosed, for example, in U.S. Patent No. 5,091,513.
- Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
- the compositions may take the form of tablets or lozenges formulated in conventional manner.
- the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.
- Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be
- the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
- Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
- the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyro gen-free water, before use.
- the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
- the compounds may also be formulated as a depot preparation.
- Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
- the compounds may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
- microspheres which offer the possibility of local noninvasive delivery of drugs over an extended period of time.
- This technology utilizes microspheres of precapillary size which can be injected via a coronary catheter into any selected part of the body, e.g., the eye, or other organs without causing inflammation or ischemia.
- the administered therapeutic is slowly released from these microspheres and taken up by su ⁇ ounding tissue cells (e.g., endothelial cells).
- Systemic administration can also be by transmucosal or transdermal means.
- penetrants appropriate to the barrier to be permeated are used in the formulation.
- penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives.
- detergents may be used to facilitate permeation.
- Transmucosal administration may be through nasal sprays or using suppositories.
- the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.
- a wash solution can be used locally to treat an injury or inflammation to accelerate healing.
- a gene delivery system for a gene therapeutic can be introduced into a patient by any of a number of methods, each of which is familiar in the art.
- a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g., by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof.
- initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized.
- the gene delivery vehicle can be introduced by catheter, See U.S. Patent 5,328,470, or by stereotactic injection, Chen et al.
- a a sequence homologous thereto can be delivered in a gene therapy construct by electroporation using techniques described, Dev et al. (1994), Cancer Treat. Rev. 20:105-115.
- the pharmaceutical preparation of the gene therapy construct or compound of the invention can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle or compound is imbedded.
- the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.
- compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
- the pack may for example comprise metal or plastic foil, such as a blister pack.
- the pack or dispenser device may be accompanied by instructions for administration.
- Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the Ld 50 (the dose lethal to 50% of the population) and the Ed 50 (the dose therapeutically effective in 50% of the population).
- the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5O /ED 50 .
- Compounds which exhibit large therapeutic indices are prefe ⁇ ed. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
- the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
- the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
- the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
- the therapeutically effective dose can be estimated initially from cell culture assays.
- a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms) as determined in cell culture.
- IC 50 i.e., the concentration of the test compound which achieves a half- maximal inhibition of symptoms
- levels in plasma may be measured, for example, by high performance liquid chromatography.
- Tissues from the a unique human donor eye repository and comprehensive donor database (CDD) have been employed for the experiments described in the Examples that follow.
- This research employs a human donor eye repository that has been developed over the past eight years.
- the repository contains over 2,000 pairs of eyes.
- Staff are on 24 hour call to retrieve and process donated tissue. Eyes are accepted only if they can be processed within four hours of death.
- a database of clinical, statistical and scientific information for each donor eye entered into the repository has been developed and will continue to be maintained.
- Sera, blood (DNA), ophthalmologic and medical histories, and family interviews are collected for as many donors as possible; these data have been collected for over 90% of the donors entered into the repository in the past two years. Over 25% of our donors in the last two years have a clinically documented history of AMD.
- Macular drusen are classified into the following categories: rare ( ⁇ 5 drusen), few (6-50 drusen), moderate (51-200 drusen), and numerous (>200-300); and sizes: small ( ⁇ 50 m), moderate (50-500 m), and large (>500 m). Additional features of age-related macular degeneration, such as macular increased RPE pigmentation, macula RPE pigment clumping, RPE atrophy, subretinal or sub RPE hemo ⁇ hage, or subretinal fibrosis are also noted.
- eye AMD is defined as 1) the presence of indistinct (“soft”) or reticular drusen, or 2) presence of any drusen type with associated visual loss, RPE degeneration, and/or abnormal retinal pigment in the macular area.
- “Late” AMD is defined as the presence of exudative AMD (RPE detachment, detachment of the retina, subretinal or subRPE hemo ⁇ hage, or subretinal fibrous scars) or geographic atrophy.
- eyes are graded based upon an adaptation of a classification system developed by The International ARM Epidemiological Study Group; this information is entered into a database containing all information available for each donor.
- the vitreous is removed and various regions excised with trephine punches; these are frozen immediately in liquid nitrogen or fixed as per the protocol.
- the neural retina is separated from the RPE/choroid in regions that are punched. Portions of every eye are processed for light and electron microscopic analyses. Wedges composed of equatorial/peripheral retina are removed with forceps and frozen similarly. Sections made from all eyes are stained with hematoxylin and eosin, Mallory trichro me, PAS, oil red O, and Sudan olack B. Histopatho logic and electron microscopic examination of all donor eyes, that includes portions of the maculas from most eyes, is performed.
- CDD Comprehensive Donor Database
- drusen size, number, and phenotype include measurements of drusen size, number, and phenotype; BLD density and distribution; RPE and photoreceptor cell densities; Bruch's membrane thickness and degree of debris accumulation; choriocapillaris density; and choroidal thickness and density of choroidal fibrils. Other parameters will be added as required.
- Fluorescein isothiocyanate- (FITC-) and rhodamine-conjugated lectins derived from Limax flavus (LFA), Triticum vulgaris (WGA), Arachea hypogea (PNA), and Ricinis communis (RCA-I) were obtained from EY Laboratories, Inc. (San Mateo, CA) and Vector Laboratories (Burlingame, CA).
- Neuraminidase isolated fro Clostridium perfringens was obtained from Boehringer-Mannheim (Indianapolis, IN) and O-glycanase (endo-a-N- acetylgalactosaminidase) was purchased from Genzyme (Cambridge, MA) and Boehringer- Mannheim (Indianapolis, IN). Sudan black B solution was obtained from Poly Scientific (Bay Shore, NY), and PNGase F and globulin-free bovine serum albumin (BSA) were purchased from Sigma Chemical Company (St. Louis, MO). Immumount was purchased from Shandon (Pittsburgh, PA).
- Human Donor Eves Eyes from 42 human donors, ranging from 35 to 101 years of age, were obtained from MidAmerica Transplant Services (St. Louis, MO) and the Iowa Lions Eye Bank (Iowa City, IA). Eyecups were preserved in 4% paraformaldehyde in lOOmM sodium cacodylate, pH 7.4, or embedded directly in Optimal Cutting Temperature compound (OCT) and frozen in liquid nitrogen, without fixation, within six hours post-mortem.
- OCT Optimal Cutting Temperature compound
- Fixation and Embedding After 2-4 hrs in fixative, eyecups were placed into buffer and embedded, as described in Example 4.
- Glycosidase Treatments Sections of fixed and unfixed tissues were incubated with lU/mL neuraminidase in 30mM sodium acetate buffer, pH 5, at 37oC, overnight in a humidified chamber. Sections from 41 eyes ⁇ 33 fixed and acrylamide-embedded and 9 unfixed and OCT-embedded — were treated with neuraminidase and subsequently labeled with FITC- or rhodamine-PNA (below). Adjacent, control sections were incubated with buffer alone.
- Lectin Histochemistry For lectin labeling, 6-8mm thick cryostat sections were cut, mounted on Superfrost Plus slides, and labeled with PNA, WGA, or LFA as described in Example 4. Unlabeled, adjacent control sections were used to distinguish between lectin binding and drusen autofluorescence.
- Drusen-containing tissues were obtained as above, and were fixed within 4 hours of death in one half-strength Kamovsky's fixative and processed as described below.
- Reagents employed in embedding tissues for transmission electron microscopy were obtained from Fluka Chemical (Milwaukee, WI). All other reagents were obtained from Electron Microscopy Sciences (Fort Wayne, PA). Tissues were fixed for at least two hours in one-half strength Karnovsky fixative (1/2K; 2% formaldehyde and 2.5% glutaraldehyde in lOOmM cacodylate buffer, pH 7.4, containing 0.025% CaCl 2 ) prior to washing 3x10 min. in lOOmM cacodylate buffer.
- Pellets or wedges were then post fixed with 2% osmium tetraoxide in cacodylate buffer for 2 hours, and were then rewashed 3x10 min. prior to dehydration through a series of graded ethanol solutions (50% ethanol 10 min. each in 70% ethanol, and 95% ethanol, followed by 2x10 min. in 100% ethanol. Tissues were then dehydrated 2x10 min. in propylene oxide, and were infiltrated overnight in a 1:1 mixture of propylene oxide:Epon 812 solution (containing 51% Epon 812, 27% dodecenyl succinic anhydride, and 22% nadic methyl anhydride, with 1.5% DMP-30 added to the solution as an accelerator). The following day the Epon solution was changed 3 times throughout the day, and the samples were cured at 40%C overnight and then at 65%C for 2 days.
- graded ethanol solutions 50% ethanol 10 min. each in 70% ethanol, and 95% ethanol, followed by 2x10 min. in 100%
- Neuraminidase Treatment Incubation of drusen-containing tissue sections with neuraminidase completely eliminated LFA labeling of drusen and other structures in the chorioretinal complex, as compared to controls. Labeling of nuclei in the choroid and neural retina persisted after neuraminidase treatment, however. This loss of labeling was used throughout the study to control for enzyme efficacy, as were changes in labeling of the interphotoreceptor matrix, as described previously (Johnson and Hageman, 1987; Kivela, 1990).
- PNA does not normally bind to drusen or any other structure within the RPE- choroid complex. Following pre-exposure to neuraminidase, the endothelial cells and/or endothelial cell basal laminae of the choriocapillaris and other choroidal vessels, as well as the basal lamina of the RPE, bound PNA intensely.
- drusen were labeled by PNA following exposure to neuraminidase. This intense labeling was typically restricted to subdomains, or "cores", within drusen. These cores were not observed in adjacent sections treated with buffer alone. Drusen cores were typically spherical, centrally located within the druse, and juxtaposed against the inner collagenous layer of Bruch's membrane. Only one core was generally observed within any given solitary druse, whereas confluent or fused drusen may possess several cores. These cores ranged from 5 to 30&m diameter, with a mean diameter of 14&m. Drusen cores were observed frequently; they were present in 32 of the 42 eyes examined in this study.
- Transmission Electron Microscopy revealed a variety of possible "core-like" structures within drusen. These include regions which exhibited subtle differences in contrast due to differences in quantity and/or electron density of particles, when compared to the rest of the drusen, as well as domains which were very dense and osmiophilic. In addition, drusen occasionally possessed regions that are electron lucent, presumably due to extraction of lipids during processing.
- Antibodies to CD3, CD 15, CD45, and anti-mouse secondary antibodies conjugated to indocarbocyanine-3 (Cy3) were purchased from Chemicon International (Temecula, CA); monoclonal antibodies to CDla, CD14, CD31, CD45, CD68, S100 and HLA- DR were purchased from Dako (Carpenteria, CA). Fluorescein-conjugated secondary antibodies were obtained from Jackson Immunoresearch Laboratories (West Grove, PA). For some experiments, the Elite Staining Kit was employed and labeling was visualized with the Vector VIP substrate (Vector, Burlingame, CA). Neuraminidase (Clostridium perfringens) was obtained from Boehringer-Mannheim (Indianapolis, IN).
- Fluorescein-conjugated peanut agglutinin was purchased from EY Laboratories, Inc. (San Mateo, CA). Other reagents used for tissue fixation and embedment were obtained from Sigma Chemical Company (St. Louis, MO), unless otherwise noted. Studies were conducted to immunophenotype the choroidal cells from which these core terminations arise and to evaluate their potential relationship to drusen biogenesis.
- Tissues Human donor eyes employed in this study were obtained from The University of Iowa Lions Eye Bank (Iowa City, IA) within four hours of death. Institutional Review Board committee approval for the use of human donor tissues was obtained from the Human Subjects Committee at The University of Iowa. Posterior poles, or wedges of posterior poles spanning between the ora se ⁇ ata and macula, were processed from 30 donors, embedded in OCT, snap frozen in liquid nitrogen, and stored at -80°C. Tissues were sectioned to a thickness of 6-8um on a cryostat. Confocal laser scanning microscopy and immunohistochemistry were employed to examine drusen-associated cores in human donor eyes. Immunolabeling of sections was performed using a battery of antibodies directed against various cell populations including endothelial cells, lymphocytes, granulocytes, monocytes/macrophages and dendritic cells.
- Immunohistochemistry Eyes employed for immunohistochemistry were dissected and embedded into Optimal Cutting Temperature compound (Miles Inc.; Elkhart, NY) without prior fixation. Cryostat sections were cut at a thickness of 6-8m and were collected on Superfrost plus slides (Fisher; Pittsburgh, PA). Neuraminidase treatment was performed in some cases; sections were digested overnight with lU/mL neuraminidase in 30mM sodium acetate buffer, pH 5.0, overnight at 37°C. For fluorescence microscopy, antibody and lectin labeling was performed as described previously in Example 1. For some experiments, the Vector Elite kit for horseradish peroxidase staining was used, according to the manufacturer's instructions.
- Double Labeling/Confocal Microscopy Cryostat sections were treated overnight with neuraminidase (above) followed by immunolabeling with a monoclonal antibodies directed against CD68 HLA-DR, and CDla. Alexa 488-or Cy-3-conjugated secondary antibodies (Chemicon; Temecula, CA: Molecular Probes; Eugene, OR) were used to visualize CD68 immunoreactivity. Sections were washed extensively and incubated with PNA conjugated to fluorescein. Confocal images of both probes were collected simultaneously using a confocal microscope (BioRad; Hercules, CA). Positive controls included choroidal and scleral leucocytes.
- cryostat sections were incubated with antibodies to CD45 (leukocyte common antigen), while alternate serial sections were digested with neuraminidase and labeled with PNA. A subset of the same cores which bound PNA are also labeled with CD45 antibodies. Anti-CD45 antibodies colocalize with PNA-binding cores in smaller drusen.
- CD45 leukocyte common antigen
- Drusen cores and the cells from which they are derived, are also strongly reactive with CDla, CD4, CD14, CD45, CD68, CD83, CD86, and HLA class II (CR3/43 and TAL.1B5) antibodies.
- the CR3/43 antibody reacts with MHC class II antigens including HLA-DP, HLA- DQ, and HLA-DR, whereas the TAL.1B5 antibody is specific to HLA-DR alpha chains.
- Both antibodies react with drusen cores, although the DR-specific clone (TAL.1B5) may react with more restricted, cell-associated domains whereas the pan-MHC-II clone (CR3/43) may label more voluminous domains within drusen.
- HLA-DR is largely confined to drusen-associated dendritic cells, in contrast to HLA-DP and HLA-DQ, which may be derivatives of membrane blebs from these cells, or exosomes.
- HLA-DP and HLA-DQ may be derivatives of membrane blebs from these cells, or exosomes.
- Ongoing studies are be directed toward determining whether there is a difference in the distribution of the various MHC class-II antigens in drusen cores, and whether other exosomal proteins are present in drusen.
- Drusen cores appear to be more prevalent in smaller drusen, and are also detected as putative drusen precursors, solitary cores within Bruch's membrane that are not su ⁇ ounded by additional drusenoid accretions.
- HLA-DR and CD68 immunoreactive drusen were determined in unfixed cryostat sections. Eighty-eight percent of all drusen were HLA-DR immunoreactive; binding was restricted to cores in some drusen, whereas in others it was observed throughout.
- HLA-DR reactive drusen The mean size of HLA-DR reactive drusen was 26um+9um.
- the mean size for HLA-DR negative drusen was 22.8um+4.8um.
- there was no significant difference in size between HLA-DR positive and negative drusen (Student's t-test).
- approximately twenty percent of all drusen in any given eye possessed anti-CD68 antibody immunoreactive cores.
- the diameters of cores that reacted with antibodies to CDla, CD45 and CD68 were measured with an eyepiece reticle. These cores measured 10.4m + 4.4m in diameter. This was somewhat smaller than the average size of PNA-binding cores (14m ) , and may suggest that the PNA-binding material in drusen surrounds the leukocyte process. This result is consistent with results from double labeling confocal microscopy experiments (above).
- Reagents Polyclonal antisera directed against vitronectin (VN) and laminin (LN) were obtained from Telios (San Diego, CA); antibodies to collagen type IV were obtained from Chemicon (Temecula, CA). Wheat germ agglutinin (WGA) and Limaxflavus agglutinin (LFA) were purchased from Vector (Burlingame, CA) and EY Laboratories (San Mateo, CA), respectively. Reagents employed in embedding tissues for immunofluorescence were obtained from Bethesda Research Laboratories (Bethesda, MD) and Sigma Chemical (St. Louis, MO).
- Human Donor Eves Human tissues were obtained from MidAmerica Transplant Services (St. Louis, MO) and the Iowa Lions Eye Bank (Iowa City, IA) within 5 hours of death. Following removal of the corneas, donor eyes were cut into quadrants. An inferior saggittal wedge from the ciliary body to the macula was removed from each eye and fixed in either 4% paraformaldehyde or one-half strength Kamovsky's fixative (1/2K) for 2 hours, to assess the presence and mo ⁇ hology of drusen in these tissues. The neural retina was removed from each eye, and individual quadrants were pinned to wax-coated Petri dishes, scleral side down.
- Microdissection Attempts were made to microdissect large drusen using number five forceps or na ⁇ ow gauge (26G) syringe needles. Drusen were gently separated from the choroid and were washed with lOmM phosphate buffered saline (PBS; pH 7.4) and placed into a Petri dish for photomicrography or into an Eppendorf tube for transmission electron microscopical or biochemical analyses.
- PBS lOmM phosphate buffered saline
- enriched drusen preparations were incubated in ice cold distilled water in order to lyse RPE cells. These preparations were then either frozen for electrophoresis or were fixed and processed for immunohistochemistry, as above.
- the RPE was removed with a stream of buffer (using a 30 gauge needle mounted to a lOcc syringe) and the Beaver #69 blade was used to debride Bruch's membrane of the remaining drusen.
- Tissue Processing In order to determine the efficacy of the scraping technique in removing RPE/drusen from Bruch's membrane, portions of the scraped material and the remaining Bruch's membrane/choroid were fixed in 4% paraformaldehyde and prepared as described in Example 4. Cryostat sections of the enriched material and the remaining choroid were collected and employed in histochemical analyses.
- Sections of drusen-enriched pellets were stained with 1% Sudan black B, WGA, LFA, and antibodies to vitronectin (VN), laminin (LN), complement C5, and collagen type IV. Lectin and antibody staining was performed as described in Examples 1 and 2.
- One enriched drusen preparation was fixed in 1/2K as above, rinsed in cacodylate, and dried down on a polylysine coated surface for subsequent examination by scanning electron microscopy.
- the tissue was dehydrated by critical-point drying, and was sputter coated, as described previously.
- LCM Laser Capture Microdissection
- Preparation of enriched drusen proteins for electrophoretic separations involved sonicating total RPE/choroid tissues or enriched RPE/drusen pellets briefly on ice, followed by boiling of samples in sample buffer, as described below. Following removal of corneas, donor eyes were cut into quadrants. Neural retinae were removed in order to reveal more precisely the extent of RPE pathology, including geographic atrophy, choroidal neovascularization, pigment clumping, and/or drusen. Tissues with advanced degeneration of the RPE were excluded from this study. The presence or absence, and extent, of drusen was determined initially under a dissecting microscope.
- Enriched drusen preparations were compared to whole RPE/choroid preparations on silver stained gels and Western blots.
- the high molecular weight aggregates at the interface of the stacking gel-separating gel, characteristic of drusen-containing preparations, were excised and analyzed by matrix assisted laser deso ⁇ tion ionization mass spectrometry (MALDI-MS).
- Amino acid sequencing was performed at the W.M. Keck Foundation's Biotechnology Resource Foundation (New Haven, CT) as described (Stone, et al., 1990).
- in- gel trypsin digestion of Coomassie-stained gel bands was performed (Stone, et al., 1990), and peptides were identified and matched to their respective proteins based on their molecular mass using matrix assisted laser deso ⁇ tion ionization mass spectrometry (MALDI-MS), as described (Williams et al., 1996).
- MALDI-MS matrix assisted laser deso ⁇ tion ionization mass spectrometry
- the European Molecular Biology Laboratory and OWL databases were then searched for masses of tryptic peptides of known, as well as conceptually- translated, proteins (Lamand and Mann, 1997). Only cases in which at least five peptides and up to 20% of the protein mass were matched to predicted tryptic fragments were the matches considered significant.
- MS/MS of Enriched Drusen As an additional approach to identify the molecular constituents of drusen and enriched RPE/drusen preparations were collected and digested with trypsin, followed by identification of resultant peptides by mass spectrometry (LC/MS MS). In other studies, these preparations were separated by two-dimensional SDS-PAGE, individual spots were collected, and analyzed, as above, employing MS/MS.
- RPE/Choroid Biochemistry Prior to the development of enrichment techniques for examining drusen constituents biochemically, proteins from the RPE/choroid complex from donors assessed to have drusen by gross and histological examination were separated and compared to those of donors without drusen. As a function of drusen status, variations in the pattern of proteins were observed with silver staining. RPE/choroid extracts from donors with drusen typically possess a doublet of approximately 35/36kDa, whereas homogenates from age- matched controls exhibit only a single band at this molecular weight. Of donors with drusen, 75% were found to possess the 35/36kDa doublet, whereas none of the donors without drusen exhibited this alteration.
- a second pattern variation coinciding with the presence of drusen is a 120kDa band which is absent in drusen-containing tissues, but is always present in age- matched controls. These bands were excised from preparative gradient gels, and their constituent proteins were identified by MALDI-MS. These analyses identified inte ⁇ hotoreceptor retinoid binding protein as being present in the 120kDa band associated with donors without drusen. The co ⁇ esponding region of the gel from drusen-containing donors contained ceruloplasmin, which was not identified in the control donor band, but did not contain ERBP.
- CRALBP Cellular retinaldehyde binding protein
- annexin II was found in samples derived from donors with and without drusen, whereas the 26S protease regulatory subunit (involved in ubiquitin-mediated proteolysis) was found only in this band from donors with drusen.
- Isolated Solitary Drusen Morphology: Large, individual drusen, relatively free of contaminants, were isolated using the techniques described. Isolated drusen were examined using bright field micrography. They were typically spherical or hemispherical, contained vesicular profiles, and were often associated with a few RPE cells or pigment. Ultrastructurally, this material was comprised of membranous debris and other structural elements characteristic of drusen in situ and fragmented RPE cells.
- SDS-PAGE Individual drusen were dissociated in sample buffer and separated by SDS- PAGE, followed by silver staining or Western blotting. Typically, we were only able to collect 5-20 drusen per eye using this approach. Although too little protein was obtained from each isolated drusen sample to run preparative gels for amino acid sequencing, sufficient material was present for analysis by silver staining and lectin labeling of Western blots. Silver stained drusen preparations typically yielded 6-7 discrete bands ranging in molecular weight from 20 to 65kDa; these preparations invariably contained a prominent band with a molecular weight of approximately 35kDa.
- Drusen in situ are typically eosinophilic when stained with hematoxylin/eosin.
- Small, hard (“hyaline”) drusen stain more intensely and uniformly than large, soft drusen, which tend to be more heterogenous.
- Drusen in enriched preparations were stained similarly. In eyes in which differences in staining between hard and soft drusen was apparent in situ, this same pattern was also noted in preparations of enriched drusen collected from the same eye. Spherical hard drusen could be discriminated from large, amo ⁇ hous soft drusen in these preparations. Layers of RPE cells were also readily apparent in these preparations.
- the RPE/drusen-debrided choroid, enriched drusen preparations, and intact, control regions from the same eye were examined using immunohistochemistry and lectin histochemistry.
- the intact basal lamina of the choriocapillaris was observed in the debrided choroid, as was the autofluorescent elastic lamina of Bruch's membrane, providing evidence that Bruch's membrane was not breached during the enrichment procedure.
- Antisera directed against VN and C5 and drusen-binding lectins were used as markers to follow drusen through the enrichment process. In the intact RPE/choroid complex, these markers labeled drusen intensely.
- the globular drusen within the enriched drusen preparations exhibited intense labeling with these probes, indicating that drusen retained VN, C5, and drusen-associated glyconjugate molecules after enrichment.
- drusen within enriched pellets bound Sudan black B and oil red O in the same manner as was seen in situ.
- Electron Microscopy Ultrastructural observations of enriched drusen preparations demonstrated that they contain RPE cells, the RPE basal lamina, free melanosomes from the RPE, and drusen that were mo ⁇ hologically identical to those observed in situ in the same eye. No contamination of the pellets with choroidal material was observed. Basal laminar deposits, that typically lie between the RPE and its basal lamina, were also present in these preparations. The drusen-debrided choroids possessed an intact Bruch's membrane. In eyes without drusen, the RPE monolayer was completely removed and much of the RPE basal lamina typically remained adherent to Bruch's membrane. The elastic lamina and inner collagenous zone of Bruch's membrane were intact and undamaged.
- enriched drusen preparations were visualized as highly heterogeneous mounds of vesicular profiles resembling drusen; RPE cell debris and melanosomes were also apparent.
- proteins were separated by SDS-PAGE.
- drusen-containing preparations contained significantly more high molecular weight protein at the gel interface than did non-drusen preparations. For this reason, stacking gel interfaces were excised and protein constituents of samples with and without drusen were identified by MALDI-MS.
- the matching putative proteins included myosin, desmoplakin I II for the RPE preparation and Myosin, beta-spectrin, alpha-spectrin, and N- acetylglucosamine (GlcNAc) transferase for the RPE/drusen preparations.
- MS/MS of Enriched Drusen A set of molecular candidates for drusen-associated molecules/molecules increased in the RPE-Bruch's membrane in association with drusen have been identified using MS/MS. Differentially-expressed proteins included an upregulation of a neutral pl, ⁇ 30kDa and a basic, 20kDa spot in the drusen-containing sample. A number of spots additionally appeared to be downregulated in the drusen-containing sample, ranging from basic to acidic and from ⁇ 15 to 80kDa. To date, these studies have conclusively identified tissue inhibitor of metalloproteases-3 (TIMP3) and vitronectin in the drusen-enriched sample(s).
- TIMP3 tissue inhibitor of metalloproteases-3
- Tissues Eyes from the human donor repository and CDD, ranging in age between 45 and 101 years, were processed within four hours of death. Many of these donors had a documented clinical diagnosis of AMD (including donors with geographic atrophy, choroidal neovascularization, and disciform scars in at least one eye) and one donor was diagnosed with cuticular drusen. Human liver was obtained within 2 hours of biopsy. RPE cells were isolated with 2% dispase within 5 hours of death and were grown in Coon's F-12 media with 10% fetal bovine serum.
- sections were rinsed (2x10 min.) in PBS/M/C, incubated in the appropriate fluorescein-conjugated secondary antibody (often adsorbed against human serum) diluted in PBS/M/C/BSA (30 min., room temperature), rinsed (2x10 min.) in PBS/M/C, and mounted in Immumount (Shandon, Pittsburgh, PA). Adjacent sections were reacted with secondary antibody alone, as negative controls. Some sections were pre-treated for 10 min with 0.5% trypsin (Sigma; St.
- Reactivities of antibodies with drusen are listed in Table 2 below. In general, all positive antibodies bound to all drusen phenotypes. Controls confirm all antibody reactivities to be specific. In addition, the majority of the antibodies utilized bound to the expected regions of sclera, choroid, RPE, retina, vitreous, and/or other "control" tissues.
- Thrombospondin (Gib/AMAC) - to +/-
- Example 6 Drusen Associated with Aging and Age-Related Macular Degeneration Contain Proteins Common to Extracellular Deposits Associated with Atherosclerosis, Elastosis. Amyloidosis.
- vitronectin is a major component of drusen. Because vitronectin is also a constituent of abnormal deposits associated with a variety of diseases, drusen from human donor eyes were examined for compositional similarities with other extracellular disease deposits.
- the sixty-three human donor eyes employed in this study were obtained from The Human Donor Repository and the CDD. All eyes were collected and processed within four hours of death; donor ages ranged from 45 to 96 years. Drusen were categorized as hard or soft. Tissues from a minimum of five donors were assayed with each antibody employed, at least two of whom had clinically-documented AMD, and each drusen phenotype was examined in at least two donors. Institutional Review Board committee approval for the use of human donor tissues was obtained from the Human Subjects Committee at The University of Iowa.
- Antibodies against human serum albumin and haptoglobin bound strongly to the choroidal stroma, but not to hard or soft drusen. Immunoreactivity of some drusen-associated proteins was frequently observed in distinct, heterogeneous patterns. For example, drusen binding by prothrombin and amyloid A antibodies, was often localized to spherical profiles within drusen. Drusen were occasionally labeled by anti-fibrinogen antibodies; this binding was generally confined to peripheral regions and/or concentric bands within drusen.
- compositional similarity between drusen and other disease deposits may be significant in view of the correlation between AMD and various systemic disorders, including atherosclerosis. These data suggest that similar pathways may be involved in the etiologies of AMD and other systemic disorders.
- Example 7 Local Sources of DRAMs Common to Extracellular Deposits Associated with Atherosclerosis, Elastosis, Amyloidosis, and Dense Deposit Disease
- DRAMs that were identified as being common to extracellular deposits associated with atherosclerosis, elastosis, amyloidosis, or dense deposit disease were produced locally in the eye by RPE, retinal, and/or choroidal cells.
- RNA Isolation Total RNA was isolated from adult human liver, RPE/choroid, retina, and enriched RPE as described by Chirgwin et. al. (1979), except that cesium trifluoroacetate was used instead of cesium chloride in the density gradient ultracentrifugation step. The resulting pellet was stored at -80oC. The quality/integrity of RNA obtained was assessed on both agarose gels and Northern blots. Total protein was determined from identically sized punches of the ocular tissue(s) from which the RNA was collected and employed as an internal reference.
- Table 4 RT-PCR results from retina, RPE/choroid, and liver.
- Ret retina
- R Ch RPE/choroid
- Gen amplification of genomic DNA by the primer pair
- * higher molecular weight genomic band detected with primer pair.
- Example 8 Local Sources of Additional DRAMs and Other Choroidal Fibrosis-Associated
- RNA Isolation Total RNA was isolated from adult human liver, RPE/choroid, enriched RPE, retina, fetal human eye, various fetal human organs, and primary cultures of human RPE cells using the RNeasy system (Qiagen; Valencia, CA). Liver and peripheral blood leukocyte RNA, as well as genomic DNA, served as positive and negative controls, respectively. The resulting pellets were stored at -80oC. The quality/integrity of RNA obtained was assessed on both agarose gels and Northern blots. Total protein was determined from identically sized punches of the ocular tissue(s) from which the RNA was collected and employed as an internal reference.
- RT-PCR Analyses Primers to nucleotide sequences were employed to amplify cDNA molecules from these tissue sources. Reaction mix without template and/or ommission of reverse transcriptase during the RT reaction were used as negative controls. PCR amplification products were separated by agarose gel electrophoresis and stained with ethidium bromide for visualization. cDNA was synthesized with reverse transcriptase using oligo(dT)i6 as a primer. Reverse transcriptase was omitted from some reactions. cDNA was amplified using molecule-specific primer pairs. PCR amplification products were separated elect, ophoretically on a 1.8% agarose gel.
- Eyes used in this study were selected from a repository of over 2,000 pairs of human donor eyes (between 0 and 102 years of age) obtained from MidAmerica Transplant Services (St. Louis, MO), the Iowa Lions Eye Bank (Iowa City, IA), the Heartland Eye Bank (Columbia, MO) and the Virginia Eye Bank (Norfolk, VA) and were processed within four hours of death.
- the RPE-choroid-sclera complex from 151 of these donors were processed for transmission electron microscopical examination. Tissues were fixed in one-half strength Kamovsky's fixative within four hours of death for a minimum of 24 hours, and transferred to 100mM sodium cacodylate buffer, pH 7.4, prior to dehydration, embedding, sectioning, and photomicrography.
- Tissues from the same eyes processed for electron microscopy were processed for light histological (Elastachrome stain; H&E) and immunohistochemical studies.
- Anti-vitronectin antibody was obtained from Telios (San Diego, CA); collagens I, III, V, and VI from Chemicon and Southern Biotech; elastin from Elastin Products; f ⁇ briHin-1 from Chemicon; and fibulins 3 and 4 from Rupert Timpl.
- Selected specimens of human donor RPE-choroid were fixed by immersion in 4% (para)formaldehyde in 0.1M sodium cacodylate buffer and processed for laser scanning confocal microscopy. Images were captured and displayed using a BioRad 1024 laser scanning confocal microscope equipped with a Nikon inverted microscope.
- the choroidal stromas of at least 30 of these individuals are filled with newly synthesized collagen, elastin, elastin-associated microfilaments, and other distinct structural proteins and fibrils as viewed by electron microscopy.
- the collagen associated with this condition appears to be largely type III and VI and typically exhibits a "spiraled", or "frayed" mo ⁇ hology that is often associated with specific hereditary and acquired diseases.
- This previously undescribed phenomenon referred to as "choroidal fibrosis" shares many pathological features that are common in arterial wall disruptive disorders.
- RT-PCR analyses of RPE-choroid complexes derived from this series of control (non-diseased) and affected (AMD/AAA, AMD, AMD/aortic stenosis; all with drusen) donors with distinct choroidal fibrosis reveal distinct patterns of up- and down-regulated gene expression between the two groups (see Table X below). These include "upregulation" of b1 integrin, elastin, collagen Vla2, collagen a3, PI-1 (antitrypsin), PI-2, human metalloelastase (and perhaps fibrillin-2) and "downregulation" of BigH3.
- Example 12 Choroidal Fibrosis - Characterization of Metalloproteinases/TIMPs in Aging and AMD Donors with Choroidal Fibrosis/Extracellular Matrix Disequilibrium
- Extracellular matrix turnover is initiated, at least in part, by the regulated secretion of members of a family of matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs).
- MMPs matrix metalloproteinases
- TRIPs tissue inhibitors of metalloproteinases
- Leukocytes including dendritic cells and macrophages, are major sources of MMP production. MMP action permits leukocyte immigration into tissues, causes tissue damage, and generates immunogenic fragments of normal proteins that may escalate autoimmune diseases [Opdenakker, 1992 #681].
- the MMP family of enzymes contributes to both normal and pathological tissue remodeling. Although the link between single MMPs and individual substrates is not as direct as once thought, it is clear that the MMPs are capable of breaking down most ECM components.
- MMPs are not constituitively expressed in normal tissues. Inflammatory cytokines (IL-1 and TNF) and growth factors (TGFb) are typically required to initiate transcription. MMPs are expressed as inactive zymogens, which are activated extracellularly by the action of enzymes such as plasmin and other MMPs. Once activated, MMPs are subject to inactivation by TIMPs and by binding to plasma proteins such as a2-macroglobulin. This balance of expression and activation, and the levels of TIMPs, govern the level of destruction mediated by MMPs. Excessive or inappropriate expression of MMPs may contribute to the pathogenesis of many tissue destructive processes, including diseases such as arthritis, multiple sclerosis, atherosclerosis, and COPD.
- MMP-1 , -2, and -9, but not MMP-3 or -8 were identified in RPE/choroid extracts and did not show changes with respect to drusen status, AMD, and/or age.
- TIMP-4 antibody bound to a band of 28 kDa in all samples, including a 2-month-old donor.
- the higher molecular weight bands may be due to smaller MMPs that have polymerized (99) or may represent novel proteases, such as the 300 kDa elastase identified in lung by broncheoalveolar lavage (100).
- Antibodies directed against TIMP-3 reacted with hard and soft drusen, whereas anti-TIMP-4 antibodies reacted with drusen cores.
- MMPs 2, 7, 9, 12, and 14 members of the MMP family that exhibit elastolytic properties, using immunohistochemistry, zymography, ELISA, and QRT-PCR.
- Example 13 Autoantibodies in the Sera of Donors with AMD and /or Drusen
- Protein extracts from an enriched drusen preparation (DR+) obtained by debridement of Bruch's membrane with a #69 Beaver blade and from a control (DR-) preparation were prepared using PBS with proteinase inhibitor cocktail and mild detergent. Proteins were separated by molecular weight using 10- 20% gradient mini SDS gels (Amresco) and transferred to PVDF membranes for Western blot analysis. PVDF strips with human retinal proteins from 50 normal human retinas were also used for detection of any anti-retinal autoantibodies in the donor sera. Sera from the same eight donors described above were screened.
- Serum from one AMD donor (#90-98) positively labeled a band in the RPE (both DR+ and DR-) and RPE/choroid preparations of approximately 35kDa.
- a second band of approximately 60kDa was labeled weakly only in the DR+ protein extract.
- Sera from an AAA donor (#189-97) reacted with a protein(s) of approximately 53kDa. This band labeled in ali three protein extracts. There was one band of approximately 64kDa that this serum sample labeled only in the DR+ sample.
- levels of the following proteins will be assayed by a clinical diagnostic laboratory. These will include Bence Jones protein, serum amyloid A, M components, C-reactive protein, mannan binding protein, serum amyloid A, C3a, C5a, other complement proteins, coagulation proteins, fibrinogen, vitronectin, CD25, interieukin 1 , interieukin 6, and apolipoprotein E. Serum protein electrophoresis, lymphocyte transformation, sedimentation rate, and spontaneous, whole blood, white cell count will also be measured.
- the presence of antibodies directed against the following proteins will also be determined: type IV collagen, giomerular basement membrane, neutrophils, cytoplasm (c-ANCA, p-ANCA), C3 convertase (C3 nephritic factor), alpha-1 anti- trypsin levels (decreased in MPGN), epsilon 4 allele, apolipoprotien E, GFAP, ANA, serum senescent cell antigen, S-100, type 2 plasminogen activator, alpha-1 -antichymotrypsin, SP-40,40, endothelial cell, parietal cell, mitochondria, Jo-1 , islet cell, inner ear antigen, epidermolysis Bullosa Acquista, endomysial IgA, cancer antigen 15-3, phospholipid, neuronal nucleus, cardiolipin, and ganglioside.
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AU33958/00A AU3395800A (en) | 1999-03-05 | 2000-03-06 | Diagnostics and therapeutics for drusen associated ocular disorders |
EP00912189A EP1161686A2 (en) | 1999-03-05 | 2000-03-06 | Diagnostics and therapeutics for drusen associated ocular disorders |
CA002363503A CA2363503C (en) | 1999-03-05 | 2000-03-06 | Diagnostics and therapeutics for drusen associated ocular disorders |
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US12305299P | 1999-03-05 | 1999-03-05 | |
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US51023000A | 2000-02-22 | 2000-02-22 | |
US09/510,230 | 2000-02-22 |
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EP1487395A2 (en) * | 2002-02-28 | 2004-12-22 | Prometheus Laboratories, Inc. | Methods of diagnosing liver fibrosis |
US7011952B2 (en) | 2000-02-22 | 2006-03-14 | University Of Iowa Research Foundation | Diagnostics and therapeutics for macular degeneration-related disorders |
US7351524B2 (en) | 2000-02-22 | 2008-04-01 | University Of Iowa Research Foundation | Diagnostics and therapeutics for macular degeneration-related disorders |
EP1937706A2 (en) * | 2005-09-09 | 2008-07-02 | University of Iowa Research Foundation | Biomarkers associated with age-related macular degeneration |
US7470660B2 (en) | 2001-12-07 | 2008-12-30 | The Regents Of The University Of California | Treatment for dark adaptation |
US7989426B2 (en) | 2002-02-15 | 2011-08-02 | Johns Hopkins University School Of Medicine | Selective induction of apoptosis to treat ocular disease by expression of PEDF |
EP2357254A1 (en) * | 2005-02-14 | 2011-08-17 | University of Iowa Research Foundation | Methods and reagents for treatment and diagnosis of age-related macular degeneration |
EP2508183A1 (en) * | 2005-05-12 | 2012-10-10 | The Texas A & M University System | Interleukin-1 blocker for use in the treatment of age-related macular degeneration |
WO2014036483A1 (en) * | 2012-08-31 | 2014-03-06 | University Of Maryland, Baltimore | Methods and compositions for detecting drusen and predicting age-related macular degeneration |
WO2014144764A2 (en) | 2013-03-15 | 2014-09-18 | Baxter International Inc. | Method for producing factor h from a plasma precipitation fraction |
WO2014151919A1 (en) | 2013-03-14 | 2014-09-25 | Baxter International Inc. | Factor h for transplantation |
WO2014151923A1 (en) | 2013-03-14 | 2014-09-25 | Baxter International Inc. | Factor h for treatment of rheumatoid arthritis |
WO2015089215A1 (en) * | 2013-12-13 | 2015-06-18 | TheraOptix, Inc. | A retinal pigment epithelial primary cell culture system producing subcellular deposits |
US9254287B2 (en) | 2012-06-11 | 2016-02-09 | Macuclear, Inc. | Therapeutic formulation and methods of treatment |
US11253614B2 (en) | 2016-08-23 | 2022-02-22 | University Of Maryland, Baltimore | Methods for detecting and/or predicting age-related macular degeneration and/or Alzheimer's disease |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
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US20060275782A1 (en) | 1999-04-20 | 2006-12-07 | Illumina, Inc. | Detection of nucleic acid reactions on bead arrays |
US8114592B2 (en) | 2008-03-18 | 2012-02-14 | Cambridge Enterprise Limited | Genetic markers associated with age-related macular degeneration, methods of detection and uses thereof |
-
2000
- 2000-03-06 AU AU33958/00A patent/AU3395800A/en not_active Abandoned
- 2000-03-06 EP EP00912189A patent/EP1161686A2/en not_active Withdrawn
- 2000-03-06 WO PCT/US2000/005858 patent/WO2000052479A2/en active Search and Examination
- 2000-03-06 CA CA002363503A patent/CA2363503C/en not_active Expired - Fee Related
Non-Patent Citations (9)
Cited By (28)
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US7351524B2 (en) | 2000-02-22 | 2008-04-01 | University Of Iowa Research Foundation | Diagnostics and therapeutics for macular degeneration-related disorders |
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US7470660B2 (en) | 2001-12-07 | 2008-12-30 | The Regents Of The University Of California | Treatment for dark adaptation |
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US7989426B2 (en) | 2002-02-15 | 2011-08-02 | Johns Hopkins University School Of Medicine | Selective induction of apoptosis to treat ocular disease by expression of PEDF |
EP1487395A4 (en) * | 2002-02-28 | 2008-09-10 | Prometheus Lab Inc | Methods of diagnosing liver fibrosis |
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US9254287B2 (en) | 2012-06-11 | 2016-02-09 | Macuclear, Inc. | Therapeutic formulation and methods of treatment |
US9694010B2 (en) | 2012-06-11 | 2017-07-04 | Macuclear, Inc. | Therapeutic formulation and methods of treatment |
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US9138466B2 (en) | 2013-03-14 | 2015-09-22 | Baxalta Incorporated | Factor H for transplantation |
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US10183976B2 (en) | 2013-03-15 | 2019-01-22 | Baxalta Incorporated | Method for producing factor H from a plasma precipitation fraction |
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US11253614B2 (en) | 2016-08-23 | 2022-02-22 | University Of Maryland, Baltimore | Methods for detecting and/or predicting age-related macular degeneration and/or Alzheimer's disease |
Also Published As
Publication number | Publication date |
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CA2363503A1 (en) | 2000-09-08 |
WO2000052479A3 (en) | 2001-02-01 |
EP1161686A2 (en) | 2001-12-12 |
CA2363503C (en) | 2009-03-10 |
AU3395800A (en) | 2000-09-21 |
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