KR20130065900A - Methods for screening therapeutic candidates of retinopathy - Google Patents

Abstract

PURPOSE: A method for screening a therapeutic agent for retinal diseases is provided to obtain a pharmaceutical composition for preventing or treating retinal diseases. CONSTITUTION: A method for screening a therapeutic agent for retinal diseases comprises: a step of contacting a test material with Arf72A(ADP(adenosine diphosphate) ribosylation factor 72A) gene or protein, or a cell containing Arl1(ADP-ribosylation factor-like protein 1) gene or protein; and a step of measuring expression level of Arf72A or Arl1 gene, protein level of Arf72A or Arl1, or activation of Arf72A or Arl1. The test material is determined as a therapeutic agent for retinal diseases when the expression level, protein level, or protein activation is down-regulated.

Description

Methods for Screening Therapeutic Candidates of Retinopathy < RTI ID = 0.0 >

The present invention relates to a screening method for candidate therapeutic agents for retinal diseases.

The endoplasmic reticulum (ER) functions as a place to synthesize secreted proteins and to confirm the folding state of the proteins (Hebert and Molinari, 2007). In cell survival, the endoplasmic reticulum is essential because it activates eukaryotic monitoring mechanisms, called ER quality control, to ensure that the protein excreted from the endoplasmic reticulum is properly folded. Some endoplasmic reticulum proteins are known to have a function of recognizing mis-folded proteins in the endoplasmic reticulum. UDP-glucose-glycoprotein glycotransferase is a recognition of the folding state of glycoproteins synthesized via hydrophilic carbohydrates (Trombetta and Helenius, 1999) and Bip / Hsc3 of the hydrophobic backbone. Known (N. ichikawa et al., 2001; Ryoo et al., 2007). Once a miss-folded protein is found, the endoplasmic reticulum is sequestered through a complex checkpoint mechanism (Vashist and Ng, 2004) and the ubiquitin / proteosome ERED (I) and autophagy / Related degradation (ERAD) pathway involving the lysosome ERAD (II) (Hiller et al., 1996; Fujita et al., 2007).

COP (II) -coated vesicles are involved in the vesicle budding of the endoplasmic reticulum. Local changes in lipid metabolism have been reported to allow the small GTPase such as Sar1 to aggregate the membrane of the adapter protein and the coat of the coat complex and to control the vesicle burding from the endoplasmic reticulum (Cai et al., 2007). Despite much work on the molecular mechanism of vesicle transport from the endoplasmic reticulum, the molecular mechanism by which cargo proteins are secreted to be degraded is not well known. That is, no coat protein and small GTPase associated with degraded cargo proteins are reported. Therefore, it is necessary to study the molecular mechanisms that determine the transport of cargo proteins to specific pathways in the endoplasmic reticulum.

Importantly, ERF quality control and malfunctioning of the delivery system for secreted proteins are closely related to many human diseases (Hebert and Molinari, 2007; Kaufman, 2002). Eukaryotic cells have a protective mechanism to prevent erroneous protein-folding ability, called Unfolded Protein Response (UPR) (Ron, 2002). However, genetic predisposition or pathological conditions can easily overwhelm the function of the UPR, including the ERAD mechanism (Travers et al., 2000), resulting in accumulation of misfolded proteins in the endoplasmic reticulum. Stress conditions are associated with many human diseases (Hebert and Molinari, 2007; Kaufman, 2002). For example, mutations in rhodopsin account for about 25% of cases of Autosomal-dominant Retinitis Pigmentosa (ADRP) cases (Inglehearn et al., 1998). Miss-folded or unstable mutant rodiocines can interfere with the maturation of wild-type rhodopsin, and these cell states are known to induce retinal degeneration. Equivalent mutations of Drosophila also induce age-dependent retinal degeneration, which means that although the Drosophila has different photoconversion (light to electrical signal) physiology, photoreceptors with this mutation (Colley et al., 1995a; Kurada and O'Tousa, 1995). This supports that the Drosophila model can be applied not only to human disease but also to study the intrinsic problems of intracellular transmission.

In Drosophila, the arf72A gene is a homolog of the mammal Arl1 (Eisman et al., 2006; Li et al., 2004). arf72A encodes an essential GTP-binding protein that is 55% homologous to Arf1 and has 70-80% homology with Arl1 in yeast and mammals, but has ARF (Acute Renal Failure) activity such as choletoxin cofactor activity or The ability to obtain the fatal dual mutation Arf1 is deficient (Tamkun et al., 1991). It is presumed that the function of Arl1 is located in the Golgi complex and shares a functional group with Arf1, and arf72A is also likely to be involved in vesicular transmission to exocytosis and endocytosis (Munro, 2005; Liu et al., 2006; Burd et al., 2004). Although arf72A is cloned first in the arf-like gene, little is known about its cellular function in Drosophila, except that it is involved in animal development and centrosome recycling (Eisman et al., 2006). Due to the mortality induced by the mutation of the arf72A gene, it is difficult to study the mutation of Drosophila. Therefore, a unique approach to explain the cellular function of arf72A has been proposed by mosaic analysis using FLP-FRT (Flippase recombination enzyme-Flippase Recognition Target) system and by knock-down using RNAi in Drosophila eyes (Stowers and Schwarz, 1999).

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

As a result of intensive efforts to improve or treat retinal diseases, the present inventors have found that when arf72A is suppressed in a photoreceptor cell of an autosomal dominant retinopathy Drosophila model through a mutation study of the arf72A gene of Drosophila similar to the mammalian gene Arl1, thereby confirming the result of inhibiting retinal degradation.

Accordingly, an object of the present invention is to provide a method for screening candidate substances for therapeutic treatment of retinal diseases.

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating retinal diseases.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for screening a candidate therapeutic substance for a retinal disease, comprising the steps of:

(a) contacting a test substance with a cell comprising (i) an Adenosine diphosphate Ribosylation Factor 72A gene or protein, or (ii) an Arl1 (ADP-ribosylation factor-like protein 1) ; And

(b) measuring the expression level of said Arf72A or Arl1 gene, the amount of Arf72A or Arl1 protein, or the activity of Arf72A or Arl1 protein;

When the amount of expression of the Arf72A or Arl1 gene, the amount of Arf72A or Arl1 protein, or the activity of Arf72A or Arl1 protein is measured to be down-regulated, the test substance is judged to be a therapeutic candidate for a retinal disease .

As a result of intensive efforts to improve or ameliorate retinal diseases, the present inventors have found that when arf72A is suppressed in a photoreceptor cell of an autosomal dominant retinopathy Drosophila model through a mutation study of a retinal-associated gene of a fruit fly similar to the mammalian gene Arl1, The results of inhibition of retinal degradation were identified.

The screening method for candidate therapeutic agents for retinal disease of the present invention will be described in detail in each step as follows:

Step (a): contacting the test substance with cells comprising (i) Arf72A gene or protein, or ( ii ) Arl1 gene or protein

According to the present invention, a test substance is contacted with cells comprising (i) the Arf72A gene or protein, or (ii) the Arl1 gene or protein.

The cells can be prepared in various ways. Preferably, the cells that may be used in the present invention are sensory transducer cells, and most preferably, photoreceptor cells.

According to a preferred embodiment of the present invention, Drosophila photoreceptor cells were used separately.

The term " test substance " used in referring to the screening method of the present invention is used in screening to examine the expression level of Arf72A or Arl1 gene, the amount of Arf72A or Arl1 protein, or the activity of Arf72A or Arl1 protein Means an unknown substance. Such samples include, but are not limited to, chemicals, nucleotides, antisense-RNA, siRNA (small interference RNA) and natural extracts.

Step (b): Measuring the expression level of Arf72A or Arl1 gene, the amount of Arf72A or Arl1 protein, or the activity of Arf72A or Arl1 protein

If the amount of expression of Arf72A or Arl1 gene, the amount of Arf72A or Arl1 protein, or the activity of Arf72A or Arl1 protein is measured to be down-regulated in the cells treated with the sample, Can be judged as candidates for the therapeutic agent of the present invention.

The measurement of the change in the expression level of the Arf72A or Arl1 gene can be carried out through various methods known in the art. For example, RT-PCR (Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)), Northern blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine (Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)) or in situ hybridization (Sambrook et al. , Molecular Cloning, A Laboratory Manual, 3rd ed., Cold Spring Harbor Press (2001)).

When performing according to the RT-PCR protocol, first, total RNA is isolated from cells treated with a sample, and then a first-chain cDNA is prepared using oligo dT primers and reverse transcriptase. Next, PCR is carried out using the first strand cDNA as a template and Arf72A or Arl1 gene-specific primer set. Then, the PCR amplification product is electrophoresed, and the band formed is analyzed to measure changes in the expression amount of the Arf72A or Arl1 gene.

Changes in the amount of Arf72A or Arl1 protein can be achieved through a variety of immunoassay methods known in the art. For example, changes in the amount of Arf72A or Arl1 protein include, but are not limited to, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or hardwood assays, and sandwich assays. It is not limited.

The candidate therapeutic agent for retinal diseases determined through the screening method of the present invention can be used for preventing or treating retinal diseases.

Preferably, the retinal disease is selected from the group consisting of retinopathy, autosomal-dominant retinopathy, Usher's syndrome, Stargardt disease, Barret-Biddle syndrome, best disease, choroidal deficiency, gyrate retinal pigment epithelium, retinal hyperplasia, Leber Congential Amaurosis (Laber's Hereditary Optic Neuropathy), BCM (Blue-cone monochromacy), retinal detachment, ML (Malattia Leventiness) Oguchi disease or Refsum disease, most preferably retinopathy or autosomal dominant retinopathy.

According to another aspect of the present invention, there is provided a pharmaceutical composition comprising (i) an inhibitor of Arf72A (Adenosine diphosphate) ribosylation factor 72A gene expression inhibitor or an inhibitor of Arf72A protein activity, or (ii) an ADP-ribosylation factor- ) Gene expression inhibitor or an Arl1 protein activity inhibitor as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating a retinal disease.

Preferably, the inhibitor normally matures and excretes rhodopsin in the endoplasmic reticulum.

Preferably, the retinal disease is retinopathy, autosomal-dominant retinopathy, Usher syndrome, Stargardt disease, Barrett-Beedley syndrome, Best disease, lack of choroid, choroidal retinal atrophy. -atrophy, retinal pigmentosa, retinal macular degeneration, Leber Congential Amaurosis; Laber's Hereditary Optic Neuropathy, Blue-cone monochromacy (BCM), interretinal separation, ML (Malattia Leventiness), Oguchi disease or Refsum disease, most preferably retinopathy or autosomal dominant retinopathy.

The pharmaceutical composition of the present invention may contain a chemical substance, a nucleotide, an antisense, an siRNA oligonucleotide and a natural product extract as an active ingredient.

According to a preferred embodiment of the present invention, the pharmaceutical composition of the present invention is an antisense or siRNA oligonucleotide having a sequence complementary to the nucleotide sequence of Arf72A (SEQ ID No. 1) or Arl 1 (SEQ ID No. 2).

The term "antisense oligonucleotide " in the present invention means DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and binds to a complementary sequence in mRNA to inhibit translation of mRNA into a protein The antisense sequence of the present invention refers to a DNA or RNA sequence which is complementary to the sequence of Arf72A or Arl1 mRNA and capable of binding to Arf72A or Arl1 mRNA, and is used for translation of Arf72A or Arl1 mRNA, translocation ), Maturation, or any other overall biological function. The length of the antisense nucleic acid is 6 to 100 bases, preferably 8 to 60 bases, more preferably 40 bases in 10 to be.

The antisense nucleic acid may be modified at one or more base, sugar or backbone locations to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol . , 5 (3): 343-55 (1995)). The nucleic acid backbone can be modified with phosphorothioate, phosphoroester, methyl phosphonate, short chain alkyl, cycloalkyl, short chain heteroatomic, heterocyclic intersaccharide linkages and the like. In addition, antisense nucleic acids may comprise one or more substituted sugar moieties. Antisense nucleic acids can include modified bases. Modified bases include hypoxanthine, 6-methyladenine, 5-me pyrimidine (particularly 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosil HMC, 2-aminoadenine, 2 Thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine, 2,6-diaminopurine, etc. There is this. In addition, the antisense nucleic acids of the present invention may be chemically bound to one or more moieties or conjugates that enhance the activity and cellular adsorption of the antisense nucleic acids. Cholesterol moieties, cholesteryl moieties, cholic acid, thioethers, thiocholesterols, fatty chains, phospholipids, polyamines, polyethylene glycol chains, adamantane acetic acid, palmityl moieties, octadecylamine, hexylamino-carbonyl-oxy Fat-soluble moieties such as a cholesterol ester moiety, and the like. Oligonucleotides, including liposoluble moieties, and methods of preparation are well known in the art (U.S. Pat. Nos. 5,138,045, 5,218,105 and 5,459,255). The modified nucleic acid can increase stability to nucleases and increase the binding affinity of the antisense nucleic acid with the target mRNA.

Antisense oligonucleotides can be synthesized in vitro by conventional methods to be administered in vivo or to allow antisense oligonucleotides to be synthesized in vivo. One example of synthesizing antisense oligonucleotides in vitro is using RNA polymerase I. One example of allowing antisense RNA to be synthesized in vivo is to allow the antisense RNA to be transcribed using a vector whose recognition site (MCS) origin is in the opposite direction. Such antisense RNAs are preferably made such that translation stop codons are present in the sequence so that they are not translated into the peptide sequence.

The term "RNAi" in the present invention preferably means "siRNA" and "siRNA" means a nucleic acid molecule capable of mediating RNA interference or gene silencing (see WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99/07409 and WO 00/44914). siRNA is provided as an efficient gene knockdown method or gene therapy method because it can inhibit the expression of the target gene. siRNAs were first found in plants, insects, fruit flies and parasites, but recently they have been applied to mammalian cell research by developing si / siRNAs (8-10).

The siRNA molecule of the present invention has a structure in which the sense strand (the corresponding sequence corresponding to the Arf72A or Arl1 mRNA sequence) and the antisense strand (the sequence complementary to the Arf72A or Arl1 mRNA sequence) . Also, according to another embodiment, the siRNA molecules of the invention may have a single stranded structure with self-complementary sense and antisense strands.

The siRNA is not limited to a complete pair of double-stranded RNA portions that are paired with each other, but is paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) May be included. The total length is 10 to 100 bases, preferably 15 to 80 bases, more preferably 20 to 70 bases.

The siRNA terminal structure is capable of blunt or cohesive termini as long as it can inhibit the expression of Arf72A or Arl1 gene by RNAi effect. The cohesive end structure is possible for both 3'-end protrusion structures and 5'-end protrusion structures.

SiRNA molecules of the invention may have a form in which a short nucleotide sequence (e.g., about 5-15 nt) is inserted between self-complementary sense and antisense strands, in which case the expression of the nucleotide sequence The formed siRNA molecules form a hairpin structure by intramolecular hybridization and form a stem-and-loop structure as a whole. This stem-and-loop structure is processed in vitro or in vivo to produce an active siRNA molecule capable of mediating RNAi.

According to a preferred embodiment of the present invention, the expression inhibitor of Arf72A gene or the expression inhibitor of Arl1 gene is an antisense oligonucleotide or siRNA for the Arf72A gene or Arl1 gene, and most preferably siRNA. The siRNA oligonucleotides of the present invention are double stranded oligonucleotides of the nucleotide sequence of Sequence Listing 3 and Sequence Listing 4.

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and agents are Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or local administration).

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.001-100 mg / kg body weight per day.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a screening method for a therapeutic agent for a retinal disease, and a pharmaceutical composition for preventing or treating a retinal disease.

(b) The present invention has identified the role of arf72A82 in the secretion protein transport and endoplasmic reticulum quality control between the ER and Golgi complexes of photoreceptor cells.

FIG. 1 shows the result of confirming that ARF72A protein is located in the Golgi complex in Drosophila photoreceptor cells. FIG. 1A is a diagram showing a cross-section (top) and a longitudinal section (bottom) of a fruit fly eye. FIG. One eye consists of eight photoreceptor cells, all of which are R1-R6 cells surrounding the centrally located R7 and R8. Figure 1b shows UAS :: RFP-arf72A / UAS :: Rab6-GFP; It is the result of observing the eye separated from the retina of Rh :: Gal4 with a confocal microscope. The arrow indicates the RFP-ARF72A (red) fluorescence, indicating that it is located with the Golgi marker Rab6-GFP (green) fluorescence.
Figure 2 shows the internal membrane-related reduction of the mosaic retina in the arf72A mutant photoreceptor. Figure 2a shows a schematic for generating arf72A mosaic retinas. Figure 2b shows an electron microscope picture of the mosaic retina. There were wild type and mutant photoreceptor cells in one eye. The existence of the pigment particles under the labdomain (b, arrow) indicates that the photoreceptor is wild type. The wild-type vesicle (c, arrow) shows a uniform vesicle membrane structure. The arf72A mutant cell (d, arrow) shows the membrane structure of the proliferated inner membrane and modified rough endoplasmic reticulum.
Figure 3 shows an over-proliferating Golgi complex due to the deficiency of arf72A. The position of Rab6-GFP was observed in the photoreceptor cells of wild-type (a) and arf72A mutant (b). One eye was visualized with a confocal microscope. Wild type photoreceptor cells show intermittent GFP staining throughout the cytoplasm, a feature of Drosophila tissue, but mutant cells show size or numerically extended GFP staining. The red line in the middle of the circle shows the autofluorescence of Rabbodimer by fixation-induced cross-linking between structural proteins.
Figure 4 shows that GFP-labeled rhodopsin migrates more rapidly from mutant cells to the destination than from wild-type cells. Figure 4a shows a schematic representation of the process by which Rorodosin reaches the final destination Rabbitomer (red) from the endoplasmic reticulum. Figures 4b and 4c illustrate eyFLP; arf72A, UAS :: Rh1-GFP, Rh1 :: GAL4, 80B FRT / TM2 (equilibrium heterozygous) and eyFLP; shows the result of vitamin A structure experiment using arf72A, UAS :: Rh1-GFP, Rh1 :: GAL4, 80B FRT / GMR :: hid and 80B FRT (mosaic flies having retinas composed of only arf72A mutant cells). Each 20 wild-type and arf72A mutant photoreceptor cells were sorted according to the intracellular distribution of GFP-labeled rhodopsin. FIG. 4b shows that the type I photoreceptor cells are Rh1-GFP mainly in the cytoplasm, and the type II photoreceptor cells are Rh1-GFP in the cytoplasm and labdomain. In the type III photoreceptor cells, Rh1-GFP is mainly present in Rabdmeyer. Figure 4c is a graph showing the results of measuring the time course of wild-type and arf72A mutant cells to Ravdorme transduction. Type II and type III photoreceptor cells were identified every 6 hours. Arrows and triangular arrows show cytoplasmic rhodopsin staining and rhabdocin staining of Rabdomere, respectively.
Fig. 5 shows the results of confirming the function of arf72A, which distinguishes the folding state of cargo molecules in the endoplasmic reticulum. Figure 5a shows that arf72A is located in the vesicle membrane of ninaE ^D1 / + photoreceptor cells. UAS :: RFP-arf72A and UAS :: Rh1-GFP were overexpressed in ninaE ^D1 / + photoreceptor cells with Rh1 :: GAL4. Unlike the wild-type, RFP-ARF72A in ninaE ^D1 / + photoreceptor cells is strongly stained in the perinuclear region corresponding to perinuclear ER and overlaps with cytoplasmic Rh1-GFP. Figure 5b shows that deficiency of arf72A in ninaE ^D1 / + photoreceptor cells bypasses ER quality control. GFP- show that the rhodopsin-labeled wild-type and ninaE ^D1 / + Rab also present only in photoreceptor cells Sami. In ninaE ^D1 / + double-mutant photoreceptor cells, the GFP-labeled cytoplasmic rhodopsin was completely transferred to Labdomie. FIG. 5c shows that rhodopsin accumulated in vesicles in arf72A-RNAi / ninaE ^D1 / + photoreceptor cells bypasses quality control of the endoplasmic reticulum. Rhodocin was detected using GFP antibody in the head of a day old fly. w ¹¹¹⁸ and ninaE ¹¹⁷ flies eyes were used as negative control. Compared to the wild type (row 3), ninaE ^D1 / + decreased the total amount of GFP-labeled rhodopsin, while immature rhodopsin increased (column 4). The level of rhodopsin decreased in the retina but the number of mature rhodopsin increased (column 5) in the retinas of the arf72A-RNAi / ninaE ^D1 / + double-mutant (arf72A-RNAi / UAS ;; Rh1-GFP, Rh1 :: Gal4, ninaE ^D1 ) .
Figure 6 shows the results of confirming that the deficiency of arf72A rescued membrane defects in ninaE ^D1 / + photoreceptor cells. To confirm the intracellular structure, the head of a fly was imbedded for electron microscopic analysis. The ninaE ^D1 / + photoreceptor cells of FIGS. 6A-6C show a large accumulation of membranes (arrows) characteristic of dominant ninaE ^D1 / + mutations. 6d to 6f show that the accumulation of ninaE ^D1 / arf72A-RNAi double mutants (+ accumulation in photoreceptor cells was significantly reduced (Fig. 6e, arrow). Also, the morphology of double- mutant photoreceptor cells is similar to that of arf72A mutant photoreceptor cells (Fig. 6F, arrows).
Figure 7 shows the result of the deficiency of arf72A to repair ninaE ^D1 -induced retinal degeneration. To determine the extent of retinal degeneration, the head of a 23-day-old fly was embedded for electron microscopy analysis. The deficiency of the arf72A function of the ninaE ^D1 / + retina shows that the integrity of the eye is significantly improved by the preservation of the photoreceptor cells at one eye. Fig. 7A is a graph showing the relationship between Rh1 :: GAL4 , UAS :: Rh1 - GFP , ninaE ^D1 / + and UAS :: arf72A - RNAi ; Rh1 :: GAL4 and UAS :: Rh1 - GFP / ninaE ^D1 .

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Experiments

Drosophila and Drosophila genotypes

Drosophila grew under standard food in a 25 ° C incubator except for the flies used in the rescue experiment. Standard genetics was used to generate the following genotypes:

w ¹¹¹⁸ was wild type, ninaE ¹¹⁷ was rhodopsin null, ninaE ^D1 was ninaE dominant mutant, arf72A ¹ was deficient in arf72A, and P [GD6768] v17826 was mainly used in arf72A. The balancer chromosome used the method reported by Lindsley and Zimm (1992). The arf72A mosaic fly was produced by the arf72A chromosome with the p [ry ⁺ ; hs-neo; FRT] 80B FLP recombinase target chromosome (Stowers and Schwarz, 1999). The chromosomes prepared are then cloned into the yweyFLP ; FRT 80B or yweyFLP ; 0.0 > FRT80B < / RTI > GMR :: hid FRT. Rh1 :: GAL4 driver insertions of the second and third chromosomes were derived from the Rh1 :: GAL4 strain produced by Tabuchi et al. (2000), and various UASs including the markers of arf72A rust-down construct and cells And used for overexpression of the target. UAS :: Rh1- GFP was prepared using prh1 :: eGFP from Pichaud and Desplan (2001). Rh1 :: GAL4 and UAS :: Rh1- GFP were recombined and inserted into the FRT arf72A strain. A new chromosome was identified with w + quantitative increase in the presence of GFP fluorescence inserted with the Rh1 :: GAL4 driver and UAS :: Rh1- GFP, and later occurrence of the Rh1 :: GAL4 driver.

Generation of transgenic animals

A primer specific for arf72A was designed using a gateway system (Invitrogen, USA). The remainder was the pENRT TOPO vector (Invitrogen, USA) and the final vector pTRW (Carnegie Institute of Washington, USA) except that the reverse primer contained an amino terminus of arf72A and a red fluorescent protein (RPP) at the stop codon. Lt; / RTI > Plasmid extraction was performed using a Qiagen kit (USA) from positive clones. After injection, the fly of the G0 generation is w ; SM1 / Sco; TM2 / Sb equilibrium. Among the offspring, mini-w ⁺ eyes were classified. Mini-w ⁺ was used as a marker to confirm the presence of the transgene. Paris (G1 family) with a Mini-w ⁺ eyes were mapped to the location of the transgene and crossed to w ^1118. w ¹¹¹⁸ After crossing of the flies, Mini-w ⁺ flies were crossed with driver stocks. In all cases, Mini-w ⁺ flies were transfected with cnbw / cnbw for transgene overexpression; Rh1 :: GAL4 , and UAS :: Rh1-GFP / Ubx130 es emc2 .

Optical microscope and electron microscope

An SV stereomicroscope (Zeiss, USA) and a Spot RTKE digital microscope camera (Diagnostic Instruments, USA) were used to photograph the fruit fly eyes. The fly eyes were prepared as samples for electron microscopy according to the method described by Wachburn and O'Tousa (1992). The sections for electron microscopy were 80-100 nm thick, stained first with 50% ethanol with 5% uranyl acetate, and placed on Renold's lead citrate. The electron microscope was a Hitachi H600 (USA) or a Tecnai G2 F30 (FEI, USA) electron microscope. All electron micrographs show cross sections cross-sectioned at the depth of the R1-R6 photoreceptor nucleus.

Whole-mount Ion separation and fluorescence microscopy

Sacrificed eclosed flies within 48 hours. For full-mount ommatidia separation, the flies head was separated from the body. The head of the fly was sagittally cut and bisected. The brain and snout were removed. The eyes were placed on a microscope slide with 1x PBS (Phosphate Buffered Saline). For ER-Tracker ^™ Blue-White DPX (Invitrogen, USA) staining, flies eyes were incubated in the ~1 μM ER-tracker for 20 minutes and washed with 1 × PBS for 30 minutes before placing on the slide. Separate the eyes using a sharp platinum needle and loosen large portions of the retina. Non-retinal tissue was removed prior to fixation (0.5% paraformaldehyde) treatment and mounting medium (Vector Laboratories, USA) was added. Each fraction was observed using a Bio-Rad MRC 1024 scanning confocal (2 channel / LaserSharp 3.2 program / networked) system (Hercules, USA) or LSM5 Pascal laser scanning microscope (Carl Zeiss, Germany). The optical image is × 100 objective.

Vitamin A structure (rescue)

According to the method reported by Nicols and Pak (1985), flies were cultured in simple medium lacking vitamin A (270 ml of distilled water, 230 ml of grape juice, 11 g of Bacto-agar, 3 g of glucose, Sucrose, 0.5 g fructose, 1 g yeast). The resulting flies were transferred to a new vial consisting of cotton soaked with water and fasted for 24 hours. The fasted flies were transferred to a glass bottle consisting of all-trans-retinol and kept in the shade for 24 hours before dissection. Total-trans-retinol (Sigma, USA) was dissolved in 95% ethanol (10 mg / ml) and diluted with 1% sucrose solution to a final concentration of 2 mg / ml.

Western Blot  analysis

The flies head was collected from 2-day old samples and homogenized in lysis buffer (1.5 M Tris pH 6.8, 10% glycerol, 2.2% SDS (Sodium Dodecyl Sulfate), 0.00125% Bromophenol Blue, 5% B- % SDS-polyacrylamide gel, and then transferred to a nylon membrane. The gel was blotted overnight at 30 V and blocked in TTBS (TBS-Tween 20) containing 5% defatted milk powder for 30 minutes. Mouse anti-rhodopsin antibody, 4C5 (Developmental Studies Hybridoma Bank, USA) were reacted in TTBS diluted 1: 1000. Rabbit anti-GFP polyclonal antibody (AB Frontier, Korea) was reacted in TTBS diluted 1: 5000. Anti-mouse or anti-rabbit HRP (horse radish peroxidase) labeled secondary antibody (Pierce, USA) was reacted in TTBS diluted 1: 10000. Chemiluminescence was measured using a Pierce super signal reagent.

result

ARF72A Location of the Golgi complex

Sequencing of the Arf27A gene from a lethal Drosophila mutation revealed that the sequence was 55% homologous to Arf1 of yeast and bovine (Tamkun et al., 1991). Phylogenetic analysis using DNA or protein from various species of the Arf family showed that arf72A was consistent with arl1 in mammals (Kahn et al., 2004). The amino acid sequence of ARF72A was 85% homologous to that of mouse or human arl1, and the G2 mistoylate portion was conserved.

Because of the clear homology between the fruit fly arf72A and the mammal Arl1, the intracellular location would be similar. In order to investigate ARF72A position in Drosophila photoreceptor cells, RFP-arf72A and the rabbit marker protein Rab6-GFP were expressed using the UAS / Rh1-Gal4 system, and then separated from the photoreceptor cells from the photoreceptor 1A). The confocal microscopy photograph of the whole-mount circle shows the RFP and GFP staining of the cytoplasm, the Golgi complex in Drosophila tissue (Figs. 1B and 1C, Kondylis et al., 2001). In addition, RFP fluorescence appeared partially overlapping with GFP fluorescence of the golgi marker protein. The position of ARF27A in the Golgi complex is consistent with the Arl1 site of yeast and mammal (Lee et al., 1997; Panic et al., 2003; Icard-Liepkalns et al., 1997; Lu et al., 2001).

Mosaic analysis

Malfunctions of the vesicle delivery system often lead to abnormal cellular structures such as hyperperfusion of the ER membrane and expansion of the Golgi complex and endosome (Colley et al., 1991; Satoh et al., 1997, 2005). The deficiency of Drosophila arf72A induces a specific change in intracellular structure, so the function of the Arf family protein in vesicular transmission is important. Due to the lethality induced by the mutation of the arf72A gene, a mosaic flies capable of examining the lethal mutant genotype in the high-differentiated photoreceptor cells were prepared using the ey-FLP-FRT system (Fig. 2a, Stowers and Schwarz, 1999) . To distinguish between wild-type clones and arf72A mutant clones, the w ⁺ gene, a pigment particle that appears only under the rhabdomere of wild-type photoreceptor cells, was used as a cell marker. Electron microscopic images of the arf72A mutant cells show an over-treated inner membrane (Fig. 2b). More prominently, the rough endoplasmic reticulum membrane was found to be significantly expanded and swollen, and it was confirmed that the homogeneous wild-type endoplasmic reticulum membrane was uneven in electron density and optical range.

Changes in the endoplasmic reticulum membrane indicate that arf72A is involved in membrane transport between the ER and the Golgi complex. Failure of the rhodopsin emission from the endoplasmic reticulum reduces the level of rhodopsin, but the decrease in the amount of rhodopsin in the retina of the arf72A mutation is less than that of rhodopsin in the intracellular transit mutation (Colley et al., 1991; Ozaki et al., 1993 Kurada and O'Tousa, 1995). A weak decrease in the amount of rhodopsin in the eyes of the arf72A mutation suggests that the vesicle phenotype is not due to the failure of vesicle excretion of secretory proteins.

arf72A Of the vesicle-ossicular membrane transport

Membrane transport between the endoplasmic reticulum and the Golgi complex is a dynamic process in which anterograde and retrograde are highly regulated, maintaining membrane balance between the two organelles (Lippincott-Schwartz, 1993). Thus, structural changes of the ER membrane due to arf27A deficiency will alter the transport kinetics between the endoplasmic reticulum and the Golgi complex. To investigate the transmembrane-vesicular transport kinetics, the eyFLP-GMR :: hid FRT system was used to produce a mosaic fly having a flies eye composed of arf72A mutant cells. Wild type cells except for arf72A mutant cells in the eye were removed by eye-specific expression of the killing reactor, hid . Golgi complexes of wild-type and arf72A mutant photoreceptor cells were visualized by expressing GFP-labeled Rab6 with an oval marker. The arf72A mutant cells exhibited a wide range of Golgi structure in size and number compared to wild type cells, indicating that the membrane balance of the Golgi complex changed (Fig. 3).

Structural changes in the endoplasmic reticulum and the Golgi complex will affect the efficiency of vesicle transport. To confirm this, GFP-labeled rhodopsin was expressed in wild-type and arf72A mutant photoreceptor cells using UAS / Rh1 Gal4. Rhodopsin is synthesized in rough endoplasmic reticulum, and is transferred via a vaginal delivery system to its destination Rabodomie (Fig. 4a, Pak, 1995). During the rhodopsin maturation process, the release from the endoplasmic reticulum requires light-induced structural changes from the pigment, retinal, and trans form to the cis form. Without retinal or light, the rhodopsin molecule will remain in the endoplasmic reticulum (Ozaki et al., 1993; Sarfare et al., 2005). Therefore, the targeting rate at which rhodopsin reaches the ribomir from the endoplasmic reticulum can be measured by supplying the pre-trans-retinol in the shade state and then exposing the retina to light. Surprisingly, the targeting rate of Rorodosin to Rabdomilla was found to move faster in the arf72A mutant photoreceptor cells than in the wild type. The GFP-labeled rhodopsin locus consists of 20 wild-type and 20 arf72A mutant photoreceptor cells of type Ⅰ (where rhodopsin is predominantly located in the cytoplasm), type Ⅱ (where rhodopsin is mainly located in the cytoplasm and labdomain) and type Ⅲ (Fig. 4B). The result was confirmed through confocal microscopy every 6 hours for 24 hours. To distinguish between type II and type III photoreceptor cells, all arf72A mutant photoreceptor cells were scored as type II or III for 6 hours (FIG. 4C). In fact, mutant photoreceptor cells were classified as 35% after 6 hours and 80% after 18 hours. In contrast, GFP-labeled rhodopsin in wild-type photoreceptor cells migrated very slowly, with only 55% of type II or type III cells even after 18 hours. In addition, when the type III cells were measured, it was confirmed that the rhodopsin in the ER of the arf72A mutant cells disappears more rapidly and is rapidly targeted. These results indicate that arf72A functions as a negative regulator, and deletion of arf72A transfers the membrane balance between the endoplasmic reticulum and the Golgi complex to the Golgi complex and accelerates the transport of the membrane from the endoplasmic reticulum to the Golgi complex.

arf72A  And endoplasmic reticulum quality control

The secretion process of proteins consists of protein folding through cell compartments, post-transcriptional modifications and transmission. Proper folding and excretion in the endoplasmic reticulum has been reported as a rate-limiting step in yeast and mammalian cell systems (Coughlan et al., 2004; Petaja Repo et al., 2000). In the present invention, the arf72A mutant photoreceptor showed rapid transfection of rhodopsin into Rabbodimer and rapid disappearance of cytoplasmic rhodopsin due to the change in membrane kinetics between the endoplasmic reticulum and the Golgi complex. These results indicate that arf72A regulates protein release from the endoplasmic reticulum. As a result, an increase in ARF72A in the endoplasmic reticulum indicates accumulation in the endoplasmic reticulum lumen to secrete or break down the newly synthesized protein.

The dominant ninaE ^D1 mutation was used to generate the same conditions. The dominant ninaE ^D1 mutant produces a miss-folded mutant rhodopsin that interferes with maturation in the wild type and inhibits the release of rhodopsin. Thus, mutations and wild-type rhodopsin accumulate in the endoplasmic reticulum. In ninaE ^D1 / + photoreceptor cells, Rh1-GFP fluorescence was mostly located with RFP fluorescence of the ERP-KDEL, an endoplasmic reticulum marker. This is an ideal system for studying the genetic elements required for the release of proteins from the endoplasmic reticulum into dominant ninaE ^D1 / + mutant cells. In this disclosure, RFP-arf72A and Rh1-GFP expression in ninaE ^D1 / + photoreceptor cells was examined by confocal microscopy (Fig. 5A). Compared with the wild type, ninaE ^D1 / + photoreceptor cells stain strong cytoplasmic staining and partially overlap Rh1-GFP staining, showing strong RFP-ARF72A staining around the nucleus, which is characteristic of endoplasmic reticulum staining in Drosophila tissue (Orso et al ., 2009). The position of ERP-ARF72A ER in ninaE ^D1 / + photoreceptor cells was confirmed by co-labeling with an endoplasmic reticulum-tracker. Therefore, more ARF72A was detected in the ER of ninaE ^D1 / + photoreceptor cells as compared to the wild type (Fig. 1B). This suggests that the functional role of arf72A in the endoplasmic reticulum is to accumulate misfolded proteins.

The results for arf72A, a negative regulator of endoplasmic reticulum-vesicle transport, suggest that the rate-limiting step of the vesicle excretion process in arf72A mutant cells will affect whether it is folding properly. In order to confirm that the deficiency of arf72A function is functioning in the vesicle quality check, the location of the GFP-labeled rhodopsin was examined to perform an epistatic analysis of arf72A with the dominant ninaE ^D1 / + mutation (Fig. 5B). Visualization of GFP-labeled rhodopsin in wild-type and arf72A-RNAi photoreceptor cells revealed staining with the exception of Rabdmeyer and ninaE ^D1 The photoreceptor cells showed dominant cytoplasmic staining (Figure 5b). On the other hand, it was confirmed that the amount of rhodopsin migrated from the cytoplasm of double-mutant to the ribomile with the eye-specific arf72A rust-down by RNAi compromises ERC quality control in the ninaE ^D1 / + photoreceptor.

Western blot analysis confirmed the epistatic results of confocal microscopy results. During maturation, rhodopsin is glycosylated in the endoplasmic reticulum and slowly changes from the 40 kDa endoplasmic reticulum form to the 34 kDa rhabdomeric form (Katanosaka et al., 1998; Webel et al., 2000). Therefore, the mature state of Rhodopsin can be confirmed in size through Western blot. GFP-labeled rhodopsin, expressed Ectopically in day-old flies, was probed with GFP-specific antibodies to confirm maturation status. Consistent with previous results, total GFP-labeled rhodopsin decreased in ninaE ^D1 / + retinas compared to wild-type retinas, whereas immature rhodopsin increased (Colley et al., 1995b) . The double-mutant (arf72A-RNAi / UAS :: Rh1-GFP, Rh1 :: Gal4, ninaE ^D1 ) retinas further decreased the level of total dopamine but increased mature rhodopsin (column 5 of FIG. These results indicate that quality control of the endoplasmic reticulum is compromised by the deficiency of arf72A function. In addition, a decrease in the level of rhodopsin in the double-mutant indicates that rhodopsin bypassed quality control in the endoplasmic reticulum, but most of the rhodopsin is denatured before reaching Rabdomere.

arf72A  rescue( rescue ) ninaE ^D1 - associated membrane damage and ninaE ^D1 - induced retinal degeneration

Dominant mutations of rhodopsin such as ninaE ^D1 in Drosophila have been used as models for studying life-dependent retinal degeneration. In a recent study, ninaE ^G69D , one of the dominant rhodopsin mutants, was rescued by regulating ER stress (Ryoo et al., 2007; Kang and Ryoo, 2009). Loss of the arf72A function responsible for ERC quality results in a reduction in the overall amount of Rhodopsin but increases the mature portion in the ninaE ^D1 / + retina. Thus, the results of the present invention improve the likelihood of rescue of the ninaE ^D1 - related phenotype through reduction of ER stress by arf72A deletion. Woo Sung ninaE ^D1 Mutant photoreceptor cells exhibit tremendous membrane buildup and accumulation of rough endoplasmic reticulum (Figs. 6A-6C). Although the vesicle-like membrane accumulation was still present in the double mutation, the abnormal membrane enhancement was significantly reduced (Figs. 6d to 6f). Interestingly, the envelope membrane of the double-mutant cells is similar to that of the arf72A mutant cell.

We confirmed that the effect of arf72A deficiency denatures ninaE ^D1 -induced retinas. The deficiency of arf72A function by introduction of rust-down construct inhibited retinal degeneration by ninaE ^D1 / + (Fig. 7). Compared with the ninaE ^D1 / + retina, which has some phagocytosis by neuronal cells and part of the rubtomaric defect, the deficiency of arf72A function of ninaE ^D1 / + effectively retards degeneration of the retina by preservation of the ribomorrhea (Figs. 7A to 7C). Interestingly, the overall size of aged dual-mutant photoreceptor cells was markedly reduced by defects in membrane balance. This suggests that arf72A deficiency in ninaE ^D1 / + assists in delaying membrane accumulation and retinal degeneration by draining the vesicle-accumulated rhodopsin from the vesicle lumen.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

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<110> Industry-University Cooperation Foundation Sogang University <120> Methods for Screening Therapeutic Candidates of Retinopathy <130> PN110418 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 543 <212> DNA <213> Drosophila arf72A <400> 1 atgggtgggg tgctcagcta ctttcgcgga ctgctcggct cccgggagat gcgcatcttg 60 atcctgggcc tggacggcgc cggcaagacc acgatcctct acagactgca ggtgggcgag 120 gtggtcacca cgatacccac catcggcttc aacgtggagc aggtcaccta caagaacctc 180 aagttccagg tctgggattt gggcggacag acaagtatta gaccttattg gcgttgctac 240 tacagcaaca cggacgcaat catctatgtg gtggactcgg cggaccggga ccgcattggc 300 atctccaagg acgagctgct gtacatgctg cgtgaggagg agctggccgg cgcgatactg 360 gtcgtcctgg cgaacaagca ggacatggac ggatgcatga ccgtcgccga ggtccatcat 420 gcgttaggac tggagaacct aaagaaccgc acatttcaaa tattcaaaac gtcggccacc 480 aagggcgagg gactcgacca ggccatggac tggctgtcca acaccctgca gagtcgcaag 540 tag 543 <210> 2 <211> 546 <212> DNA <213> Human arl1 <400> 2 atgggtggct ttttctcaag tatattttcc agtctgtttg gaactcggga aatgagaatt 60 ttaattttgg gattagatgg agcaggaaaa accacaattt tgtacagatt acaagtggga 120 gaagttgtta ctactatacc taccattgga tttaatgtag agacggtgac gtacaaaaac 180 cttaaattcc aagtctggga tttaggagga cagacaagta tcaggccata ctggagatgt 240 tactattcaa acacagatgc agtcatttat gtagtagaca gttgtgaccg agaccgaatt 300 ggcatttcca aatcagagtt agttgccatg ttggaggaag aagagctgag aaaagccatt 360 ttagtggtgt ttgcaaataa acaggacatg gaacaggcca tgacttcctc agagatggca 420 aattcacttg ggttacctgc cttgaaggac cgaaaatggc agatattcaa aacgtcagca 480 accaaaggca ccggccttga tgaggcaatg gaatggttag ttgaaacatt aaaaagcaga 540 cagtaa 546 <210> 3 <211> 31 <212> RNA <213> arf72A RNAi-F <400> 3 gcgtctagac ttgcgactct gcagggtgtt g 31 <210> 4 <211> 35 <212> RNA <213> arf72A RNAi-R <400> 4 cgcgaattca ccttattggc gttgctacta cagca 35

Claims (9)

A screening method for a therapeutic agent candidate for a retinal disease comprising the steps of:
(a) contacting a test substance with a cell comprising (i) an Adenosine diphosphate Ribosylation Factor 72A gene or protein, or (ii) an Arl1 (ADP-ribosylation factor-like protein 1) ; And
(b) measuring the expression level of said Arf72A or Arl1 gene, the amount of Arf72A or Arl1 protein, or the activity of Arf72A or Arl1 protein;
When the amount of expression of the Arf72A or Arl1 gene, the amount of Arf72A or Arl1 protein, or the activity of Arf72A or Arl1 protein is measured to be down-regulated, the test substance is judged to be a therapeutic candidate for a retinal disease .
2. The method of claim 1, wherein the cell is a photoreceptor cell.
The method of claim 1, wherein the retinal disease is selected from the group consisting of retinopathy, autosomal-dominant retinopathy, Usher's syndrome, Stargardt disease, Barret-Biddle's syndrome, best disease, choroidal abscess, retinal pigment epithelium, retinal hyperplasia, Leber Congential Amaurosis (Laber's Hereditary Optic Neuropathy), BCM (Blue-cone monochromacy), retinal detachment, ML (Malattia Leventiness) Wherein the disease is Oguchi disease or Refsum disease.
4. The method of claim 3, wherein the retinal disease is retinopathy or autosomal dominant retinopathy.
(i) an inhibitor of the expression of Arf72A [Adenosine diphosphate (ADP) Ribosylation Factor 72A] or an inhibitor of the activity of Arf72A protein, or (ii) an inhibitor of the expression of Arl1 (ADP-ribosylation factor-like protein 1) gene or an inhibitor of Arl1 protein. Pharmaceutical composition for the prevention or treatment of retinal diseases comprising as an active ingredient.
6. The composition of claim 5, wherein the inhibitor normally matures and releases rhodopsin in the endoplasmic reticulum.
The method of claim 5, wherein the retinal disease is retinopathy, autosomal-dominant retinopathy, Usher syndrome, Stargardt disease, Barrett-Bead syndrome, Best disease, choroidal defect, choroidal atrophy (gyrate-atrophy), retinitis pigmentosa, retinal macular degeneration, Leber Congential Amaurosis; Laber's Hereditary Optic Neuropathy, Blue-cone monochromacy (BCM), interretinal separation, ML (Malattia Leventiness) A composition characterized in that the disease (Oguchi disease) or Lefsum disease (Refsum disease).
4. The composition of claim 3, wherein the retinal disease is retinopathy or autosomal dominant retinopathy.
The composition of claim 5, wherein the expression inhibitor of Arf72A gene or the expression inhibitor of Arl1 gene is an antisense oligonucleotide or siRNA for the Arf72A gene or Arl1 gene.

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