KR20180109278A - Composition for treating retinal vascular disease comprising mTOR targeting siRNA with cross-species activity - Google Patents

Abstract

The present invention relates to a composition for treating retinal vascular diseases containing a siRNA targeting mTOR having interspecies cross-activity. The present invention provides an siRNA targeting mTOR having interspecies cross-activity, a recombinant vector containing the same, and a pharmaceutical composition containing the same as an active ingredient. The mTOR targeting siRNA is composed of a sequence complementary to a part of the mTOR gene so that the mRNA of the mTOP gene can be decomposed or translation can be inhibited. Therefore, various retinal vascular diseases targeting mTOR, for example, a selected disease from a group consisting of macular degeneration, diabetic retinopathy, retinopathy of prematuritv, occlusion of retinal vessels, and ocular ischemic syndrome can be prevented or treated.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for treating retinal vascular disease, which comprises siRNA targeting mTOR having interspecific cross-reactivity,

The present invention relates to a composition for treating retinal vascular disease containing an siRNA targeting mTOR having interspecies cross-reactivity.

Various retinal vascular diseases such as diabetic retinopathy, retinal vein occlusion, and wet age-related macular degeneration are common retinal pathologies that cause severe vision loss. The major pathological processes of these vascular diseases include new blood vessel formation after retinal ischemia and vascular leakage from new blood vessels. Based on this pathological mechanism, current standard therapies include intravitreal injection or laser treatment of anti-vascular endothelial growth factor (anti-VEGF) in the avascular area . Recent clinical studies have shown that patients with diabetic retinopathy who received sustained anti-vascular endothelial growth factor have improved vascular occlusion and severity, but the current practice for prevention or reduction of angiogenic zone formation in retinal vascular disease There is no standard treatment.

The mammalian target of rapamycin (mTOR) is a serine-threonine protein known to play an important role in a variety of cellular functions including protein synthesis, cell growth, cell proliferation, cell motility and autophagy Kinase. As an essential component of the mTOR signaling pathway, mTOR is present in both mTOR complex 1 (mTOR complex 1; mTORC1) and mTOR complex 2 (mTORC2). As an mTOR inhibitor, rapamycin has been originally used as an antifungal agent and is known to have anti-tumor and anti-angiogenic properties. Rapamycin specifically binds to the FK507 binding protein 12 (FKBP12) of mTORC1 and interferes with the signal transduction pathway. Anti - angiogenic activity by rapamycin is known to be associated with decreased VEGF production and vascular endothelial cell response by VEGF. Recent studies have shown that rapamycin inhibits proliferation of hypoxia-induced retinal pigment epithelial cells through a mechanism associated with the target of mTOR / hypoxia-inducible factor-1 (HIF-1) / VEGF signaling There is a bar. Based on these results, targeting mTOR signaling may be a new treatment for choroidal neovascularization. The use of systemic rapamycin in retinopathy of premature hyperoxia / hypoxia model as well as choroidal vascular disease has been reported to inhibit retinal neovascularization.

As a mediator, RNA interference with small interfering RNAs (siRNAs) is an evolutionary mechanism associated with post-transcriptional gene silencing (PTGS), which is a sequence-specific manner ) Provides a new therapeutic method for treating various human diseases by interfering with the causative genes associated with the development of the pathological process. Considering the basic concept of siRNA, siRNA technology has great potential as a new therapeutic method for various hereditary congenital diseases or retinal diseases. However, the current major limitation for applying siRNA to human disease is that it is difficult for siRNAs to reach the cytoplasm of target tissues through the cell membrane. In order to overcome this limitation associated with siRNA delivery systems, numerous non-viral carriers such as polymers, lipids, protein nanocarriers and dendrimers as well as virus-based vectors have been used as siRNA It is being tried for delivery. Among these, recombinant adeno-associated virus (rAAV) is a safe, effective, and continuous tool for gene expression delivery.

Oxygen-induced retinopathy (OIR) is a good experimental tool for studying vascular changes in retinal vascular disease. Previous studies have shown that retinal vascular growth is inhibited at high oxygen concentrations and that neovascularization is activated at normal oxygen concentrations. Oxygen-induced retinopathy is also a reproducible model of angiogenesis in animal experiments (in vivo). The degree of neovascularization can be easily quantified by measuring abnormal blood vessel morphology, vascular occlusion and retinal ischemia. Oxygen-induced retinopathy is therefore a useful tool to demonstrate the effectiveness of molecular and cellular mechanisms of angiogenic disease, and anti-angiogenic agent testing.

In vitro studies are carried out in the presence of various other external factors as much as possible, by examining how a part of a tissue or cell of a living body is separated and reacted to a specific substance or environment. However, it is very difficult to grasp how cell-level experimental results are related to the health status of the whole population. Therefore, in order to evaluate the effect on the human body, ultimately, a comprehensive understanding of epidemiological studies and animal experiments (in vivo) and consistent results are required in addition to cell experiments. Even though the results of animal experiments can not be directly linked to humans, the common biologic responses of animals and humans are so numerous that the results of structured and reproducible animal experiments can be evaluated for efficacy and adverse effects on specific substances rather than cell- It can be very useful in assessing the impact of exposure to humans ultimately when analyzed in aggregate with epidemiological studies.

Korean Patent Publication No. 10-2016-0120028 (published Oct. 17, 2016)

It is an object of the present invention to provide a siRNA having interspecies cross activity capable of degrading mRNA of mTOR gene or inhibiting translation or a recombinant vector containing siRNA as an active ingredient And to provide a pharmaceutical composition for preventing or treating retinal vascular disease.

In order to accomplish the above object, the present invention provides a siRNA comprising a nucleotide sequence of SEQ ID NO: 1 and having interspecies cross activity that binds to mTOR and inhibits its expression, or a retina containing as an active ingredient a recombinant vector comprising the siRNA There is provided a pharmaceutical composition for preventing or treating vascular disease.

The present invention relates to an siRNA targeting mTOR having interspecific cross-activity, a recombinant vector containing the siRNA, and a pharmaceutical composition containing the siRNA as an effective ingredient, wherein the mTOR target siRNA comprises a sequence complementary to a part of the mTOR gene, Since the mTOR gene can be degraded or inhibited in translation, it can be used in a variety of retinal vascular diseases targeting mTOR, such as macular degeneration, diabetic retinopathy, retinopathy of prematurity, retinal vascular occlusion and ocular ischemic syndrome The selected disease can be prevented or treated.

Figure 1 shows the secondary structure of mTOR siRNA.
Fig. 2 shows the cleavage map of the recombinant vector rAAV-GFP-simTOR according to the present invention.
FIG. 3 shows the gene silencing effect of siRNA targeting mTOR in ARPE19 cells or HeLa cells. The results are shown by real-time PCR and Western blotting of (A) mRNA and (B) protein expression of mTOR.
FIG. 4 shows the result of analysis of retinopathy by angiographic and confocal microscopy in oxygen-induced retinopathy, showing (A) a boundary mask of the blood vessel region, (B) a hollow region in the blood vessel region, and (C) .
FIG. 5 shows a typical flat mount image of the retina of oxygen-induced retinopathy, wherein (A) the vehicle treated group, (B) rAAV-siCon treated group and (C) rAAV-GFP-simTOR treated group . Retinal mounts were stained to check for vascular development status, the presence and extent of aneurysmal retinal vessels. (Yellow: a vein area in the retina, blue: retinal vascular boundary, red: retinal vasculature)
FIG. 6 shows the effect of reducing vascular occlusion by inhibiting mTOR signaling in an oxygen-induced retinopathy model.
FIG. 7 shows the effect of suppressing the expression of HIF-1 and VEGF by mTOR siRNA in a hypoxic state.

The inventors of the present invention found that treatment of rAAV vector expressing mTOR siRNA in an oxygen-induced retinopathy animal model resulted in inhibition of angiogenesis by reducing the expression of abnormally increased HIF-1 and VEGF, , Suggesting that inactivation of the mTOR signaling pathway by mTOR siRNA may be a new therapeutic strategy in the treatment of various retinal vascular diseases including retinal vascular occlusion, ischemic syndrome, and diabetic retinopathy Thus completing the present invention.

The present invention provides a pharmaceutical composition for the prevention or treatment of retinal vascular disease comprising an siRNA having the interspecies cross-activity which is composed of the nucleotide sequence of SEQ ID NO: 1 and inhibits its expression by binding to mTOR as an active ingredient.

Preferably, the retinal vessel disease is selected from the group consisting of, but not limited to, macular degeneration, diabetic retinopathy, retinopathy of prematurity, retinal vascular occlusion, and ischemic syndrome.

Preferably, the siRNA can inhibit the expression of HIF-1 and VEGF.

In the present invention, the siRNA having interspecies crossing activity that binds to mTOR and inhibits its expression is composed of 100% complementary sequence with a part of the mTOR gene of human, monkey, and rat, and degrades mRNA of mTOR gene or inhibits translation can do. When the complementarity is 80 to 90%, translation of mRNA can be inhibited, and when it is 100%, mRNA can be degraded.

In addition, the siRNA targeting mTOR according to the present invention may include a base sequence having a homology of 80%, preferably 90%, more preferably 100%, to the nucleotide sequence shown in SEQ ID NO: 1. The nucleotide sequence may be linked by a loop region of 5 to 15 bp to form a hairpin structure.

The siRNA of the present invention can be produced by a method for producing an RNA molecule known in the art. As a method of producing the RNA molecule, a chemical synthesis method and an enzymatic method can be used. For example, the chemical synthesis of RNA molecules can be performed using the methods described in the literature (Verma and Eckstein, Annu. Rev. Biochem. 67, 99-134, 1999) And SP6 RNA polymerase (Milligan and Uhlenbeck, Methods Enzymol. 180: 51-62, 1989).

The present invention also provides a pharmaceutical composition for the prevention or treatment of retinal vascular disease comprising a recombinant vector comprising the nucleotide sequence of SEQ ID NO: 1 and comprising an siRNA having interspecies crossing activity which binds to mTOR and inhibits its expression, as an active ingredient .

Preferably, the recombination vector may have the cleavage map shown in Fig.

Preferably, the retinal vessel disease is selected from the group consisting of, but not limited to, macular degeneration, diabetic retinopathy, retinopathy of prematurity, retinal vascular occlusion, and ischemic syndrome.

Preferably, the siRNA can inhibit the expression of HIF-1 and VEGF.

The recombinant vector of the present invention can be produced by a recombinant DNA method known in the art.

Viral or viral vectors useful in the present invention for delivering siRNA to mTOR include baculoviridiae, parvoviridiae, picornoviridiae, herepesviridiae, But not limited to, poxviridiae, adenoviridiae, and the like.

In the present invention, it is most preferable to use Adeno Associated Virus (AAV). Adeno-associated virus is known to cause little immunological reaction and cytotoxicity. In particular, the adeno-associated virus serotype 2 is capable of efficient gene transfer to the central nervous system (CNS) neurons and can also effectively express the transgene in the nervous system for a long period of time .

The non-viral vectors useful for delivering siRNA to mTOR in the present invention include all vectors conventionally used for gene therapy except for the viral vectors described above. For example, various plasmids capable of being expressed in eukaryotic cells plasmids) and liposomes.

In the present invention, mTOR-targeted siRNA is preferably operably linked to at least a promoter for proper transcription in the transfected cells. The promoter may be any promoter capable of functioning in eukaryotic cells, more preferably a human H1 polymerase-III promoter. For the efficient transcription of mTOR-targeted siRNAs, regulatory sequences including leader sequences, polyadenylation sequences, promoters, enhancers, upstream activation sequences, signal peptide sequences and transcription termination factors, May also be included.

When an siRNA having interspecies crossing activity that inhibits its expression in combination with the mTOR according to the present invention or a recombinant vector containing the siRNA is used as a pharmaceutical composition, a suitable carrier, excipient or diluent commonly used in the production of a pharmaceutical composition .

Examples of carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

The composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose sucrose), lactose, gelatin, and the like.

In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

The amount of the composition to be used may vary depending on the age, sex, and body weight of the patient, but may be administered in an amount of 0.1 to 2.0 mg / kg once to several times per day.

In addition, the dosage of such a composition may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The composition can be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

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  One: mTOR siRNA  Design and analysis

Target sequences were selected from the completely conserved sequence pattern of mTOR of various species. At this time, the species used include human (LOCUS: NM_004958), monkey (XR_014791), rat (NM_019906) and mouse (NM_020009).

For effective siRNA design, we used siRNA design software developed on-campus called MWG Biotech AG software (www.mwgbiotech.com) or CAPSID (Convenient Application Program for siRNA Design). The mTOR siRNA sequence (SEQ ID NO: 1) and siRNA score are shown in Table 1 below.

  name Target sequence
(5 '- >3') location
(Nucleotides)   target siRNA
score
mTOR siRNA
GAAUGUUGACCAAUGCUAU
7221
Kinase
13

As shown in Table 1, when the score of mTOR siRNA was measured using CAPSID, it was confirmed that it had an excellent score of 13 points. Generally, if the siRNA score is 8 or more, it belongs to the superior side.

Also, referring to FIG. 1, the secondary structure of mTOR siRNA was analyzed, and the energy value was -64.3. In view of the structure, mTOR had excellent accessibility to and binding to the target mRNA Respectively.

Example  2: Cell culture

HeLa cells as human cervical cancer cells and ARPE19 cells as human retinal pigment epithelial cells were purchased from ATCC (American Type Culture Collection, Manassas, Va.). The cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS, Gibco, Middleton, WI), GlutaMAX-1 (2 mM) and penicillin (100 IU / ml) / streptomycin (Invitrogen, Carlsbad, Calif.) At 37 ° C in a 5% CO ₂ incubator.

For hypoxic treatment, 100 μM of hypoxia-mimetic chemical Cobalt (II) chloride (Sigma, St. Louis, Mo.) was added to the culture medium for 24 hours.

Example  3: siRNA / rAAV  Manufacture and transfection

siRNA (5'-GAAUGUUGACCAAUGCUAU-3 '; SEQ ID NO: 1) targeting mTOR was purchased from Genolux (Seoul, Korea). A non-specific scrambled control siRNA (5'-AUUCUAUCACUAGCGUGAC-3 '; SEQ ID NO: 2) used as a negative control was provided by Dharmacon Inc.

For transient transfection, the cells were incubated with 100 nM siRNA and serum-free medium for 4 hours using oligofectamine reagent (Invitrogen), and then incubated with fresh 10% fetal bovine serum The medium was added and incubated for a specific time.

For animal experiments, autologous complementary recombinant adeno-associated genes expressing the corresponding mTOR siRNA (rAAV-simTOR) or control siRNA (rAAV-siCon) induced by green fluorescent protein (GFP) and the H1 promoter The recombinant adeno associated virus 2 (rAAV2) vector was prepared by reference to a previously reported paper (Cell Mol Life Sci 2012; 69: 3147-3158). The cleavage map of the recombinant vector rAAV-GFP-simTOR is shown in Fig.

Example  4: Real time Reverse transcription  Polymerase chain reaction analysis

RNA was extracted using trizol (Invitrogen) reagent, and extracted RNA was synthesized with cDNA using Superscript III (Invitrogen) kit. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed using SyBr Green I and a primer: mTOR primer, 5'-CAC AGT GCC AGA ATC TAT TC-3 '(forward; SEQ ID NO: 3) and 5'-GAG AAG TCC CGA CCA GTG AG-3' (reverse; SEQ ID NO: 4); VEGF primer, 5'-CCC ATG GCA GAA GGA GG-3 '(forward; SEQ ID NO: 5) and 5'-GGA AGA TGT CCA CCA GG-3' (reverse; (Reverse direction; SEQ ID NO: 7) and 5'-TGC TTG CTG ATC CAC ATC TG-3 '(reverse direction, SEQ ID NO: 7) and 5'- TGA AGA TCA AGA TCA TTG CTC- 8). The reaction was repeated 39 times at 95 ° C for 3 minutes (initial denaturation / polymerase activity) after one cycle, at 95 ° C for 15 seconds, at 60 ° C for 30 seconds, and at 72 ° C for 30 seconds. β-actin was used for normalization and relative gene expression was assessed using GeneXpression Macro chromo4 software (Bio-Rad, Hercules, Calif.).

Example  5: Western Blat  analysis

Proteins were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Western blotting was performed. The protein separated by electrophoresis was transferred to polyvinylidene fluoride (PVDF) at 350 mA for 120 minutes using a transfer buffer (20% methanol, 25 mM Tris-HCl, 192 mM glycine) Membrane. Protein-transferred membranes were blocked with TBS (Tris-buffered saline) containing 0.1% tween 20 and 5% fetal bovine serum, then reacted with primary antibody and HRP-conjugated secondary antibody Jackson ImmunoResearch Laboratories, West Grove, Pa.). The membrane was coated with a mixed solution of 1: 1 mixture of color development reagents I and II of ECL (enhanced chemiluminescence) detection kit (Advansta), and X-ray film (CP-G plus, Agfa Healthcare Ltd, New Orleans , LA, USA), and the degree of protein expression on the film was observed. Antibodies to mTOR (2983S), HIF-1 (610958) and β-actin (A5441) used in the experiments were obtained from cell signaling (Boston, MA), BD Biosciences (San Jose, Calif. Sigma).

Example  6: Oxygen-induced retinopathy (Oxygen-induced retinopathy; OIR ) Model

All experiments were carried out in accordance with ARVO (Association of Research In Vision and Ophthalmology Statement for the Use of Ophthalmics and Vision Research), and the Asan Life Science Institute, University of Ulsan College of Medicine, Seoul, Korea) was approved by the Internal Review Board (IRB).

Sprague-Dawley rats (SD rats) were grown in an oxygen chamber at 80 ± 5% from day 7 postnatally (P8) to P12, and then grown for 5 days in an indoor air environment. The animals were divided into three groups: Group 1; Vehicle treated sham group (n = 5), group 2; rAAV-GFP-siCon treated group (n = 5), Group 3; rAAV-GFP-simTOR treated group (n = 5). SD rats were anesthetized by intraperitoneal injection of alfaxan (20 mg / kg) and tiletamine (12.5 mg / kg). Pupils were dilated with Mydrin-P (tropicamide 5 mg / mL and phenylephrine 5 mg / mL, Santen Pharmaceuticals, Japan), and a 33 gauge RAAV-GFP-siCon (2.5 X 10 ⁸ TPs), rAAV-GFP-simTOR (2.5 X 10 ⁸ TPs) or vehicle 5 L were injected into the vitreous using a Hamilton syringe (Hamilton, NE) Sclerotomy was performed at the posterior part of the limbus approximately 1 mm away from the limbus using a 33-gauge Hamilton syringe and care was taken to prevent damage to the lens. Respectively.

Example  7: Fluorescein Angiogram ( 분광제 angiogram ) analysis

Rat retina preparation and vascular staining methods were previously described (Connor et al) and were performed with some modification of the method. Obtained eyes were immersed in 4% paraformaldehyde (PFA) and fixed at room temperature for 1 hour. The sclera, uveal tissue, cornea and lens were then removed from the retina and the detached retina was immersed in phosphate buffered saline (PBS) for 5 minutes, And washed.

To stain the retinal vessels, the retina was placed in isolectin (Isolectin, B4-594, Molecular Probes, Carlsbad, Calif.) And reacted at 4 ° C for two days. The retina was then mounted flat on the slide using gelatin, covered with a cover slip, and then sealed with a manicure. Green fluorescent protein (GFP) expression was analyzed using a fluorescein angiogram and confocal microscope system (LSM 780, Zeiss, NY, NY).

Example  8: Retinopathy in an animal model of oxygen induced retinopathy

The following three steps were performed to obtain a boundary mask of the blood vessel area, an empty area in the blood vessel area and an entire eye cell area. As preprocessing, all image files were divided into RGB-color channel images. First, we segmented the blood vessel region by applying the automatic threshold method in the red channel image. The boundary mask was extracted by the 'particle analysis' method of image J (ImageJ), which is performed to detect a large object with the restriction of the object size option together with the vascular area mask. If more than a single object, multiple objects were created to create a single vascular area mask using a boundary 'OR' operation. Second, to obtain an empty area in the blood vessel area, the blood vessel area mask was invert. Then, an insufflated blood vessel mask was extracted by a 'particle analysis' method performed to obtain a small object in the blood vessel region. As with the first step, multiple objects were created to create a single blank area mask using a boundary 'OR' operation. Finally, we apply the automatic threshold method in the blue channel image to subdivide the outer boundary mask of the entire eye region. If the outer boundary mask is not a closed circular shape, a manual drawing is performed to make it a closed circular shape. Next, the outer boundary was detected as a whole eye cell region using the 'particle analysis' method. Based on the three masks, each area was measured based on the total number of pixels. The severity of retinopathy was quantified using the ratio of the total number of pixels in the retinal vascular region to the total retinal region. After reviewing all the images that were automatically segmented, the obvious errors were corrected manually by a specialist.

Example  9: Statistical analysis

All results were expressed as mean ± SD and values of P <0.05 were considered significant. Comparison of the mean retinopathy score between groups was analyzed by Mann-Whitney U-test, and comparison of VEGF expression between groups was analyzed by Kruskal-Wallis test. Statistical analysis was performed using SPSS V21 (IBM SPSS, Inc., Chicago, IL, USA).

Experimental Example  One : mTOR target To siRNA  by mTOR  Gene silencing effect

To confirm the effect of mTOR-specific siRNAs on mTOR expression, mTOR siRNAs synthesized in ARPE19 cells or nonspecific scrambled control siRNAs were treated.

As a result, referring to FIG. 3, mTOR mRNA and protein expression was remarkably decreased after treatment with mTOR siRNA. On the other hand, no changes were observed in mTOR mRNA and protein expression after scrambled control siRNA treatment. The gene knockdown level of mTOR siRNA in ARPE19 cells, a retinal pigment epithelial cell, was similar to that of cervical cancer cell line HeLa cells.

In addition, rAAV expressing the corresponding mTOR siRNA (rAAV-GFP-simTOR) or scrambled control siRNA (rAAV-GFP-siCon) with GFP as a reporter protein was prepared and analyzed. As a result, GFP expression was rapidly observed, whereas mTOR expression was decreased by rAAV2-GFP-simTOR. This means that simTOR effectively down-regulates endogenous reduced mTOR mRNA and protein expression levels.

Experimental Example  2 : mTOR  Retinal vasculature by inhibition of signal transduction vasculature ) Formation effect

For the analysis of retinopathy in an oxygen-induced retinopathy animal model, all mount images (Group 1, Group 2 and Group 3) were analyzed by automatic 'particle analysis' method.

Referring to FIG. 4, retinal angiograms show a wide range of non-vascular regions around and in the retina in the vehicle treated group and the rAAV-GFP-siCon treated group. However, referring to FIG. 5, it was confirmed that the area free of blood vessels of the retina was remarkably reduced in rAAV-GFP-simTOR-treated group. 6, when the blood vessel area was measured in the entire mounting area, the average blood vessels of 50.66.9% (group 1), 52.05.4% (group 2), and 62.810.3% (group 3) Area was observed.

In addition, other signs of retinal vascular insufficiency, such as retinal vasodilation and flexion, were observed in all subjects. There are numerous retinal vascular bundles near the bloodless area, but it was difficult to discriminate clearly from the stained remnants. Although retinal neovascularization was not quantified in this experiment, retinal angiogenesis was observed in most subjects, similar to retinal detachment. Angiogenesis was identified between the vascular occlusion area and the vascularized retina. This pathologic new vessel showed an unorganized form with small diameter and numerous retinal vascular bundles. Taken together, this means that mTOR siRNA treatment significantly reduces the vascular area in the OIR model.

Experimental Example  3: In the hypoxic state mTOR To siRNA  by HIF -1 and VEGF  Inhibitory effect

To determine whether the decrease in mTOR expression in hypoxic conditions affects the expression of HIF-1 protein and VEGF mRNA, ARPE19 cells were treated with mTOR siRNA or Control siRNA and cultured in normal or hypoxic conditions.

As a result, referring to FIG. 7, it was confirmed that expression of HIF-1 protein and VEGF mRNA was significantly increased under hypoxic conditions. mTOR siRNA treatment significantly inhibited the expression of HIF-1 protein and VEGF mRNA in a hypoxic state as well as a normal state. mTOR siRNA treatment rapidly destroyed the stabilization of HIF-1 protein by hypoxic conditions and decreased VEGF mRNA expression.

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

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

<110> University of Ulsan Foundation for Industry Cooperation          THE ASAN FOUNDATION          CdmoGen co., Ltd <120> Composition for treating retinal vascular disease comprising mTOR          targeting siRNA with cross-species activity <130> ADP-2016-0662 <160> 8 <170> KoPatentin 3.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> mTOR siRNA <400> 1 gaauguugac caaugcuau 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> scrambled control siRNA <400> 2 auucuaucac uagcgugac 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for mTOR <400> 3 cacagtgcca gaatctattc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for mTOR <400> 4 gagaagtccc gaccagtgag 20 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for VEGF <400> 5 cccatggcag aaggagg 17 <210> 6 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for VEGF <400> 6 ggaagatgtc caccagg 17 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for beta-actin <400> 7 tgaagatcaa gatcattgct c 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for beta-actin <400> 8 tgcttgctga tccacatctg 20

Claims (7)

1. A pharmaceutical composition for preventing or treating retinal vascular disease, comprising an siRNA having the interspecies cross-activity which is composed of the nucleotide sequence of SEQ ID NO: 1 and binds to mTOR and inhibits its expression, as an active ingredient. The pharmaceutical composition for preventing or treating retinal vascular disease according to claim 1, wherein the retinal vascular disease is selected from the group consisting of macular degeneration, diabetic retinopathy, retinopathy of prematurity, retinal vascular occlusion, and ischemic syndrome. The pharmaceutical composition for preventing or treating retinal vascular disease according to claim 1, wherein the siRNA inhibits the expression of HIF-1 and VEGF. 1. A pharmaceutical composition for the prevention or treatment of retinal vascular disease comprising a recombinant vector comprising the nucleotide sequence of SEQ ID NO: 1 and comprising an siRNA having interspecies cross activity that binds to mTOR and inhibits its expression. 5. The pharmaceutical composition for preventing or treating retinal vascular disease according to claim 4, wherein the recombinant vector has the cleavage map shown in Fig. [Claim 5] The pharmaceutical composition according to claim 4, wherein the retinal vascular disease is selected from the group consisting of macular degeneration, diabetic retinopathy, retinopathy of prematurity, retinal vascular occlusion and ischemic syndrome. The pharmaceutical composition for preventing or treating retinal vascular disease according to claim 4, wherein the siRNA inhibits the expression of HIF-1 and VEGF.

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