JPWO2005063984A1 - Double-stranded RNA that suppresses gene expression - Google Patents

Abstract

The present invention relates to a double-stranded RNA that suppresses gene expression, and particularly provides a small interfering RNA or a small hairpin RNA that is effective in suppressing the expression of the VEGF receptor. Also provided is a pharmaceutical composition comprising the small interfering RNA / small hairpin RNA. [Selection figure] None

Description

  The present invention relates to a double-stranded RNA that suppresses gene expression, and particularly to a small interfering RNA or a small hairpin RNA that is effective in suppressing the expression of the VEGF receptor. The present invention also relates to a pharmaceutical composition containing the above-mentioned small interfering RNA / low molecular hairpin RNA.

  Recently, angiogenesis is a research topic that has received much attention from the viewpoint of basic research and clinical application.

  This angiogenesis is a tissue morphological reaction in which branching from existing blood vessels causes vascular endothelial cells to proliferate and form a strong vascular network surrounded by smooth muscle cells. Angiogenesis is a vital phenomenon essential for the development and development of individuals, but it is rarely seen in mature individuals except for the ovary associated with the sexual cycle.

  On the other hand, recent studies have reported that pathological angiogenesis occurs in various diseases such as cancer. As shown by Folkman et al., The supply of nutrients and oxygen from tumor blood vessels plays an extremely important role in the growth of solid cancers of several centimeters in diameter. In addition, solid cancers use this tumor blood vessel as a metastatic pathway and induce angiogenesis again at the metastasis destination. Furthermore, it has been clarified that this pathological angiogenesis is deeply involved in the progression and malignant development of many diseases such as diabetic retinopathy and rheumatoid arthritis. Therefore, it has been considered clinically very important to control pathological angiogenesis, but its molecular mechanism has long been unclear.

  Recently, however, the vascular endothelial growth factor VEGF (vasoreceptor growth factor) and its receptor VEGFR (VEGF receptor: flt kinase), which are thought to be involved in the proliferation and differentiation of endothelial cells, have been clarified. .

  VEGF was discovered in 1983 as a new vascular permeability factor (VPF) by Senger et al. At the same time, Ferrara et al. Of Genetique, USA, found a growth factor that specifically acts on endothelial cells and named it VEGF (basal endothelial growth factor). Later studies revealed that VPF and VEGF were products of the same gene, even though they were obtained using different biological activities as indicators. This VEGF did not show any proliferative activity on fibroblasts and epithelial cells, while it showed proliferative activity on all endothelial cells such as umbilical vein endothelial cells and aorta-derived endothelial cells. Thus, the discovery of a growth factor with strict specificity for endothelial cells was the first time, and there was great interest in its structure and receptors.

  Subsequent studies have revealed the signaling mechanism of the VEGF / VEGF receptor family. For example, in tumor cells, the expression of VEGF mRNA is induced in response to hypoxia, and the produced VEGF binds to the receptor tyrosine kinase VEGF receptor (VEGFR) -1, -2 that is expressed almost exclusively in endothelial cells. And induces autophosphorylation of the receptor. In response to the activated receptor signal, the endothelial cells degrade the extracellular basement membrane and proliferate and migrate. The paracrine system by these VEGF and VEGF receptor signals is positioned at the center of the angiogenic signal. The VEGF receptor is composed of three kinds of genes, and it has been clarified so far that VEGFR-2 plays a central role in angiogenesis, proliferation and migration of vascular endothelial cells.

  As described above, since VEGF plays a central role in pathological angiogenesis, VEGF and its receptor (VEGFR) have been considered as important targets for angiogenesis inhibition. To date, anti-VEGF antibodies (Hurwitz, H. et al. (2004) N. Engl. J. Med. 350, 2335-2342), neutralizing antibodies against VEGFR-2 (Witte, L. et al (1998) Cancer) Metastasis Rev 17, 155-61; Zhu, Z. et al. (2003) Lukemia 17, 604-611), a specific inhibitor of tyrosine kinases of VEGFR (Fong, TA et al. (1999) Cancer Res. 59, 99-106), and the trap of VEGF by free VEGFR-1 (Holash, J. et al. (2002) Proc Natl Acad Sci USA 99, 11393-8) and the like have been carried out. However, the present situation is that it has not been put into practical use because it is expensive to produce and purify, requires large doses, has low specificity, and may cause side effects.

  Therefore, the present invention has been made in view of such problems, and is a novel composition for effectively inhibiting angiogenesis, which is costly to produce and purify and has a high specificity. Is intended to provide.

  In order to achieve the above object, the present inventors have focused on RNA interference (RNAi) technology. That is, paying attention to the feature that (1) the inhibition specificity of gene expression of RNA interference (RNAi) technology is extremely high, (2) the synthesis method and expression system are established, the dosage is low and the cost is low, Obtained knowledge that small interfering RNAs / small hairpin RNAs against VEGFR may have an effective angiogenesis inhibitory action, and sincerely investigating and experimenting based on these findings, suppressed expression of the VGEF receptor The present inventors have succeeded in identifying an RNA sequence effective for this purpose, and obtained a small interfering RNA (siRNA) and a small hairpin RNA (shRNA) having this sequence.

  That is, according to the first main aspect of the present invention, it has an RNA sequence that is the same as or complementary to a part of the nucleic acid sequence of the VEGF receptor and is effective for suppressing the expression of the VEGF receptor. A small interfering RNA (siRNA) or a small hairpin RNA (shRNA) is provided. In particular, the VEGF receptor is VEGF receptor 2 (VEGFR-2), although not limited thereto.

  According to such a configuration, for example, by introducing into a target, a low molecular interference RNA / low molecular hairpin RNA that effectively and specifically suppresses the expression of VEGFR in the target can be obtained.

  Further, the RNA sequence of the small interfering RNA / small hairpin RNA has the sequence of SEQ ID NO: 1.

  In one embodiment of the present invention, the small interfering RNA / small hairpin RNA has an RNA sequence identical or complementary to the 3'-UTR sequence of the VEGF receptor 2. Here, the 3'-UTR sequence is not limited to this, but is preferably 4375 to 5830 bases of the DNA of SEQ ID NO: 2.

  The RNA sequence is a double-stranded RNA and is not limited thereto, but the double-stranded region preferably has 15 bases or more. Furthermore, the RNA sequence has a single-stranded region protruding at the 3 'end.

  According to the second main aspect of the present invention, an amount of the small interfering RNA / low molecular hairpin RNA that effectively suppresses the expression of the VEGF receptor in the target, and a pharmaceutically acceptable carrier thereof. A pharmaceutical composition is provided. According to the pharmaceutical composition thus obtained, it becomes possible to treat diseases associated with pathological angiogenesis.

  Further features and remarkable effects of the present invention will become apparent to those skilled in the art from the description of the embodiments of the present invention described below.

  Reference materials and scientific literature referred to herein are referred to in order to establish the knowledge of those skilled in the art, the contents of each disclosure being incorporated by reference.

RNA Interference (RNAi) Technology In recent years, RNA interference (RNAi) technology has attracted attention not only in the bio industry but also in the medical industry as a new gene function analysis. That is, attempts have been made to use this RNA interference technology as a new tool for preventing the expression of genes causing disease (as well as protein synthesis). RNA interference is a phenomenon in which mRNA having the same sequence is specifically decomposed when double-stranded RNA (dsRNA) is introduced into a cell, and was first reported in 1998 by a nematode (Fire). A. et al. (1998) Nature 391, 806-811). Subsequently, it has been suggested that RNA interference is a biological defense mechanism at the nucleic acid level preserved in insects, plants, and the like.

  In nematodes and the like, dsRNA is processed into a 21-base double-stranded small interfering RNA (siRNA) having a 2-base overhang at the 3 ′ end by a protein called Dicer, and this small interfering RNA is It is incorporated into a complex called RNAi-inducing silencing complex (RISC). Double-stranded small interfering RNA is believed to dissociate into single strands after being incorporated into RISC. RISC is believed to bind to and degrade mRNA having a sequence complementary to this single strand.

  Application of RNA interference to mammalian cells has been considered difficult, but Tuschl et al. In 2001 revealed that RNA interference is possible by introducing double-stranded small interfering RNA into cells. (Elbashir, SM et al. (2001) Nature 411, 494-498).

  Furthermore, for the purpose of performing RNAi, small hairpin RNA (shRNA) having a hairpin structure transcribed using a promoter system transcribed by RNA polymerase III has been developed. This shRNA is thought to have a structure similar to that of siRNA because the loop portion is degraded in the cell.

  This RNA interference, also called RNA silencing, is highly expected for application in the medical field because it can specifically suppress the expression of the target protein by promoting specific degradation of the target mRNA. ing. However, no actual treatment has been reported to date.

  However, the present inventors have (1) extremely high inhibition specificity of gene expression in the above-described RNA interference (RNAi) technology, (2) a synthesis method and expression system have been established, and the dosage is low and the cost is low. Focusing on the feature of being cheap, we obtained knowledge that small interfering RNA / small hairpin RNA against VEGFR may show effective angiogenesis inhibitory action, and based on this, sincerity studies and experiments were repeated As a result, an RNA having several properties described below has been successfully produced.

  That is, according to the present invention, there is provided a small interfering RNA or a small hairpin RNA characterized by having an RNA sequence effective for suppressing the expression of VEGF receptor.

VEGF receptor small interfering RNA / small hairpin RNA
Recently, many studies have revealed that the progression and malignancy of various diseases such as cancer depend on pathological angiogenesis. That is, if this pathological angiogenesis could be effectively suppressed, it was expected that a new treatment method for a disease related to pathological angiogenesis could be established.

  VEGF (Vascular Endothelial Growth Factor) has been found to play a central role in in vivo angiogenesis signals, and its receptors are considered to be important targets for inhibiting angiogenesis. I came. So far, angiogenesis inhibitors targeting VEGF / VEGF receptors, such as antibodies and specific inhibitors, have been developed and clinical trials have been conducted, but they are expensive, have low specificity and have side effects. There is a problem and it has not yet been put into practical use. On the other hand, the present inventors paid attention to the recently reported RNA interference (RNAi) technology, and found that small interfering RNA / small hairpin RNA for VEGF receptor may exhibit an effective angiogenesis inhibitory action. As a result of conducting sincerity studies and experiments based on this, we succeeded in producing RNA having several properties described below.

Previous studies have shown that the biological activity of VEGF and its receptor, the VEGF receptor, is mostly confined to endothelial cells.
Two molecules of VEGF receptor 1 (Flt-1) and VEGF receptor 2 (KDR / Flk-1) have been identified as VEGF high affinity receptors on endothelial cells. Therefore, it was considered that the basic action of VEGF is caused through autophosphorylation of these two receptor tyrosine kinases.

  In this VEGF receptor family, VEGF receptor 2 plays a central role in angiogenesis, proliferation and migration of vascular endothelial cells. That is, the phosphorylation of VEGF receptor 1 was considerably weaker than that of VEGF receptor 2, and the contribution of signals to the proliferation / migration of vascular endothelial cells was considered to be low. VEGF receptor 3 is expressed mainly in lymphatic endothelial cells and is not involved in the proliferation of vascular endothelial cells in the living body.

  In order to verify whether angiogenesis can be effectively inhibited by suppressing the expression of VEGF receptor 2 using RNA interference technology, the present inventors are identical to a part of the nucleic acid sequence of VEGF receptor 2. Alternatively, a small interfering RNA or a small hairpin RNA having a complementary RNA sequence is prepared and introduced into human umbilical vein endothelium (HUVE) cells, so that the expression of the VEGF receptor 2 is effectively suppressed. Clarified that it will be.

  According to one embodiment of the present invention, the small interfering RNA or the small hairpin RNA has a sequence identical or complementary to an RNA sequence effective for suppressing the expression of VEGF receptor 2, and the RNA The sequence is the 3′-UTR (untranslated region) sequence of the VEGF receptor 2. In the present invention, using the 3'-UTR sequence as a target sequence is also beneficial from the viewpoint of a control experiment. That is, when the expression of the VEGF receptor 2 is suppressed using a small interfering RNA and a physiological effect is observed, this effect is counteracted when the VEGF receptor 2 is further introduced as a functional control. It is desirable to consider whether or not. In the present invention, by using 3'-UTR as a target sequence, it is possible to easily perform a function control experiment by expressing cDNA of only the translation region not containing 3'-UTR.

  However, in the present invention, the RNA sequence is not limited to the 3'-UTR sequence, and may be any sequence that can effectively suppress the expression of VEGF receptor 2 in the target.

  The small interfering RNA or small hairpin RNA prepared according to the present invention can analyze which amino acid of the VEGF receptor 2 is important for its function by introducing a cDNA introduced with a point mutation or deletion. Is possible. Furthermore, the sequence of the small interfering RNA or small hairpin RNA of the present invention can be used as an index for searching for useful dsRNA by creating a database together with many other small interfering RNAs.

  According to one embodiment of the present invention, a method for producing a small interfering RNA effective in suppressing the expression of VEGF receptor 2 provided in the present invention includes a method of annealing a single-stranded RNA prepared by chemical synthesis. is there. The structure of the small interfering RNA thus prepared has a single-stranded overhang at the 3 'end, and is preferably a 2-base overhang. Furthermore, the double-stranded portion is 15 bases or more, preferably 18 bases or more, and most preferably 19 bases.

  Furthermore, in the present invention, the method for introducing the small interfering RNA into the target is performed, for example, using a commercially available transfection reagent such as Oligofectamine, Lipofectamine, TransIT-TKO. As an introduction method to an animal, injection into an egg, living body administration using atelocollagen, administration using cationic liposomes, and the like are performed. However, not only this method but also other known techniques can be used.

  In addition, as a method for producing a small hairpin RNA effective for suppressing the expression of VEGF receptor 2 of the present invention, there is a method of transcription using a promoter transcribed by RNA polymerase III. According to one embodiment of the present invention, for example, an H1 promoter, a U6 promoter, or a CMV promoter is used as the promoter. Furthermore, plasmids, retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses are used as the types of vectors. Moreover, in producing these, in addition to a ligation (ligation) reaction using a restriction enzyme, a recombination reaction using Cre-loxP can also be used. Among these vectors, plasmids can be introduced into cells using commercially available transfection reagents such as Lipofectamine and Effectene. Other viral vectors can be introduced into cells by standard infection procedures in the field of the present invention. In addition, these vectors can be administered to a living body by local administration, intravenous injection, or the like.

  Furthermore, a drug obtained by the present invention, comprising a small interfering RNA / small hairpin RNA in an amount that effectively suppresses the expression of a VEGF receptor in an object, and a pharmaceutically acceptable carrier. A composition is provided. The pharmaceutical composition may be used to treat diseases associated with pathological angiogenesis, such as cancer, diabetic retinopathy, rheumatoid arthritis.

  In one example, the small interfering RNA (siKDR) expression vector obtained in one embodiment of the present invention was examined. The present inventors have shown that this viral vector inhibits two-dimensional lumen formation, which is one of the indicators of angiogenesis, in human umbilical vein endothelial (HUVE) cells in a co-culture system with fibroblasts. I made it. This result strongly suggests that the small interfering RNA / small hairpin RNA obtained in one embodiment of the present invention is useful for introducing RNA interference technology in anti-angiogenic therapy.

  Regarding the viewpoint of a pharmaceutical composition, the small interfering RNA / small hairpin RNA of the present invention is as follows: (1) The target depends strictly on the gene sequence, so that it has high specificity, (2) Intracellular The small interfering RNA incorporated into the DNA lasts for about a week, and therefore is expected to have a high effect per administration frequency. (3) Different action from other existing VEGF receptor 2 inhibitors Because of the mechanism, it is considered that the synergistic effect by the combination is high. (4) When used as a short hairpin RNA using a vector, it is considered that it has a beneficial effect in that the cost is low. .

  In addition, (1) VEGF receptor 2 is expressed in hematopoietic stem cells using the small interfering RNA / small hairpin RNA that effectively suppresses the expression of VEGF receptor 2 obtained in one embodiment of the present invention. Therefore, it has been suggested that (2) the VEGF receptor 2 functions in the nervous system, which suppresses the differentiation of these stem cells into blood cells, and it is speculated that such functions can also be controlled.

  Next, in the following examples, the present invention has an RNA sequence that is the same as or complementary to a part of the nucleic acid sequence of the VEGF receptor, and has an RNA sequence effective for suppressing the expression of the VEGF receptor. An example of the production of the small interfering RNA / low molecular hairpin RNA and the effect thereof will be described.

dsRNA, Transfection dsRNA used in a preferred embodiment of the present invention was purchased from Nippon Bioservice. For labeling (labeling) of this dsRNA, a LabelT siRNA Tracker Cy3 kit (Murus) was used. dsRNA transfection was performed using TransIT-TKO Transfection Reagent (Mirus) according to the instruction manual.

RNA isolation and RT-PCR
Human umbilical vein endothelial (HUVE) cells (Clonetics) were routinely maintained in Humedia EG2 (Kurabo). HUVECs were seeded at 1.6 × 10 ⁵ cells / 3.5 cm plate and siRNA transfection was performed the next day. After 48 hours, total RNA was isolated by standard protocol means using guanidine thiocyanate phenol chloroform. CDNA was synthesized from 1 μg RNA using M-MLV reverse transcriptase (Invitrogen) according to the instruction manual. PCR was then performed using the primers described below:
VEGFR-2 translation region 5'-TTGGAGCATCTCATCTGTTACAGC-3 '
5'-CTTCTGAGGCAAGAAACCATACCAC-3 '
3 'untranslated region 5'-GGAGCCAGTCTTTCTAGGCATA-3' of VEGFR-2
5'-CCCGCATCAGATCTCCAGACAT-3 '
VEGFR-1
5'-AGCTGGCAAGCGGTCTTACC-3 '
5'-GAGTCAGCCCACACACAGAGGT-3 '
VEGFR-3
5'-AGCAGCTGTGACACCGTGCC-3 '
5'-GTCTCTCACGCGCTGGCAGA-3 '
GAPDH
5'-TCACCATTCTTCAGGAGCGAGA-3 '
5'-GTGGGGTGTCGCTGTTGAAGTCAG-3 '
PCR conditions are 94 ° C for 15 seconds, 55 ° C for 30 seconds, and 72 ° C for 90 seconds for 25 cycles. In the case of VEGFR-1, 40 cycles of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 60 seconds were performed.

VEGF stimulation and cell extraction 1.6 × 10 ⁵ cells / 3.5 cm plate HUVEC were transfected under the same conditions as described above. After 48 hours, the cells were cultured for 14 hours in growth factor-free medium (EGM2, 0.4% fetal bovine serum). To stimulate VEGFR-2, hr-VEGF-A165 (Genzyme Techne) was added to the cells at a final concentration of 50 ng / ml for 5 minutes, washed twice with PBS and solubilized. As the total cell lysate, HNTG buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1.5 mM MgCl ₂ , 1 mM EGTA, 1 mM vanadic acid, 1 mM PMSF, 1% Trasylol) is used. Prepared, separated by 7.5% SDS-PAGE, transferred to Immobilon (Millipore) and subjected to Western blot. The antibody against VEGFR2 used was the antibody previously described (Takahashi et al. EMBO J 20pp 2768-2778). Anti-PLCγ (Santa Cruz), anti-phosphate MAPK, and anti-MAPK (Cell Signaling) were purchased. The protein was detected by chemiluminescence using Western blotting luminol reagent (Santa Cruz) means.

MTS assay 5 × 10 ³ HUVECs were seeded in 95 well plates and transfected with siRNA. After 4 hours, the medium was replaced with EGM2 containing 0.1% fetal bovine serum containing 10 ng / ml hr-VEGF-A165 or the same concentration of BSA. Forty-eight hours later, live cells were quantified by the Aquaone One Solution Proliferation Assay (Promega). Absorbance was measured at 492 nm.

Results Inhibition of VEGFR2 mRNA Expression by siKDR The VEGF-A and VEGFR-2 (KDR / Flk-1) systems are known to be important for angiogenic signaling (FIG. 1). In order to suppress VEGFR2 gene expression in endothelial cells by RNA interference, the present inventors designed siKDR dsRNA specifically targeting the 3′UTR region of human VEGFR-2 mRNA (FIGS. 2 and 4). The siKDR was introduced into human umbilical vein endothelial (HUVE) cells, and after 48 hours, gene expression was analyzed by RT-PCR to analyze whether it acts as siKDR having the ability to silence the VEGFR2 gene. Using several commercially available transfection reagents, we tested the ability to carry dsRNA, and in subsequent experiments TransIT-TKO was used so that the transfection efficiency of HUVE cells exceeded 90%. Using. The amount of 800 bp RT-PCR product of VEGFR2 mRNA detected by the primers in the coding sequence was reduced by more than 90% compared to mock-treated cells (FIG. 3).

  RT-PCR analysis using primers in the 3'UTR region showed similar results, confirming down-regulation by siKDR. To confirm that this silencing effect is mediated by the unique sequence specificity of RNAi, we have two mutations in its central region that are consistent with siKDR. Another dsRNA (siKDRmut) was designed (Figure 4). This siKDRmut was expected to have no effect on VEGFR2 mRNA levels. The expression of two other members of the VEGF receptor family, VEGFR1 and VEGFR3, and the housekeeping gene, GAPDH, remained unchanged when treated with siKDR, further confirming the sequence specificity of RNAi. These results demonstrated that siKDR is specifically targeting VEGFR2 mRNA and can effectively reduce its expression in endothelial cells.

Knockdown of VEGFR2 protein and downstream signaling by siKDR We next analyzed the expression of VEGFR2 protein and its downstream signaling, consistent with a significant decrease in VEGFR2 mRNA in siKDR-treated cells. In siKDR-transfected HUVE cell lysate, the amount of VEGFR2 detected as 230 kD and 200 kD bands decreased to below detectable levels with the antibody. On the other hand, the expression levels of other proteins, PLCγ and MAP kinase, did not change (FIG. 5). When siKDRmut was introduced into HUVE cells as a control, no decrease in VEGFR2 protein was observed. Stimulation with VEGF-A ₁₆₅ resulted in weak activation of MAP kinase in siKDR treated cells, but related activation was also observed in mock or siKDRmut treated HUVE cells. This result means that the introduction of siKDR into endothelial cells can be an effective means for inhibiting VEGFR2-mediated signaling.

Inhibition of Endothelial Cell Proliferation and / or Apoptosis by siKDR In order to resolve the biological causal relationship regarding the effect of siKDR on endothelial cells, the present inventors have said that VEGF-dependent proliferation and / or HUVE cells after introduction of siRNA. Survival was examined. Live cells were quantified 48 hours after transfection with siKDR or siKDRmut in the presence of VEGF-A using the MTS assay. The number of mock or siKDRmut-treated cells is 1.8-fold higher in the presence of VEGF, the number of siKDR-treated cells is 1.2-fold (FIG. 6), and the RNAi of VEGFR2 mediated by siKDR is responsible for endothelial cell proliferation and It has been demonstrated that / or survival can be inhibited.

Angiogenesis inhibitory action by siKDR
In order to express a small hairpin RNA (shRNA) that matches the adenovirus production siKDR, we synthesized the oligonucleotides described below,
SEQ ID NO: 1;
5'-gatccgtagagttcgttgtgtgctttcaagagagagacacaactacactcttattttttggaaaa-3 '
5'-agctttttccaaaaaaagtaggagtcgtttgctgtttctcttgaaaaacagcacaacgaactctac-3 '
It was annealed to produce double stranded DNA and ligated to pSilencer 2.1-U6 hygro (Ambion) that promotes expression of the shRNA.

The oligonucleotide corresponding to siKDRmut was synthesized as follows:
5'-gatccgtagagttGgttCtgctgttcaagagaagacacaacacactacttttttttggaaaa-3 '
5′-agctttttccaaaaaaagtaggtGgttCtgctgtttcttttgaaaaaccacaacacactactac-3 ′
It was introduced into pSilencer 2.1-U6 hygro in the same manner as above.

  Both shRNA expression cassettes were cleaved with HindIII and EcoRI, the 5 ', 3' ends were blunt ended (blunt ends) and ligated to the SwaI site of pAxcw (Adenovirus Expression Vector Kit (Takara Bio)). This cosmid vector was co-transfected into 293 cells with DNA-TPC using Effectene (Qiagen). This recombinant adenovirus having the shRNA expression cassette was screened according to the manufacturer's instructions to produce adenosiKDR and adenosiKDRmut expressing siKDR and KDRmut from the U6 promoter, respectively.

  Infection of HUVE cells with adenovirus was performed according to the method described in the instructions for the adenovirus expression vector kit. Briefly, sub-confluent HUVE cells in 6-well culture plates were incubated for 1 hour in Dulbecco's Modified Eagle Medium (DMEM) containing 5% FBS with adenovirus, and further incubated for the time indicated with Humdia EG2. For time course experiments, HUVE cells were infected with virus at MOI = 40 and cells were made into total lysate at the times indicated in the figure. In order to assess the effective MOI (infection efficiency) against silence of the VEGFR-2 expression, the adenovirus solution was diluted with DMEM containing 5% FBS and used for infection against HUVE cells at the indicated MOI. Used. Cells were RNA conditioned 48 hours after infection.

In vitro angiogenesis assay In vitro angiogenesis was assessed by the formation of capillary-like structures in HUVE cells co-cultured with human diploid fibroblasts. This experimental procedure was in accordance with the instructions accompanying the Angiogenesis Kit (Kurabo). Briefly, HUVE cells were infected with shRNA expressing adenovirus for 3-7 days and fixed. Subsequently, HUVE cells were stained with anti-human CD31 antibody (Kurabo) according to the protocol according to the kit. To measure the formation of a capillary-like network, microscopic images of CD31 staining were analyzed by Angiogenesis Image Analyzer software.

Results siKDR Inhibits HUVE Cell Capillary Formation To assess the biological effects of siKDR in angiogenesis, we have examined whether the siKDR is a tube of endothelial cells in a co-culture system with diploid fibroblast support cells. We investigated whether to prevent cavity formation. First, the present inventors created an adenovirus that expresses a short hairpin RNA (shRNA) that matches siKDR or siKDRmut. From day 2 post infection, adeno siKDR was able to knock down the expression of VEGFR-2 mRNA, whereas in the case of adeno siKDRmut, VEGFR-2 mRNA levels did not change (FIG. 7). This effect of adenosiKDR lasted at least 4 days after infection and was evident even when MOI = 0.2 (FIG. 8).

The inventors then added siKDR or siKDRmut to the endothelial cell and fibroblast co-culture system, and as described in the experimental procedure, the capillary network joints and branches (paths). ) Was measured. The number of junctions and branches of adenosiKDR-infected HUVE cells was significantly reduced compared to siKDRmut (FIGS. 9 and 10). This means that adenosiKDR is effective in inhibiting endothelial cell tube formation.

The figure which showed the signal transduction of angiogenesis of a VEGF-VEGF receptor system, especially VEGF-A and VEGFR-2 (KDR / Flk-1) system. The figure which showed the site | part of siKDR dsRNA in the 3'UTR area | region of preferable human VEGFR2 mRNA based on this invention, and the site | part of the primer used in experiment. The figure which analyzed the influence with respect to the expression of VEGFR-2, VEGFR-1, and VEGFR-3 gene by siKDR which concerns on this invention, control siKDR-mut, and mock by RT-PCR. The figure which showed the iRNA arrangement | sequence of siKDR which concerns on this invention, and another dsRNA (siKDRmut) which has two variation | mutations in the center area | region. The figure which analyzed the expression of VEGFR-2 protein and its downstream signaling by siKDR which concerns on this invention, control siKDR-mut, and mock. The figure which examined the VEGF-dependent proliferation and / or HUVE cell survival after siRNA (siKDR, siKDR-mut, and mock) introduction | transduction using the MTS assay. It is the figure which examined the influence on the expression of VEGFR-2 mRNA of adenosiKDR and adenosiKDRmut on the 2nd, 3rd and 4th day after infection. The figure which evaluated effective MOI (infection efficiency) with respect to silence of VEGFR-2 expression of adenosiKDR and adenosiKDRmut. The figure which evaluated the influence with respect to the formation of the capillary-like structure of the HUVE cell co-cultured with the human diploid fibroblast of adenoadenosiKDR and adenosiKDRmut. The figure which measured the number of the junction part (joints) (top figure) and branch part (paths) (bottom figure) of a capillary-like structure in adenosiKDR infection and adenosiKDRmut infection HUVE cell.

Claims (10)

  A small interfering RNA comprising an RNA sequence that is the same as or complementary to a part of a nucleic acid sequence of a VEGF receptor and is effective for suppressing the expression of the VEGF receptor.   A small hairpin RNA comprising an RNA sequence identical or complementary to a part of a nucleic acid sequence of a VEGF receptor, wherein the RNA sequence is effective for suppressing the expression of the VEGF receptor.   The small interfering RNA / small hairpin RNA according to claim 1 or 2, wherein the VEGF receptor is VEGF receptor 2.   4. The small interfering RNA / small hairpin RNA according to claim 3, which has an RNA sequence identical or complementary to the 3'-UTR sequence of the VEGF receptor 2.   4. The small interfering RNA / small hairpin RNA according to claim 3, wherein the RNA sequence has the sequence of SEQ ID NO: 1.   The small interfering RNA / low molecular hairpin RNA according to claim 4, wherein the 3'-UTR sequence is 4375-5830 bases of the DNA of SEQ ID NO: 2.   3. The small interfering RNA / low molecular hairpin RNA according to claim 1 or 2, wherein the RNA sequence is a double-stranded RNA, and the double-stranded region has 15 bases or more.   4. The small interfering RNA / low molecular hairpin RNA according to claim 1, wherein the RNA sequence has a single-stranded region protruding at the 3 'end. The small interfering RNA / small hairpin RNA according to claim 1 or 2 in an amount that effectively suppresses the expression of the VEGF receptor in an object;
A pharmaceutical composition comprising the pharmaceutically acceptable carrier.   The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition is for treating a disease associated with pathological angiogenesis.

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