WO2024045251A1 - 一种治疗新生血管性视网膜疾病的小干扰rna及其dna四面体复合物 - Google Patents

一种治疗新生血管性视网膜疾病的小干扰rna及其dna四面体复合物 Download PDF

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WO2024045251A1
WO2024045251A1 PCT/CN2022/121379 CN2022121379W WO2024045251A1 WO 2024045251 A1 WO2024045251 A1 WO 2024045251A1 CN 2022121379 W CN2022121379 W CN 2022121379W WO 2024045251 A1 WO2024045251 A1 WO 2024045251A1
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sirna
seq
interfering rna
small interfering
group
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French (fr)
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罗德伦
徐枭潇
吴大颖
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成都景润泽基因科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present invention relates to the field of drugs for neovascular ophthalmic diseases, specifically, to the field of gene drugs for silencing the Runt-related transcription factor 1 (RUNX1) gene.
  • RUNX1 Runt-related transcription factor 1
  • Neovascularization is a common pathological change in many eye diseases. It often occurs in the cornea, iris, choroid and retina. The resulting diseases include corneal neovascularization, choroidal neovascularization, retinal neovascularization and other diseases. Common causes Eye diseases caused by this include, for example: diabetic retinopathy (DR), retinopathy of prematurity (ROP), age-related macular degeneration (AMD) (including wet AMD) And dry AMD, the pathological changes of wet AMD are choroidal neovascularization), neovascular glaucoma, etc.
  • DR diabetic retinopathy
  • ROP retinopathy of prematurity
  • AMD age-related macular degeneration
  • AMD age-related macular degeneration
  • AMD age-related macular degeneration
  • AMD dry AMD
  • the pathological changes of wet AMD are choroidal neovascularization
  • neovascular glaucoma etc.
  • New blood vessels can appear in almost all tissues in the eye, such as the cornea, iris, ciliary body, choroid, retina, macula, and optic disc. It can cause a series of pathological changes such as bleeding, exudation, and proliferation of tissues in these parts, thus causing damage to the structure of the eyeball. and functional damage, seriously damaging visual function.
  • the number of patients with fundus neovascular diseases exceeds 40 million, and as the population ages, the number of patients continues to rise. Patients with fundus neovascular diseases require follow-up visits or injections of related drugs almost every month, otherwise the damage to their vision will seriously affect their lives.
  • VEGF vascular endothelial growth factor
  • PDGF platelet-derived growth factor
  • RUNX1 also known as AML1
  • AML1 is the most common target site for chromosomal translocations in human leukemias.
  • RUNX1 is a very important transcription factor that is widely expressed in a variety of hematopoietic cell lines. It plays a key role in the differentiation of hematopoietic cells and can also regulate the expression of hematopoietic-related genes. Abnormal expression and mutations of RUNX1 gene are often associated with the occurrence of human leukemia. Many studies have shown that RUNX1 is an important regulatory factor in the process of hematopoietic cell production.
  • the RUNX1 protein can accept a variety of post-translational modifications, including phosphorylation, acetylation, etc. Its activity can be affected by these post-translational modifications, thereby regulating the differentiation of hematopoietic cells. , apoptosis and self-renewal.
  • RUNX1 retinal microvascular endothelial cells
  • Ro5-3335 (RUNX1-CBF ⁇ interaction inhibitor) was associated with a significant reduction in neovascular cluster area compared with vehicle-treated pups. This was not associated with significant changes in ischemic areas, suggesting independent regulation between normal and abnormal vascularization of the retina and that inhibition of RUNX1 function with Ro5-3335 effectively reduces pathological retinal neovascularization.
  • One object of the present invention is to provide small interfering RNA (siRNA) that targets the Runt-related transcription factor 1 (RUNX1) gene and its isotype mRNA for degradation;
  • Another object of the present invention is to provide a pharmaceutical composition containing the siRNA
  • Another object of the present invention is to provide the use of the siRNA or the pharmaceutical composition containing siRNA;
  • Another object of the present invention is to provide a DNA tetrahedron-siRNA complex and its use.
  • a small interfering RNA specifically targets and causes RNAi-induced mRNA degradation of Runt-related transcription factor 1 (RUNX1) and its isoforms.
  • the siRNA double strand can be connected to the RNA-induced gene silencing complex (RISC), and after binding to RISC, it targets and cuts specific mRNA into small fragments of 10 to 11 bases, thereby Interrupt the translation process of RUNX1 mRNA, silence the expression of its complementary genes, down-regulate the expression of RUNX1, thereby inhibiting new blood vessel formation.
  • RISC RNA-induced gene silencing complex
  • the small interfering RNA of the present invention specifically targets the Runt-related transcription factor 1 gene and its isotype mRNA, and down-regulates the expression of RUNX1 by silencing the expression of its complementary gene.
  • the small interfering RNA is selected from:
  • the pharmaceutical composition of the present invention contains a therapeutically effective amount of the small interfering RNA.
  • the siRNA comprises a sense RNA strand and an antisense RNA strand forming an RNA duplex.
  • the sense RNA strand contains the same nucleotide sequence as the target sequence of about 19 to about 25 contiguous nucleotides in the target mRNA.
  • the siRNA sequences while possibly sharing some sequence homology with the non-target sequence, are sufficiently distinct that RNA silencing does not occur with respect to the non-target sequence.
  • the siRNA may comprise partially purified RNA, substantially pure RNA, synthetic RNA or recombinantly produced RNA and differ from naturally occurring RNA by the addition of one or more nucleotides, Deleted, substituted and/or altered RNA.
  • Such alterations may include the addition of non-nucleotide materials such as to the termini of the siRNA or to one or more internal nucleotides of the siRNA, including modifications that render the siRNA resistant to nuclease degradation.
  • the siRNA nucleotide sequence is the sense strand as shown in SEQ ID NO: 1, the antisense strand as shown in SEQ ID NO: 2; or the sense strand as shown in SEQ ID NO: 3 , the antisense strand as shown in SEQ ID NO:4; or the sense strand as shown in SEQ ID NO:5, or the antisense strand as shown in SEQ ID NO:6.
  • siRNA of the present invention can be obtained using conventional techniques known to those skilled in the art, including but not limited to chemical synthesis or recombinant production.
  • siRNA can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and conventional DNA/RNA synthesizers, or siRNA can also be expressed from recombinant circular or linear DNA plasmids using any appropriate promoter .
  • siRNA expressed from a recombinant plasmid can be isolated from a cultured cell expression system using standard techniques, or can be expressed in vivo within cells at or near areas of neovascularization.
  • a second aspect of the present invention provides the use of the siRNA of the present invention or a pharmaceutical composition containing the same in the preparation of drugs for preventing and/or treating neovascular ophthalmic diseases.
  • the neovascular ophthalmic diseases include, but are not limited to: diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, periretinal vein inflammation, age-related macular degeneration, high myopia macular hemorrhage, central exudative retinochoroidopathy and Other angiogenic diseases.
  • the third aspect of the present invention provides a pharmaceutical composition for treating neovascular ophthalmic diseases (neovascular ophthalmic diseases), including a therapeutically effective amount of siRNA compound and pharmaceutically acceptable excipients (vehicle, carrier),
  • the siRNA nucleotide sequence includes the sense strand of RNA as shown in SEQ ID NO:1, the antisense strand of RNA as shown in SEQ ID NO:2 (corresponding to siRNA-3 in Table 1); or as shown in SEQ ID NO:3
  • one skilled in the art can readily determine the effective amount of siRNA to be administered to a given subject by considering factors such as the size and weight of the subject.
  • the effective amount of siRNA is an amount sufficient to cause RNAi-mediated degradation of the target mRNA in the cell.
  • a clinically effective amount is an amount that, when administered to a subject, will inhibit the progression of angiogenesis in the subject by RNA silencing.
  • the present invention also provides a method for treating neovascular ophthalmic diseases, especially retinal neovascular ophthalmic diseases (neovascular retinal diseases), comprising adding a preventive and/or therapeutic effective amount
  • neovascular ophthalmic diseases especially retinal neovascular ophthalmic diseases (neovascular retinal diseases)
  • the siRNA or a pharmaceutical composition containing the siRNA is administered to an individual in need thereof.
  • siRNA or a drug combination containing siRNA can be administered to the entirety or part of the affected eye, particularly the vitreous body, at a preventive and/or therapeutically effective dose.
  • the formulation may be any dosage form suitable for topical administration to the eye, including but not limited to injections, eye drops, etc.
  • Administration of the pharmaceutical composition can reduce the gene expression of RUNX1, thereby inhibiting the formation of new blood vessels in relevant parts of the affected eye, and promoting the normalization of blood vessels in the affected eye.
  • siRNA drugs that have been studied for fundus neovascular diseases such as AMD and DME, all of which target VEGF.
  • siRNA drugs there are two major challenges in using siRNA drugs for treatment: one is that exposure of siRNA to the blood will cause stability problems and cause immunogenicity, and the other is that siRNA with large molecular weight and negative charge cannot cross the membrane and enter the cell by itself. Therefore, siRNA often needs to be delivered into the cell through a vector to function.
  • the most widely used delivery platform is the adeno-associated viral vector (AAV).
  • AAV adeno-associated viral vector
  • LNP non-viral carrier nanoparticles
  • TDN DNA tetrahedral
  • a tetrahedral structure formed by denaturation and renaturation of four single-stranded DNA and then complementary base pairing between strands. It is easy to synthesize and has high biocompatibility.
  • Previous patents have disclosed the use of TDNs in ophthalmic diseases.
  • the patent CN109646450B discloses the use of TDNs in the preparation of drugs for the treatment of corneal damage.
  • the patent CN112007044B discloses the TDNs-miR155 complex and its use in the preparation of prevention or treatment of corneal damage.
  • patent CN112843085B discloses the TDNs-miR22 complex and its use in preparing medicines for treating optic nerve damage. So far, TDNs carrying siRNA have not been seen for the treatment of ophthalmic diseases.
  • the present invention uses DNA tetrahedrons as a carrier system to carry siRNA targeting RUNX1 mRNA. It has excellent stability and safety and is not easily dissolved by nucleases. It does not require transfection and can easily penetrate the cell membrane, thereby enhancing the cellular uptake effect of the drug. Improve the degradation efficiency of siRNA on mRNA and prevent the formation of new blood vessels. At the concentrations used, various types of living cells (such as RAW264.7 cells and L929 fibroblast-like cells) showed no obvious cytotoxicity or adverse reactions.
  • the present invention provides a DNA tetrahedral drug complex for treating neovascular ophthalmic diseases, the DNA tetrahedral drug complex comprising:
  • the small interfering RNA specifically targets the Runt-related transcription factor 1 gene and its isotype mRNA, silences the expression of the RUNX1 complementary gene, and downregulates the expression of RUNX1.
  • the small interfering RNA is selected from:
  • a DNA tetrahedron the DNA tetrahedron is formed by complementary base pairing of four single-stranded DNAs; the nucleotide sequences of the four single-stranded DNAs are selected one-to-one from SEQ ID NO. 7 to SEQ The sequence shown in ID NO.10;
  • the small interfering RNA is connected to at least one single strand of the DNA tetrahedron.
  • the sense strand of the small interfering RNA is connected to at least one single strand of the DNA tetrahedron through a chemical bond.
  • a linker is further included between the sense strand of the small interfering RNA and at least one single strand of the DNA tetrahedron.
  • the linker is a linker sequence -TTTTT-.
  • the four single-stranded DNAs forming the DNA tetrahedron are denatured in an equal molar ratio at a temperature sufficient to denature them, and then the temperature is lowered to It is annealed to form a DNA tetrahedral structure through complementary inter-strand base pairing; at least one of the four single-stranded DNAs is connected to the small interfering RNA.
  • the DNA tetrahedrons of the present invention are formed by complementary base pairing of four single-stranded DNAs; the sequences of the four single-stranded DNAs correspond to SEQ ID NO.7 in sequence
  • the sequence of -10 can be assembled to form the target product TDNs through denaturation and annealing processes. Specifically, the four single-stranded DNAs of TDNs were maintained at a temperature sufficient to denature them for 10 minutes, and then the temperature was lowered to 2-8°C for more than 20 minutes.
  • the DNA tetrahedron and siRNA are formed in a molar ratio of 1: (1-4).
  • the end of at least any one DNA single strand in the DNA tetrahedron is connected to the sense strand of the connected siRNA.
  • the single-stranded DNA of the DNA tetrahedron and the sense strand of siRNA also contain a connecting sequence
  • the connecting sequence is a nucleotide sequence, preferably a deoxyribonucleotide sequence, and more preferably - TTTTT-, the -TTTTT- sequence is five consecutive thymidine deoxynucleotide sequences.
  • a method for preparing TDNs-siRNA nucleic acid molecules which involves placing the four DNA single strands of the DNA tetrahedron at a temperature sufficient to denature them for more than 10 minutes, The temperature is then lowered to 2-8°C and maintained for more than 20 minutes, wherein at least one of the four single strands is connected to siRNA targeting silencing of RUNX1.
  • the DNA tetrahedron is prepared by denaturing the four DNA single strands at 90 to 98°C for 10 to 15 minutes and annealing at 2 to 8°C for 20 to 30 minutes, one of which is connected to an siRNA targeting the silencing of RUNX1 .
  • the DNA tetrahedron is prepared by denaturing the four DNA single strands at 95°C for 10 min and annealing at 4°C for 20 min, one of which is connected to an siRNA targeting the silencing of RUNX1.
  • Another aspect of the present invention also provides the use of the above-mentioned TDNs-siRNA nucleic acid molecule in the preparation of medicines for preventing and/or treating neovascular ophthalmic diseases, especially in the preparation of medicines for preventing and/or treating retinal neovascular diseases. Use in medicines for diseases.
  • the neovascular ophthalmic diseases include, but are not limited to: diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, periretinal phlebitis, age-related macular degeneration, high myopia macular hemorrhage, central exudative retinochoroidopathy and other angiogenic diseases.
  • the present invention also provides a pharmaceutical composition for treating neovascular ophthalmic diseases, which pharmaceutical composition contains the above-mentioned TDNs-siRNA nucleic acid molecules for preventing and/or treating neovascular ophthalmic diseases and pharmaceutically acceptable excipients. (medium, carrier).
  • TDNs-siRNA the effective amount of TDNs-siRNA to be administered to a given subject by considering factors such as the size and weight of the subject.
  • the effective amount is an amount sufficient to cause RNAi-mediated degradation of the target mRNA in the cell.
  • a clinically effective amount is an amount that, when administered to a subject, will inhibit the progression of angiogenesis in the subject by RNA silencing.
  • the present invention also provides a treatment method for neoangiogenic ophthalmic diseases, including applying a preventive and/or therapeutically effective amount of TDNs-siRNA or a pharmaceutical composition containing TDNs-siRNA to a patient in need. individual.
  • TDNs-siRNA or a drug combination containing TDN-siRNA can be administered to the whole or part of the affected eye, especially the vitreous body, at an effective dose for prevention and/or treatment.
  • the formulation may be any dosage form suitable for topical administration to the eye, including but not limited to injections, eye drops, etc.
  • the siRNA provided by the invention can cause the RNAi-mediated translation process of Runt-related transcription factor 1 (RUNX1) mRNA, thereby inhibiting or silencing the expression of its complementary gene, down-regulating the expression of RUNX1, and using the siRNA to prepare and treat neoangiogenic ophthalmology.
  • Drugs for diseases have very good application prospects.
  • this invention designs siRNA and TDNs into TDNs-siRNA nucleic acid molecules with a spatial tetrahedral structure, which increases stability, helps siRNA to efficiently enter cells, and improves the degradation efficiency of siRNA on RUNX1 mRNA, thereby reducing the expression of RUNX1 and preventing new blood vessels. of generation.
  • Figure 1 shows the agarose gel electrophoresis detection picture of RUNX1 mRNA PCR products after treatment with each candidate siRNA (siRNA-2/siRNA-3/siRNA-4/siRNA-6).
  • Lanes 1-3 are siRNA-2 samples.
  • Lanes 4-6 are siRNA-3 samples, lanes 7-9 are siRNA-4 samples, lanes 10-12 are siRNA-6 samples, and lanes 13-15 are blank control samples;
  • Figure 2 is a quantitative analysis of the RUNX1 gene silencing effect after each candidate siRNA treatment
  • Figure 3 is the polyacrylamide gel electrophoresis pattern of TDN and TDN-siRNA, in which lane 1 is the TDN sample and lanes 2-6 are the TDN-siRNA-3 sample;
  • Figure 4 is the capillary electrophoresis pattern of TDN and TDN-siRNA
  • Figure 5 shows the transmission electron microscope images of TDN and TDN-siRNA
  • Figure 6 shows the particle size/potential results of TDN and TDN-siRNA
  • TDN left
  • TDN-siRNA right
  • Figure 7 shows the cell entry efficiency of siRNA, siRNA+Lipo, TDN, and TDN-siRNA complexes in different cells (HEK-293 cells, HUVEC cells, and HREC cells);
  • Figure 8 shows the RUNX1 gene silencing effect in different cells after each administration group, including the blank group (control), siRNA group, siRNA+Lipo group, and different concentrations of TDN groups (5 nmol/L, 10 nmol/L, 15 nmol/ L), different concentrations of TDN-siRNA groups (5nmol/L, 10nmol/L, 15nmol/L);
  • Figure 9 shows the statistics of RUNX1 protein expression inhibition levels in different cells (HEK-293 cells, HUVEC cells and HREC cells) after each administration group, including the blank group (control), siRNA group, siRNA+Lipo group, and TDN group , TDN-siRNA group;
  • FIG 10 shows the RUNX1 protein WB detection bands of each experimental group of HREC cells
  • Figure 11 shows the inhibition level of new blood vessels after each administration group was applied to the chick chorioallantoic membrane model, including the blank group (control), Aflibercept (positive control) group, siRNA group, siRNA+Invivofectamine (transfection reagent), TDN group, TDN-siRNA group;
  • Figure 12 shows the expression levels of RUNX1 protein in the retina of mice in each group after each administration group was administered to the mouse OIR model, including the blank group (control), siRNA group, siRNA+Invivofectamine group, TDN group, and TDN-SiRNA group. ;
  • Figure 13 shows the statistical results of the new blood vessel area in the retina of mice in each group after each administration group was administered to the mouse OIR model, including the blank group (control), Aflibercept (positive control) group, siRNA+Invivofectamine group, TDN group, TDN -siRNA group;
  • Figure 14 shows the retinal neovascularization and recovery of avascular areas of mice in each administration group in the OIR model
  • FIG. 15 is a schematic diagram of TDN-siRNA-3 connection.
  • HEK-293 was purchased from Shanghai Jingze Biotechnology Co., Ltd.;
  • HREC cells were purchased from angiopromie company
  • HUVEC cells were purchased from Aucells Biotechnology;
  • LipoRNAiMAX was purchased from Thermo Fisher;
  • Invivofectamine reagent was purchased from Thermo Fisher;
  • Aflibercept injection (Aflibercept): purchased from Bayer Healthcare Co., Ltd., the specification is 40mg/ml/bottle;
  • C57/BL mice were purchased from Spefford (Beijing) Biotechnology Co., Ltd.
  • HUVEC cells Take out HUVEC cells from the liquid nitrogen tank, recover them, culture them stably for 2-4 generations, and then use them for transfection experiments.
  • the siRNA and Lipofectamine RNAiMAX mixture can be prepared as follows: add candidate siRNA to 250 ⁇ l of serum-free growth medium and mix gently. Then add 5 ⁇ l Lipofectamine RNAiMAX to 250 ⁇ l growth medium to dilute and incubate at room temperature for 5 minutes. Lipofectamine RNAiMAX must be mixed before use. Mix the diluted siRNA and Lipofectamine RNAiMAX and incubate at room temperature for 20 minutes.
  • RNA was taken from each group and reverse transcription was synthesized according to the steps of the reverse transcription kit.
  • cDNA store at -80°C.
  • PCR amplification was performed using cDNA as a template, and each set of PCR products was detected using agarose gel electrophoresis.
  • Real Time-PCR amplification was performed using the standard three-step method in the kit. Obtain the Ct value, use GAPDH as the internal reference, and use the relative quantification method to calculate the 2- ⁇ Ct value.
  • siRNA-1 and siRNA-5 were not significantly different from the control group, and the gene silencing effect was poor (not shown).
  • siRNA-2/siRNA-3/siRNA- 4/siRNA-6 showed a certain degree of gene silencing effect compared to the control group.
  • siRNA-3/siRNA-4/siRNA-6 had a better gene silencing effect. Therefore, siRNA-3, siRNA-4, and siRNA- were selected. 6. Perform Real Time-PCR testing.
  • siRNA-3, siRNA-4 and siRNA-6 all showed a certain gene silencing effect, among which siRNA-3 had the best effect, so siRNA-3 was selected for subsequent experiments.
  • S1 ATTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGACGAACATTCCTAAGTCTGAA(SEQ ID NO.7);
  • the 5' end of S1 is optionally connected to a Cy5 fluorescent labeling group for tracking TDNs.
  • Example 1 replace the S1 sequence with S1-siRNA-3, in which S1 is chemically connected to the sense strand of siRNA-3 through the connecting sequence -TTTTT-, and S1-siRNA-3, S2, S3, and S4 are Add 95 ⁇ l of TM buffer (10mM Tris-HCl, 50mM MgCl 2 , pH 8.0) to a 200 ⁇ l EP tube at an equal molar ratio (1 ⁇ l of 100 ⁇ M stock solution per single chain), and heat the reaction solution to 95 °C for 10 min, and then rapidly cooled to 4 °C and maintained for 20 min to synthesize TDN-siRNA-3 (TDN-siRNA).
  • TM buffer 10mM Tris-HCl, 50mM MgCl 2 , pH 8.0
  • TDN-siRNA Use capillary electrophoresis and PAGE electrophoresis to detect the synthesized TDN-siRNA; use transmission electron microscopy to detect the appearance of TDNs and TDN-siRNA; use dynamic light scattering to detect the zeta potential and particle size of TDNs and TDNs-siRNA.
  • the electrophoresis results indicate that the molecular weight of the TDN-siRNA band is consistent with the complex situation of TDN and siRNA, indicating that siRNA has been successfully connected to TDN.
  • the tetrahedral structure particles of TDNs and TDN-siRNA can be observed in the transmission electron microscope image.
  • the particle sizes of TDNs particles and TDN-siRNA particles are approximately 15-20nm, with the zeta potential of the former being -6.41mV and the zeta potential of the latter being -21.9mV.
  • HREC cells human embryonic kidney cells 293 (HEK-293), human retinal endothelial cells (HREC cells) and human umbilical vein endothelial cells (HUVEC) as experimental cells
  • mice blank group, siRNA (Cy5 labeled) group, siRNA (Cy5 labeled) + Lipo RNAiMAX (transfection reagent) group, TDN (Cy5 labeled) group, TDN-siRNA (Cy5 labeled) group.
  • the method of using the transfection reagent is as follows: change the cell medium and add 1.7 mL of culture medium with 3% serum. Then add 3 ⁇ l siRNA to 147 ⁇ l of serum-free medium and mix well. Add 9 ⁇ l of transfection reagent to 141 ⁇ l of serum-free medium and mix evenly. Mix the two tubes of liquid, incubate for 5 minutes, and add dropwise to the cells.
  • the cell entry results of each cell were similar. No obvious fluorescent signal was detected in the blank group (not shown). An obvious positive fluorescent signal was detected in the samples of the siRNA plus transfection reagent treatment group, and the cell entry efficiency was 90.0%. On the left and right, only a weak fluorescence signal was detected in the siRNA-free transfection reagent-treated group, and the cell entry efficiency did not exceed 2%, indicating that siRNA itself has poor cell entry ability and can only effectively enter cells with the assistance of transfection reagents. . Strong positive fluorescence signals were detected in both the TDN group and the TDN-siRNA treatment group, indicating that TDN loading can achieve efficient entry of siRNA into cells without adding transfection reagents.
  • HREC cells human embryonic kidney cells 293 (HEK-293), human retinal endothelial cells (HREC cells) and human umbilical vein endothelial cells (HUVEC) as experimental cells
  • mice blank group, siRNA group, siRNA+Lipo (transfection reagent) group, 5nmol/L TDN group, 10nmol/L TDN group, 15nmol/L TDN group, 5nmol/L TDN-siRNA group, 10nmol/L TDN- siRNA group and 15nmol/L TDN-siRNA group
  • the trend of gene silencing in each cell is roughly the same.
  • the siRNA group treated without adding transfection reagent did not show obvious differential effects, while the siRNA+Lipo group was different from the blank control.
  • it showed a certain gene silencing effect.
  • the TDN-siRNA group showed obvious gene silencing effect as the concentration increased, which was equivalent to or even better than the siRNA+Lipo group at the same concentration.
  • the TDN-siRNA group showed obvious superiority at the same concentration.
  • HREC cells human embryonic kidney cells 293 (HEK-293), human retinal endothelial cells (HREC cells) and human umbilical vein endothelial cells (HUVEC) as experimental cells
  • mice blank group, siRNA group, siRNA+Lipo (transfection reagent) group, TDN group, TDN-siRNA group
  • the protein quantification results are basically consistent with the aforementioned gene silencing effects.
  • the TDN-siRNA group showed a comparable or even lower expression of RUNX1 than the siRNA+Lipo group, especially in HREC cells, the TDN-siRNA group showed At the same concentration, the RUNX1 expression inhibition level was significantly better than that of the siRNA+Lipo group, as shown in Figure 10.
  • Experimental groups blank group, Aflibercept (positive control) group (1nmol/L), siRNA group (1nmol/L), siRNA (1nmol/L)+Invivofectamine (transfection reagent), TDN group (1nmol/L), TDN- siRNA group (1nmol/L)
  • Wipe the eggs with 1:1000 Xinjie Er Killer Clean the surface of the purchased SPF eggs. After drying, use an egg camera to check whether the eggs are intact. Mark the name of the experiment with a pencil. Then put the eggs in the incubator to hatch. The conditions are set to 37.0 ⁇ 0.5°C, relative humidity 60%, the instrument is set to transfer eggs every two hours, and continue to incubate for 4-5 days.
  • the chicken embryos used in the experiment were randomly divided into 6 groups, 5 in each group.
  • Use a marker pen to mark the air chamber under the egg illuminator and draw the window opening position.
  • Make a window about 1cm in diameter use a grinding wheel, and then use ophthalmic tweezers to gently peel off the egg membrane to expose the chorioallantoic membrane of the chicken embryo.
  • Angiogenesis inhibition rate (MVD value of blank group - MVD value of experimental group)/MVD value of blank group * 100%
  • the aflibercept positive control group showed obvious neovascularization inhibitory effect, while the siRNA+Invivofectamine group showed better neovascularization inhibitory activity than the siRNA group, indicating the role of siRNA in the transfection reagent. It can effectively enter cells and produce a certain inhibitory effect on new blood vessels by silencing the expression of RUNX1.
  • TDN alone exhibited neovascular inhibitory activity almost equivalent to that of the positive control group, and its specific mechanism of action remains to be further explored.
  • the TDN-siRNA group showed extremely excellent neovascularization inhibitory activity, which was not only significantly better than the positive control group, but also better than the siRNA+Invivofectamine group and the TDN group.
  • OIR vasculoproliferative retinopathy
  • C57/BL mice and mothers on the 7th day after birth were placed in a breeding box with an oxygen volume fraction of 75% ⁇ 3% for 5 consecutive days, and the breeding temperature was maintained at (25 ⁇ 2)°C. Lighting was provided for 12 hours every day, and an automatic oxygen analyzer was used to monitor the oxygen content in the box.
  • P12 the mice were put back into normal air and raised. At this time, the mouse retina was in a relatively hypoxic state.
  • P17 On the 17th day after birth (P17), a large number of new blood vessels were formed in the retina.
  • Intravitreal injection is performed when P12 is released from the oxygen box.
  • the groups are as follows:
  • siRNA group Intravitreal injection of 1 ⁇ L of 75 ⁇ mol/L siRNA into both eyes
  • siRNA+Invivofectamine group 1 ⁇ L of siRNA-Invivofectamine complex was injected into the vitreous cavity of both eyes, in which the concentration of siRNA was 75 ⁇ mol/L.
  • TDN group intravitreal injection of 1 ⁇ L 75 ⁇ mol/L TDN in both eyes
  • TDN-siRNA group Intravitreal injection of 1 ⁇ L 75 ⁇ mol/L TDN-siRNA complex into both eyes
  • mice from each group were taken. After anesthesia and sacrifice, the eyeballs of both eyes of a total of 6 mice were removed and placed in 250 ⁇ L of cell lysis solution. They were crushed by ultrasonic and ultracentrifuged at low temperature for 30 min. The supernatant was collected and stored in a low-temperature refrigerator. ELISA kit was used to detect the expression of RUNX1 in retinal protein extract.
  • the siRNA-Invivofectamine group showed a significant inhibitory effect on RUNX1 expression compared with the blank group, while the TDN-siRNA group showed a better inhibitory effect on RUNX1 protein expression than the siRNA-Invivofectamine group, indicating that the TDN vector was used to carry Loading siRNA can effectively promote its entry into cells and silence the expression of RUNX1 gene, thereby reducing its protein expression.
  • Intravitreal injection is performed when P12 is released from the oxygen box.
  • the groups are as follows:
  • Aflibercept (positive control) group vitreous injection of 1 ⁇ L 40 mg/mL aflibercept in both eyes;
  • siRNA+Invivofectamine group 1 ⁇ L of siRNA-Invivofectamine complex was injected into the vitreous cavity of both eyes, in which the concentration of siRNA was 75 ⁇ mol/L;
  • TDN group intravitreal injection of 1 ⁇ L 75 ⁇ mol/L TDN in both eyes;
  • TDN-siRNA group Intravitreal injection of 1 ⁇ L 75 ⁇ mol/L TDN-siRNA complex into both eyes;
  • mice from each group were taken. After anesthesia and sacrifice, a total of 6 eyeballs from both eyes were taken out and fixed in 10% formaldehyde at room temperature for half an hour. The cornea, iris and lens were removed under a microscope, and the retina was carefully and completely peeled off. A radial incision was made from the ora serrata of the retina to the equator of the four quadrants. The retina was laid flat on a glass slide, sealed with water-soluble mounting agent, and covered with a coverslip. Fluorescence microscopy was used to detect the tiled retina, and Image-Pro Plus (Media Cybernetics, USA) software was used to measure the area of retinal neovascularization.
  • Image-Pro Plus Media Cybernetics, USA
  • the pictures of the blank group show that the retinas of mice that underwent OIR modeling had obvious lesions at P17, and the retina formed an avascular area (in a small circle) and a neovascular area (inside a small circle) at the posterior pole ( between small and large circles).
  • the Aflibercept (positive control) group After administration in the Aflibercept (positive control) group, it can inhibit the formation of new blood vessels to a certain extent and promote the normalization of blood vessels in the avascular area.
  • the TDN group showed a neovascularization inhibitory effect close to that of the Aflibercept (positive control) group. It is worth noting that the TDN-siRNA group showed a significantly better inhibitory effect on neovascularization than the positive control group, and could significantly promote the normalization of the avascular zone, showing an excellent repair effect on the diseased retina.

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Abstract

一种小干扰RNA及其用途，所述小干扰RNA特异性靶向Runt相关转录因子1基因及其同型mRNA，通过沉默其互补基因的表达，下调RUNX1表达量。将该siRNA用于制备治疗新血管生成性眼科疾病的药物，具有十分良好的应用前景。将该siRNA与TDNs设计成具有空间四面体结构的TDNs siRNA核酸分子，能够增加稳定性，帮助siRNA高效入胞，提高siRNA对RUNX1 mRNA的降解效力，从而减少RUNX1的表达，阻止新生血管的生成。

Description

一种治疗新生血管性视网膜疾病的小干扰RNA及其DNA四面体复合物 技术领域
本发明涉及针对新生血管性眼科疾病的药物领域，具体而言，其涉及沉默Runt相关转录因子1(RUNX1)基因的基因药物领域。
背景技术
新生血管形成(新血管生成)是很多眼部疾病的共同病理改变，常发生于角膜、虹膜、脉络膜和视网膜，导致的疾病包括角膜新生血管、脉络膜新生血管、视网膜新生血管等病变，常见的由此导致的眼部疾患包括例如：糖尿病性视网膜病变(diabetic retinopathy，DR)、早产儿视网膜病变(retinopathy of prematurity，ROP)、年龄相关性黄斑变性(age-related macular degeneration，AMD)(包括湿性AMD和干性AMD，湿性AMD的病理改变为脉络膜新生血管)、新生血管性青光眼等。新生血管可以在角膜、虹膜睫状体、脉络膜、视网膜、黄斑及视盘等几乎眼内所有组织中出现，它能够引起这些部位的组织出血、渗出及增生等一系列病理改变，因而造成眼球结构和功能的破坏，严重损害视功能。眼底新生血管性疾病患者人数在4000万以上，且随着人口老龄化程度加重，患者人数还在不断上升。眼底新生血管性疾病的患者几乎每月都要复诊或注射相关药物，否则视力的受损会严重影响生活。已有研究对于上述疾病的病因探究主要集中于因缺血、缺氧等因素触发炎性反应并刺激该区域促血管生成因子如血管内皮生长因子(vascular endothelial growth factor，VEGF)、血小板源性生长因子(platelet-derived growth factor，PDGF)等产生，诱导脉络膜、视网膜新生血管形成，并迅速增加微血管的通透性，从而导致黄斑水肿、视网膜出血、玻璃体积血等严重危害视力的并发症。
RUNX1又称为AML1，是人类白血病中染色体易位最常见的靶位点。RUNX1是十分重要的转录因子，在多种造血细胞系中广泛表达，在造血细胞的分化中起着关键作用，亦可调节造血相关基因的表达。RUNX1基因异常表达和突变常与人类白血病的发生相关。许多研究表明RUNX1是造血细胞生成过程中重要的调节因子，RUNX1蛋白可接受多种翻译后修饰，包括磷酸化、乙酰化等，其活性可受这些翻译后修饰的影响，从而调节造血细胞的分化、凋亡及自我更新。
近年来，相关研究挖掘出RUNX1靶点与眼新生血管之间的关系，如文献Identification of RUNX1 as a Mediator of Aberrant Retinal Angiogenesis[J].Diabetes,2017:1950-1956.中将Runt相关转录因子1(RUNX1)鉴定为通过转录组学分析从人PDR纤维血管膜(FVM)获得的CD31(+)血管内皮细胞中上调的基因。 使用人视网膜微血管内皮细胞(HRMECs)的体外研究表明，在高葡萄糖反应中，RUNX1RNA和蛋白质表达增加，而RUNX1抑制降低了HRMEC的迁移，增殖和管形成。RUNX1的免疫组织化学染色显示，氧诱导视网膜病变小鼠视网膜中患者来源的FVM血管和血管生成簇的血管生成血管具有反应性，这表明RUNX1上调是视网膜血管生成异常的标志。文献Inhibition of Runx1 by the Ro5-3335 benzodiazepine derivative reduces aberrant retinal angiogenesis[.Investigative Ophthalmology&Visual Science,2017中通过RUNX1染色定位为新生血管簇表明，它是视网膜血管生成异常中内皮细胞功能的新型特异性调节因子。玻璃体内注射Ro5-3335(RUNX1-CBFβ相互作用抑制剂)与载体处理的幼崽相比，新生血管簇面积显着减少有关。这与缺血区域的显著变化无关，这表明视网膜的正常和异常血管形成之间存在独立调节，用Ro5-3335抑制RUNX1功能可有效减少病理性视网膜新生血管形成。
发明内容
本发明的一个目的是提供靶向Runt相关转录因子1(RUNX1)基因及其同型mRNA降解的小干扰RNA(siRNA)；
本发明的另一个目的是提供含有所述siRNA的药物组合物；
本发明的再一个目的是提供所述siRNA或所述含有siRNA的药物组合物的用途；
本发明的再一个目的是提供一种DNA四面体-siRNA复合物及其用途。
根据本发明的一方面，一种针对特异性靶向并引起RNAi诱导的Runt相关转录因子1(RUNX1)及其同种型的mRNA降解的小干扰RNA(siRNA)。该siRNA双链可与RNA诱导的基因沉默复合物(RNA-induced silencing complex，RISC)连接在一起，并在与RISC结合后，靶向切割特定mRNA为10～11个碱基的小片段，从而中断RUNX1 mRNA的翻译过程，沉默其互补基因的表达，下调RUNX1表达量，从而抑制新血管生成。
因此，本发明所述的小干扰RNA特异性靶向Runt相关转录因子1基因及其同型mRNA，通过沉默其互补基因的表达，下调RUNX1表达量，所述的小干扰RNA选自：
(a)如SEQ ID NO.1所示的核苷酸组成的有义链和如SEQ ID NO.2所示的核苷酸组成的反义链；
(b)如SEQ ID NO.3所示的核苷酸组成的有义链和如SEQ ID NO.4所示的核苷酸组成的反义链；和
(c)如SEQ ID NO.5所示的核苷酸组成的有义链和如SEQ ID NO.6所示的核苷酸组成的反义链。
本发明所述的药物组合物，含有治疗有效量的所述的小干扰RNA。
在某些实施方式中，所述siRNA包含形成RNA双链体的正义RNA链和反义RNA链。正义RNA链包含与靶mRNA中约19至约25个连续核苷酸的靶序列相同的核苷酸序列。
在某些实施方式中，所述siRNA序列，虽然可能与非靶序列共有一定的序列同源性，但仍然充分不同以致对于非靶序列不发生RNA沉默。包括与非靶序列具有90％同源性、85％同源性、80％同源性、75％同源性、70％同源性、65％同源性、60％同源性、55％同源性、50％同源性、45％同源性、40％同源性、35％同源性、30％同源性、25％同源性、20％同源性、15％同源性、10％同源性、5％同源性、2％同源性和1％同源性的序列。与非靶序列完全不同源的序列被认为是与序列不互补的。
在某些实施方式中，所述siRNA可包含部分纯化的RNA、大体上纯的RNA、合成的RNA或重组产生的RNA以及与天然存在的RNA相异在于一个或多个核苷酸的添加、缺失、置换和/或改变的RNA。此类改变可包括非核苷酸材料例如至siRNA的末端或至siRNA的一个或多个内部核苷酸的添加，包括使siRNA抗核酸酶降解的修饰。
在某些实施方式中，siRNA核苷酸序列为如SEQ ID NO:1所示的正义链，如SEQ ID NO:2所示的反义链；或如SEQ ID NO:3所示的正义链、如SEQ ID NO:4所示的反义链；或如SEQ ID NO:5所示的正义链、如SEQ ID NO:6所示的反义链。
本发明的siRNA可使用本领域技术人员已知的常规技术获得，包括但不限于化学合成或重组产生。在某些实施方式中，siRNA可使用适当地保护的核糖核苷亚磷酰胺和常规DNA/RNA合成仪来化学合成，或者还可使用任何适当的启动子从重组环状或线性DNA质粒表达siRNA。在某些实施方式中，可利用标准技术从培养的细胞表达系统分离从重组质粒表达的siRNA，或可在体内在新血管形成的区域或附近细胞内表达siRNA。
本发明第二方面是提供本发明的siRNA或含有其的药物组合物在制备预防和/或治疗新生血管性眼科疾病的药物中的应用。所述新生血管性眼科疾病包括但不限于:糖尿病视网膜病变、早产儿视网膜病变、视网膜静脉阻塞、视网膜静脉周围炎、年龄相关性黄斑变性、高度近视黄斑出血、中心性渗出性视网膜脉络膜病变和其他血管生成性疾病。
本发明第三方面提供了一种治疗新血管生成性眼科疾病(新生血管性眼科疾病)的药物组合物，包括治疗有效量的siRNA化合物和药学上可接受的辅料(媒介、载体)，所述siRNA核苷酸序列包含如SEQ ID NO:1所示的RNA正义链，如SEQ ID NO:2所示的RNA反义链(对应表1中siRNA-3)；或如SEQ  ID NO:3所示的RNA正义链、如SEQ ID NO:4所示的RNA反义链(对应表1中siRNA-4)；或如SEQ ID NO:5所示的RNA正义链、如SEQ ID NO:6所示的RNA反义链(对应表1中siRNA-6)。
在某些实施方式中，通过考虑诸如受试者的大小和体重等因素,本领域技术人员可以容易地确定要给予给定受试者的siRNA的有效量。所述有效量的siRNA是足以引起细胞中的靶mRNA的RNAi介导的降解的量。临床上有效量是，当给受试者施用时，将通过RNA沉默抑制受试者的血管生成的进展的量。
根据本发明的再一方面，本发明也提供新血管生成性眼科疾病的治疗方法，特别是视网膜新血管性眼科疾病(新生血管性视网膜疾病)的治疗方法，包括将预防和/或治疗有效量的siRNA或含有siRNA的药物组合物施用于有需要的个体。
在某些实施方式中，可以将siRNA或含有siRNA的药物组合以预防和/或治疗的有效剂量施用于患眼的整体、局部，特别是玻璃体。制剂方式可以是任何适于眼部局部施用的剂型，包括但不限于注射剂、滴眼剂等。
施用所述药物组合物能够降低RUNX1的基因表达，进而抑制患眼相关部位新血管生成，并促进患眼血管的正常化。
DNA四面体-siRNA复合物
目前已有研究的针对AMD、DME等眼底新生血管疾病的siRNA药物有3款，针对的靶点均为VEGF。然而，siRNA药物用于治疗有两大挑战：一个是siRNA暴露血液会有稳定性问题并造成免疫原性，一个是大分子量负电的siRNA无法自己跨膜进入胞内。因此，siRNA往往需要通过载体递送入胞来发挥功能，其中使用最丰富的是腺相关病毒载体(AAV)递送平台，然而，不断有权威研究表明AAV可将外源基因片段插入到染色体上，其是否会影响细胞增殖凋亡等功能甚至最终导致癌变，成为其作为载体平台使用中的隐患。又如非病毒载体质纳米粒(LNP)在目前的药物开发中也有较多使用，但由于存在较严重的过敏反应，因而在注射使用LNP递送系统的药物之前，患者需要使用抗组胺和激素药物控制，因此只适用于罕见病和癌症等严重疾病，对于慢性病使用限制较大。
DNA四面体(Tetrahedral DNA Nanostructures,TDN)是一种由4条单链DNA通过变性和复性进而通过链间碱基互补配对形成的一种四面体结构，它易于合成，生物相容性高，在先的专利对于TDNs在眼科疾病中的用途进行了公开，如专利CN109646450B中披露了TDNs在制备治疗角膜损伤的药物中的用途，专利CN112007044B中公开了TDNs-miR155复合物及其在制备预防或治疗湿性黄斑病变的药物中的用途，专利CN112843085B中公开了TDNs-miR22复合物及其在制备治疗视神经损伤的药物中用途。目前尚未见TDNs携载siRNA进行眼科疾病的治疗。
本发明采用DNA四面体作为携带靶向RUNX1 mRNA的siRNA的载体系统，其稳定性和安全性优异，不易被核酸酶溶解；无需转染，易于穿过细胞膜，能够增强药物的细胞摄取效果，进而提高siRNA对mRNA的降解效力，阻止新生血管的生成。在使用浓度下，各种类型的活细胞(如RAW264.7细胞和L929成纤维细胞样细胞)均未显示出明显的细胞毒性或不良反应。
因此，本发明提供用于治疗新生血管性眼科疾病的DNA四面体药物复合物，所述DNA四面体药物复合物包括：
(1)小干扰RNA，所述小干扰RNA特异性靶向Runt相关转录因子1基因及其同型mRNA，沉默RUNX1互补基因的表达，下调RUNX1表达量，所述的小干扰RNA选自：
(a)如SEQ ID NO.1所示的核苷酸组成的有义链和如SEQ ID NO.2所示的核苷酸组成的反义链；
(b)如SEQ ID NO.3所示的核苷酸组成的有义链和如SEQ ID NO.4所示的核苷酸组成的反义链；和
(c)如SEQ ID NO.5所示的核苷酸组成的有义链和如SEQ ID NO.6所示的核苷酸组成的反义链；以及
(2)一DNA四面体，所述DNA四面体由四条单链DNA经碱基互补配对形成；所述四条单链DNA的核苷酸序列分别一对一地选自如SEQ ID NO.7～SEQ ID NO.10的所示序列；
其中所述小干扰RNA与DNA四面体的至少一条单链连接。
优选地，其中将所述小干扰RNA的有义链通过化学键与所述DNA四面体的至少一条单链连接。
优选地，其中在所述小干扰RNA的有义链与所述DNA四面体的至少一条单链之间还包含一接头。
更优选地，其中所述接头为连接序列-TTTTT-。
本发明所述的的DNA四面体药物复合物的制备中，将形成所述DNA四面体的四条单链DNA以等摩尔比置于足以使其变性的温度下使其变性，然后将温度降低至使其退火进而通过链间碱基互补配对形成DNA四面体结构；将所述四条单链DNA的至少一条连接所述小干扰RNA。
在一个较佳实施方案中，本发明所述的DNA四面体，也即TDNs，是4条单链DNA经碱基互补配对形成；所述4条单链DNA的序列依次对应SEQ ID NO.7-10的所述序列，其可通过变性、退火过程组装形成目标产物TDNs。具体而言，将TDNs的4条单链DNA置于足以使其变性的温度下维持10min，再将温度降低至2-8℃维持20min以上。
在某些实施方式中，DNA四面体和siRNA按照1:(1～4)的摩尔比构成。所 述DNA四面体中的至少任意一条DNA单链的末端与所连接siRNA的正义链连接。
在某些实施方式中，所述DNA四面体的单链DNA与siRNA正义链之间还含有连接序列，所述连接序列为核苷酸序列，优选为脱氧核糖核苷酸序列，更优选为-TTTTT-,所述-TTTTT-序列即连续5个胸腺嘧啶脱氧核苷酸序列。
在本发明的一个较佳实施方案中，还提供了一种TDNs-siRNA核酸分子的制备方法，它是将DNA四面体的4条DNA单链置于足以使其变性的温度下维持10min以上，再将温度降低到2～8℃维持20min以上，其中所述4条单链的至少1条连接有靶向沉默RUNX1的siRNA。
优选地，所述DNA四面体是由所述4条DNA单链经90～98℃变性10～15min、2～8℃退火20～30min制备而成，其中1条连接有靶向沉默RUNX1的siRNA。
更优选地，所述DNA四面体是由所述4条DNA单链经95℃变性10min、4℃退火20min制备而成，其中1条连接有靶向沉默RUNX1的siRNA。
本发明的又一方面，还提供了上述的TDNs-siRNA核酸分子在制备预防和/或治疗新血管生成性眼科疾病的药物中的用途，特别是在制备预防和/或治疗视网膜新血管生成性疾病的药物中的用途。
在某些实施方式中，所述新血管生成性眼科疾病包括但不限于：糖尿病视网膜病变、早产儿视网膜病变、视网膜静脉阻塞、视网膜静脉周围炎、年龄相关性黄斑变性、高度近视黄斑出血、中心性渗出性视网膜脉络膜病变和其他血管生成性疾病。
本发明还提供了一种治疗新血管生成性眼科疾病的药物组合物，所述药物组合物含有上述预防和/或治疗新血管生成性眼科疾病的TDNs-siRNA核酸分子和药学上可接受的辅料(媒介、载体)。
在某些实施方式中，通过考虑诸如受试者的大小和体重等因素,本领域技术人员可以容易地确定要给予给定受试者的TDNs-siRNA的有效量。所述有效量是足以引起细胞中的靶mRNA的RNAi介导的降解的量。临床上有效量是，当给受试者施用时，将通过RNA沉默抑制受试者的血管生成的进展的量。
根据本发明的再一方面，本发明也提供与新血管生成性眼科疾病的治疗方法，包括将预防和/或治疗有效量的TDNs-siRNA或含有TDNs-siRNA的药物组合物施用于有需要的个体。
在某些实施方式中，可以将TDNs-siRNA或含有TDN-siRNA的药物组合以预防和/或治疗的有效剂量施用于患眼的整体、局部，特别是玻璃体。制剂方式可以是任何适于眼部局部施用的剂型，包括但不限于注射剂、滴眼剂等。
有益的效果
本发明所提供的siRNA能引起RNAi介导的Runt相关转录因子1(RUNX1) mRNA的翻译过程，从而抑制或沉默其互补基因的表达，下调RUNX1表达，将siRNA用于制备治疗新血管生成性眼科疾病的药物，具有十分良好的应用前景。
本发明首次将siRNA与TDNs设计成具有空间四面体结构的TDNs-siRNA核酸分子，增加了稳定性，帮助siRNA高效入胞，提高siRNA对RUNX1 mRNA的降解效力，从而减少RUNX1的表达，阻止新生血管的生成。
附图说明
图1为经各候选siRNA(siRNA-2/siRNA-3/siRNA-4/siRNA-6)处理后其RUNX1 mRNA PCR产物琼脂糖凝胶电泳检测图，其中泳道1-3为siRNA-2样品、泳道4-6为siRNA-3样品、泳道7-9为siRNA-4样品、泳道10-12为siRNA-6样品、泳道13-15为空白对照组样品；
图2为经各候选siRNA处理后，其RUNX1基因沉默效果定量分析图；
图3为TDN以及TDN-siRNA的聚丙烯酰胺凝胶电泳图，其中泳道1为TDN样品、泳道2-6为TDN-siRNA-3样品；
图4为TDN以及TDN-siRNA的毛细管电泳图；
图5为TDN以及TDN-siRNA的透射电镜图；
图6为TDN以及TDN-siRNA的粒径/电位结果图；
TDN(左)、TDN-siRNA(右)
图7为siRNA、siRNA+Lipo、TDN、TDN-siRNA复合物在不同细胞(HEK-293细胞、HUVEC细胞以及HREC细胞)中的入胞效率；
图8为各给药组施用后，不同细胞中RUNX1基因沉默效果，其中包括空白组(control)、siRNA组和siRNA+Lipo组、不同浓度的TDN组(5nmol/L、10nmol/L、15nmol/L)、不同浓度的TDN-siRNA组(5nmol/L、10nmol/L、15nmol/L)；
图9为各给药组施用后，不同细胞(HEK-293细胞、HUVEC细胞以及HREC细胞)中RUNX1蛋白表达抑制水平统计，其中包括空白组(control)、siRNA组、siRNA+Lipo组、TDN组、TDN-siRNA组；
图10为HREC细胞各实验组RUNX1蛋白WB检测条带；
图11为各给药组施用于鸡胚绒毛尿囊膜模型后，其新生血管抑制水平，包括空白组(control)、Aflibercept(阳性对照)组、siRNA组、siRNA+Invivofectamine(转染试剂)、TDN组、TDN-siRNA组；
图12为各给药组施用于小鼠OIR模型后，各组小鼠视网膜中RUNX1蛋白表达量的水平，包括空白组(control)、siRNA组、siRNA+Invivofectamine组、TDN组、TDN-SiRNA组；
图13为各给药组施用于小鼠OIR模型后，各组小鼠视网膜中新生血管面积 统计结果，包括空白组(control)、Aflibercept(阳性对照)组、siRNA+Invivofectamine组、TDN组、TDN-siRNA组；
图14为OIR模型中各给药组小鼠视网膜新生血管生成情况及无血管区恢复情况；
图15为TDN-siRNA-3连接示意图。
具体实施方式
以下通过对本发明较佳实施方式的描述，详细说明但不限制本发明。
材料来源：如非特别说明，本发明所使用的材料均为市售购买。
HEK-293购自上海景泽生物技术有限公司；
HREC细胞购自angiopromie公司；
HUVEC细胞购自澳赛尔斯生物；
LipoRNAiMAX购自赛默飞；
Invivofectamine试剂购买自赛默飞；
阿柏西普注射液(Aflibercept)：购自拜耳医药保健有限公司，规格为40mg/ml/瓶；
C57/BL小鼠购买自斯贝福(北京)生物技术有限公司
实施例1 siRNA筛选及其基因沉默
(1)siRNA合成
根据RUNX1 mRNA设计并委托金斯瑞合成公司合成了6种siRNA，序列如下表1所示：
Figure PCTCN2022121379-appb-000001
(2)细胞转染
1)将HUVEC细胞从液氮罐中取出复苏，经培养稳定传代2-4代后用于转染实验。
2)转染前一天，在6孔板中每个孔内接种3.0x10^5个细胞，每孔加入2.5mL 不含抗生素的生长培养基，保证转染时会有50-60％的融合率。
3)从细胞中去除生长培养基。每孔加入1.5mL新鲜的无血清的生长培养基，用培养基稀释各候选siRNA+Lipofectamine RNAiMAX的复合物加入到各待测细胞组中，使其siRNA浓度为15nmol/L。在37℃的二氧化碳培养箱中培养细胞5-6小时后更换含有血清的培养基并培养细胞48小时后检测基因沉默效果。
其中，siRNA和Lipofectamine RNAiMAX混合物可如下方法准备：在250μl的无血清生长培养基中加入候选siRNA，柔和混匀。然后在250μl生长培养基中加入5μl Lipofectamine RNAiMAX进行稀释，室温下孵育5分钟。Lipofectamine RNAiMAX使用前要先混匀，混合稀释好的siRNA和Lipofectamine RNAiMAX，室温下孵育20分钟。
(3)检测
上述各组细胞接受处理后按照总RNA提取试剂盒步骤过柱提取，提取后测定RNA浓度并用琼脂糖凝胶电泳验证纯度后，每组取等量RNA参照逆转录试剂盒步骤，进行逆转录合成为cDNA，-80℃保存。以cDNA为模板进行PCR扩增，使用琼脂糖凝胶电泳检测各组PCR产物。同时，使用q-PCR染料试剂盒，严格按照步骤，以cDNA为模板进行实时荧光定量PCR扩增。采用试剂盒中标准三步法进行Real Time-PCR扩增。得到Ct值，以GAPDH为内参，使用相对定量法，计算2 -△△Ct值。
(4)实验结果
基于琼脂糖凝胶电泳的检测，siRNA-1与siRNA-5与对照组无显著差异，基因沉默效果不佳(未呈现)，而如图1所示，siRNA-2/siRNA-3/siRNA-4/siRNA-6相较于对照组展现出一定程度的基因沉默效果，其中siRNA-3/siRNA-4/siRNA-6的基因沉默效果更好，因而选siRNA-3、siRNA-4、siRNA-6进行Real Time-PCR检测。
如图2所示，与空白对照组相比，siRNA-3、siRNA-4和siRNA-6均表现出一定的基因沉默效果，其中siRNA-3效果最佳，因此选择siRNA-3进行后续实验。
实施例2 TDN-siRNA复合物
(1)TDNs的合成
将四条单链(S1，S2，S3，S4)按照等摩尔比(每条单链加入1μl浓度为100μM的储存液)加入到含有96μl的TM buffer(10mM Tris-HCl，50mM MgCl2，pH 8.0)的200μl EP管中，将反应液加热到95℃维持10min，然后快速降温到4℃维持20min合成了TDNs。
4条单链的序列(5′→3′)如下：
S1:ATTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGACGAACATTCCTAAGTCTGAA(SEQ ID NO.7)；
S2:ACATGCGAGGGTCCAATACCGACGATTACAGCTTGCTACACGATTCAGACTTAGGAATGTTCG(SEQ ID NO.8)；
S3:ACTACTATGGCGGGTGATAAAACGTGTAGCAAGCTGTAATCGACGGGAAGAGCATGCCCATCC(SEQ ID NO.9)；
S4:ACGGTATTGGACCCTCGCATGACTCAACTGCCTGGTGATACGAGGATGGGCATGCTCTTCCCG(SEQ ID NO.10)；
其中S1的5’端可选地连接一个Cy5荧光标记基团用于TDNs的示踪。
(2)TDNs-siRNA复合物的合成
在实施例1的基础上，将S1序列替换为S1-siRNA-3，其中S1通过连接序列-TTTTT-与siRNA-3的正义链以化学键连接，将S1-siRNA-3、S2、S3、S4按照等摩尔比(每条单链加入1μl浓度为100μM的储存液)加入到含有95μl的TM buffer(10mM Tris-HCl，50mM MgCl 2，pH 8.0)的200μl EP管中，将反应液加热到95℃维持10min，然后快速降温到4℃维持20min合成了TDN-siRNA-3(TDN-siRNA)，其连接方式如示意图(图15)所示。
实施例3 TDN-siRNA的表征
(1)鉴定方法
使用毛细管电泳、PAGE电泳检测合成得到的TDN-siRNA；使用透射电镜检测TDNs与TDN-siRNA的外形；使用动态光散射检测TDNs与TDNs-SiRNA的zeta电位及粒径。
(2)结果
如图3-4所示，电泳结果指示TDN-siRNA的条带分子量符合TDN与siRNA的复合情况，表示siRNA已成功连接至TDN。
如图5所示，透射电镜图中可观测到TDNs以及TDN-siRNA的四面体结构颗粒。
如图6所示，TDNs颗粒和TDN-siRNA颗粒的粒径约为15-20nm，其中前者的zeta电位为-6.41mV，后者的zeta电位为-21.9mV。
实施例4 细胞实验
(1)荧光标记细胞摄取实验
细胞：人胚胎肾细胞293(HEK-293)、人视网膜内皮细胞(HREC细胞)和人脐静脉内皮细胞(HUVEC)作为实验细胞
实验分组：空白组、siRNA(Cy5标记)组、siRNA(Cy5标记)+Lipo RNAiMAX (转染试剂)组、TDN(Cy5标记)组、TDN-siRNA(Cy5标记)组。
实验方法：
1)在转染实验的前一天，在每个孔的2ml生长培养基中加入3x10^5个细胞(确保转染时细胞密度在60-80％)。
2)使用DMEM培养基稀释各组样品至终体积250μl。
3)将待测样品添加到细胞中：每孔加样250ul，其最终样品浓度为15nmol/L。
4)37℃静置培养细胞24h(小时)，之后收样使用流式细胞仪检测入胞情况。
其中转染试剂的使用方法为：给细胞换液，加3％血清的培养基1.7mL。然后用147μl的无血清培养基加3μl siRNA混匀，141μl的无血清培养基加9μl转染试剂混匀，把两管液体混合，孵育5min,滴加至细胞中
实验结果：
如图7所示，各细胞的入胞结果类似，空白组未检测到明显荧光信号(未呈现)，siRNA加转染试剂处理组的样本检测到明显的阳性荧光信号，入胞效率为90.0％左右，siRNA无转染试剂处理组的样本则仅检测到微弱的荧光信号，入胞效率不超过2％，说明siRNA自身的入胞能力不佳，在转染试剂的辅助下才能够有效入胞。TDN组与TDN-siRNA处理组均检测到较强的阳性荧光信号，说明在不添加转染试剂的情况下TDN的携载能够实现siRNA的高效入胞。
(2)RUNX1基因沉默实验
细胞：人胚胎肾细胞293(HEK-293)、人视网膜内皮细胞(HREC细胞)和人脐静脉内皮细胞(HUVEC)作为实验细胞
实验分组：空白组、siRNA组、siRNA+Lipo(转染试剂)组、5nmol/L TDN组、10nmol/L TDN组、15nmol/L TDN组、5nmol/L TDN-siRNA组、10nmol/L TDN-siRNA组和15nmol/L TDN-siRNA组
实验方法：
1)在转染实验的前一天，在每个孔的2ml生长培养基中加入3×10^5个细胞(确保转染时细胞密度在60-80％)。
2)使用DMEM培养基稀释各组样品至终体积250μl。
3)将待测样品添加到细胞中：每孔加样250μl，其中siRNA组、siRNA+Lipo(转染试剂)组中siRNA的终浓度均为15nmol/L，其余给药组按照实验分组的浓度梯度为终浓度。
4)37℃静置培养细胞24h，之后收样进行qPCR实验检测基因沉默效果。
实验结果：
如图8所示，各细胞中基因沉默的作用趋势大致相同，其中不添加转染试剂处理的siRNA组与空白对照相比，并未展现出明显的差异性效果，而siRNA+Lipo组与空白组相比展现出一定的基因沉默作用。而TDN-siRNA组随浓度增大展现出明显的基因沉默作用，在相同浓度下与siRNA+Lipo组相当甚至更优，尤其是在HREC细胞中，其TDN-siRNA组在相同浓度下展现明显优于siRNA+Lipo组的基因沉默效果。
(3)RUNX1蛋白表达抑制实验
细胞：人胚胎肾细胞293(HEK-293)、人视网膜内皮细胞(HREC细胞)和人脐静脉内皮细胞(HUVEC)作为实验细胞
实验分组：空白组、siRNA组、siRNA+Lipo(转染试剂)组、TDN组、TDN-siRNA组
实验方法：
1)在转染实验的前一天，在每个孔的2ml生长培养基中加入3×10^5个细胞(确保转染时细胞密度在60-80％)。
2)使用0％FBS的培养基换液后，将各组样品添加到细胞中，六孔为一组，控制其终浓度为15nmol/L。
3)37℃静置培养细胞24h后收样，提取蛋白，检测蛋白浓度后进行WB实验，检测其RUNX1的蛋白表达量。
实验结果：
如图9所示，蛋白定量结果与前述基因沉默效果基本一致，TDN-siRNA组展现出与siRNA+Lipo组相当甚至更低的RUNX1的表达量，尤其是在HREC细胞中，其TDN-siRNA组在相同浓度下展现明显优于siRNA+Lipo组的RUNX1表达抑制水平，见图10。
实施例5 体内外模型实验
(1)鸡胚绒毛尿囊膜(CAM)新生血管抑制实验
实验分组：空白组、Aflibercept(阳性对照)组(1nmol/L)、siRNA组(1nmol/L)、siRNA(1nmol/L)+Invivofectamine(转染试剂)、TDN组(1nmol/L)、TDN-siRNA组(1nmol/L)
实验方法：
用1：1000新洁尔灭液擦拭鸡蛋，将购买的SPF种鸡蛋表面清洁，拭干后用照蛋器检查种蛋是否完好，用铅笔标记实验名称，之后将鸡蛋放入孵化箱中孵化，条件设定为37.0±0.5℃，相对湿度60％，仪器设定每两个小时转蛋一次，继续孵育4-5天。
等鸡蛋孵育5-6d(天)的时候，将实验用的鸡胚随机分为6组，每组5只。在照蛋器下用马克笔标记气室，画出开窗位置，用75％酒精消毒后，用注射器针头轻轻将蛋壳钻出一个小孔，之后用眼科镊在蛋壳上慢慢撕出一个直径约1cm窗口(用砂轮，然后用眼科镊轻轻揭掉蛋膜，暴露鸡胚绒毛尿囊膜，注意不要损伤血管。)滴加待测样品药液于绒毛尿囊膜上，后用透明封口膜封闭窗口，孵育48h后，用酒精消毒接种部位及四周，撕去封闭处卵壳，滴入10％福尔马林液固定10min，待CAM上的血管凝固后，剥离假气室周围的蛋壳，使CAM充分暴露，用眼科剪取3cm×3cm的CAM，脱水后用石蜡包埋，平行于CAM方向，连续切片，厚度8μm，0.5％甲苯胺蓝染色。取标本于250倍视野下计数微血管的横截面，分别随机6个不重复的高倍视野，取其平均值(四舍五入)作为该标本的单位面积下的MVD(微血管密度)，并计算器血管生成抑制率，计算方法如下：
血管生成抑制率＝(空白组MVD值-实验组MVD值)/空白组MVD值*100％
表2 实验结果：
Figure PCTCN2022121379-appb-000002
如图11所示，阿柏西普(Aflibercept)阳性对照组展现出明显的新生血管抑制作用，而siRNA+Invivofectamine组展现出优于siRNA组的新生血管抑制活性，指示siRNA在转染试剂的作用下能够有效进入细胞并通过对RUNX1的沉默表达作用产生一定的新生血管抑制效果。令人惊讶的是，单独的TDN展现出几乎与阳性对照组相当的新生血管抑制活性，其具体作用原理还有待进一步探究。同时，TDN-siRNA组展现出极其优异的新生血管抑制活性，不仅显著优于阳性对照组，也优于siRNA+Invivofectamine组和TDN组。
(2)氧诱导的血管增殖性视网膜病变(OIR)小鼠模型
造模过程：
将出生后第7天(P7)的C57/BL小鼠与母鼠放入含氧体积分数为75％±3％的饲养箱内连续饲养5天，饲养温度维持在(25±2)℃，每天照明12h，用自动氧气分析仪监控箱内氧气含量。出生后第12天(P12)放回正常空气中饲养，这 时小鼠视网膜处于相对缺氧状态，出生后第17天(P17)视网膜内有大量新生血管形成。
1)小鼠视网膜RUNX1蛋白表达量检测
实验分组及给药方式：
于P12出氧箱时进行玻璃体腔注射，各分组如下：
空白组：不进行玻璃体腔给药
siRNA组：双眼玻璃体腔注射1μL的75μmol/L的siRNA
siRNA+Invivofectamine组：双眼玻璃体腔注射1μL siRNA-Invivofectamine复合物，其中siRNA的浓度为75μmol/L。
TDN组：双眼玻璃体腔注射1μL 75μmol/L TDN
TDN-siRNA组：双眼玻璃体腔注射1μL 75μmol/L TDN-siRNA复合物
实验方法：
给药两天后，取各组小鼠3只，麻醉处死后取出双眼眼球共6只放入250μL细胞裂解液中，超声粉碎，低温超速离心30min，收集上清液，置于低温冰箱中保存。采用ELISA试剂盒检测视网膜蛋白提取液中RUNX1表达。
实验结果：
如图12所示，siRNA-Invivofectamine组相较于空白组展现出明显的RUNX1表达抑制作用，而TDN-siRNA组展现出更优于siRNA-Invivofectamine组的RUNX1蛋白表达抑制效果，说明采用TDN载体携载siRNA能够有效促进其入胞并沉默RUNX1基因表达进而降低其蛋白表达量。
2)小鼠视网膜新生血管生成抑制实验
实验分组及给药方式：
于P12出氧箱时进行玻璃体腔注射，各分组如下：
空白组(negative CTRL)：不进行玻璃体腔给药
Aflibercept(阳性对照)组：双眼玻璃体注射1μL 40mg/mL的阿柏西普；
siRNA+Invivofectamine组：双眼玻璃体腔注射1μL siRNA-Invivofectamine复合物，其中siRNA的浓度为75μmol/L；
TDN组：双眼玻璃体腔注射1μL 75μmol/L TDN；
TDN-siRNA组：双眼玻璃体腔注射1μL 75μmol/L TDN-siRNA复合物；
实验方法：
于P17，取各组小鼠3只，麻醉处死后取出双眼眼球共6只，在10％的甲醛中室温固定半小时。显微镜下去除角膜、虹膜和晶体，小心完整地剥离视网膜，从视网膜锯齿缘到4个象限的赤道部做放射状切开，视网膜平铺在玻片上，水溶性封片剂封口，加盖玻片。用荧光显微镜检测平铺的视网膜，Image-Pro Plus(Media Cybernetics，美国)软件测量视网膜新生血管的面积。
实验结果：
如图13-14所示，空白组图片显示经过OIR造模的小鼠，其视网膜在P17产生了明显的病变，视网膜在后极部形成无血管区(小圈内)，以及新生血管区(小圈和大圈之间)。Aflibercept(阳性对照)组给药之后能够在一定程度上抑制新生血管的生成，并促进无血管区的血管正常化。此外，TDN组展现出与Aflibercept(阳性对照)组接近的新生血管生成抑制效果。值得注意的是，TDN-siRNA组展现出明显优于阳性对照组的新生血管抑制作用，并能够明显促进无血管区的正常化，展现出对病变视网膜优异的修复作用。
本发明不限于以上实施方式，本领域技术人员可以根据说明书的描述对本发明做出各种改变或变形，只要不脱离本发明的精神，均属于本发明的范围。
序列列表：
Figure PCTCN2022121379-appb-000003

Claims (12)

  1. 一种小干扰RNA，所述小干扰RNA特异性靶向Runt相关转录因子1基因及其同型mRNA，通过沉默其互补基因的表达，下调RUNX1表达量，所述的小干扰RNA选自：
    (a)如SEQ ID NO.1所示的核苷酸组成的有义链和如SEQ ID NO.2所示的核苷酸组成的反义链；
    (b)如SEQ ID NO.3所示的核苷酸组成的有义链和如SEQ ID NO.4所示的核苷酸组成的反义链；和
    (c)如SEQ ID NO.5所示的核苷酸组成的有义链和如SEQ ID NO.6所示的核苷酸组成的反义链。
  2. 一种药物组合物，含有治疗有效量的权利要求1所述的小干扰RNA。
  3. 权利要求1所述的小干扰RNA或权利要求2所述的药物组合物在制备预防和/或治疗新生血管性眼科疾病的药物中的应用。
  4. 根据权利要求3所述的应用，其中所述的新生血管性眼科疾病选自糖尿病视网膜病变、早产儿视网膜病变、视网膜静脉阻塞、视网膜静脉周围炎、年龄相关性黄斑变性、高度近视黄斑出血或中心性渗出性视网膜脉络膜病变。
  5. 一种用于治疗新生血管性眼科疾病的DNA四面体药物复合物，所述DNA四面体药物复合物包括：
    (1)小干扰RNA，所述小干扰RNA特异性靶向Runt相关转录因子1基因及其同型mRNA，通过沉默其互补基因的表达，下调RUNX1表达量，所述的小干扰RNA选自：
    (a)如SEQ ID NO.1所示的核苷酸组成的有义链和如SEQ ID NO.2所示的核苷酸组成的反义链；
    (b)如SEQ ID NO.3所示的核苷酸组成的有义链和如SEQ ID NO.4所示的核苷酸组成的反义链；和
    (c)如SEQ ID NO.5所示的核苷酸组成的有义链和如SEQ ID NO.6所示的核苷酸组成的反义链；以及
    (2)一DNA四面体，所述DNA四面体由四条单链DNA经碱基互补配对形成；所述四条单链DNA的核苷酸序列分别一对一地选自如SEQ ID NO.7～SEQ ID NO.10的所示序列；
    其中所述小干扰RNA与DNA四面体的至少一条单链连接。
  6. 根据权利要求5所述的DNA四面体药物复合物，其中将所述小干扰RNA的有义链通过化学键与所述DNA四面体的至少一条单链连接。
  7. 根据权利要求6所述的DNA四面体药物复合物，其中在所述小干扰RNA的有义链与所述DNA四面体的至少一条单链之间还包含一接头。
  8. 根据权利要求7所述的DNA四面体药物复合物，其中所述接头为连接序列-TTTTT-。
  9. 根据权利要求5～8任一项所述的DNA四面体药物复合物，其中将形成所述DNA四面体的四条单链DNA以等摩尔比置于足以使其变性的温度下使其变性，然后将温度降低至使其退火进而通过链间碱基互补配对形成DNA四面体结构；将所述四条单链DNA的至少一条连接所述小干扰RNA。
  10. 一种药物组合物，含有权利要求5所述的用于治疗新生血管性眼科疾病的DNA四面体药物复合物。
  11. 权利要求5所述的DNA四面体药物复合物或权利要求10所述的药物组合物在制备预防和/或治疗新生血管性视网膜疾病药物中的应用。
  12. 根据权利要求11所述的应用，其中所述的新生血管性视网膜疾病选自糖尿病视网膜病变、早产儿视网膜病变、视网膜静脉阻塞、视网膜静脉周围炎、年龄相关性黄斑变性、高度近视黄斑出血或中心性渗出性视网膜脉络膜病变。
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