Tetrahedral framework nucleic acid related to miR-22 as well as preparation method and application of tetrahedral framework nucleic acid

Abstract

The invention provides a tetrahedral framework nucleic acid related to miR-22 as well as a preparation method and application thereof. The tetrahedral framework nucleic acid is formed by a first single-stranded oligonucleotide, a second single-stranded oligonucleotide, a third single-stranded oligonucleotide and a fourth single-stranded oligonucleotide through base complementation. The tetrahedral framework nucleic acid provided by the invention is a nucleic acid drug delivery system with high delivery efficiency, good structural stability and low immunogenicity, and provides an efficient, accurate and low-toxicity new scheme for the treatment of eye lesions.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid technology, and further relates to a tetrahedral framework nucleic acid related to miR-22, and a preparation method and use thereof. Background Art

[0002] Fundus lesions refer to a series of diseases occurring in the posterior structures of the eyeball, involving key parts such as the retina, optic nerve, and choroid. These lesions may cause serious impacts on visual function. Among them, age-related macular degeneration (AMD) is a blinding eye disease characterized by degenerative lesions in the macular area, mainly affecting people over 50 years old, and is the leading cause of irreversible visual impairment in the elderly and middle-aged worldwide. AMD is divided into two types: dry (atrophic) and wet (exudative). Dry AMD is characterized by atrophy of the retinal pigment epithelium (RPE) and deposition of drusen, with a slow progression of the disease but may develop into advanced atrophy. Wet AMD is caused by abnormal proliferation of choroidal neovascularization (CNV), accompanied by bleeding, exudation, and scar formation, which can lead to a sharp decline in vision. Common symptoms in patients include blurred central vision, metamorphopsia (such as straight lines being distorted), and abnormal color vision. Permanent loss of central vision may occur in the late stage. The etiology of AMD has not been fully clarified and is currently considered to be the result of the combined action of genetic, environmental, and metabolic factors. Oxidative stress, inflammatory response, and abnormal choroidal blood flow are the core pathological mechanisms of this disease. At present, there is no specific therapy for dry AMD, and the progression is mainly delayed by supplementing antioxidants (such as the AREDS2 formula); the first-line treatment for wet AMD is intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) drugs (such as ranibizumab, aflibercept), which can effectively inhibit neovascularization and improve vision. However, the treatment requires long-term repeated injections, and some patients experience problems such as drug resistance or economic burden. There is an urgent need for a new therapy to fill the current treatment gap.

[0003] MicroRNA (miRNA, miR) is a class of endogenous non-coding RNA molecules approximately 20 - 24 nucleotides in length, which have various important regulatory functions within cells. In recent years, miRNA therapy has attracted significant attention in the field of nucleic acid drugs. miRNA therapy can be achieved through miRNA replacement therapy using miRNA mimics or by inhibiting the function of miRNAs with miRNA inhibitors. miRNA molecules are easily degraded by nucleases in the blood and tissues, and are negatively charged, making it difficult for them to enter cells on their own. Therefore, the implementation of miRNA therapy depends on a suitable delivery system. Existing miRNA delivery systems, such as lipid nanoparticles, polymer nanoparticles, viral vectors, etc., have various problems, including insufficient delivery efficiency, immunogenicity or cytotoxicity, unclear metabolic pathways, poor stability, and complex production processes, which limit the application of miRNA therapy.

[0004] Tetrahedral framework nucleic acid (tFNA) is a novel nucleic acid nanomaterial that can freely penetrate cell membranes and enter cells, exhibits stability in a complex serum environment, and has biocompatibility. tFNA can enter cells through the caveolin-mediated endocytosis pathway, then be transported within cells via a microtubule-dependent pathway, and finally be orderly transported into cell lysosomes. Moreover, tFNA can maintain a stable structure in the cell cytoplasm for up to 12 hours. Therefore, tFNA is expected to become a good drug delivery carrier. However, the method of loading other molecules onto tFNA to achieve a satisfactory loading effect is still a problem that technicians are striving to explore. Summary of the Invention

[0005] In the present invention, the relevant terms have the following meanings: As used herein, "oligonucleotide" refers to a polymer of nucleotide monomers, that is, a linear polynucleotide fragment formed by connecting multiple nucleotide residues through phosphodiester bonds. The oligonucleotides referred to herein can be deoxyribonucleic acid chains (DNA) formed by connecting several deoxynucleotide residues, ribonucleic acid chains (RNA) formed by connecting several ribonucleotide residues, or chimeric nucleic acid chains (DNA / RNA chimeras) formed by connecting several deoxynucleotide residues and several ribonucleotide residues. A DNA / RNA chimera means that any number of ribonucleotide residues and any number of deoxynucleotide residues coexist in the same nucleic acid chain.

[0006] The typical length range of the oligonucleotides referred to herein can be within 100 nucleotide residues (nt), for example, 20 - 70 nucleotide residues.

[0007] When oligonucleotides are represented by a letter sequence, it should be understood that the nucleotides are in the 5'→3' direction from left to right. In this text, unless otherwise specified, "A" represents deoxyadenosine monophosphate, "C" represents deoxycytidine monophosphate, "G" represents deoxyguanosine monophosphate, "T" represents thymidine monophosphate, "a" represents adenosine monophosphate, "c" represents cytidine monophosphate, "g" represents guanosine monophosphate, and "u" represents uridine monophosphate.

[0008] As used herein, "complementary" refers to the ability of two oligonucleotide strands to form base pairs with each other. Base pairs are usually formed by hydrogen bonds between nucleotides in antiparallel oligonucleotide strands. Complementary oligonucleotide strands can form base pairs in the Watson-Crick manner, such as A / a-T / u, C / c-G / g.

[0009] When two oligonucleotides are complementary to form a double strand, it can be two single-stranded DNAs complementary to form a double-stranded structure, two single-stranded RNAs complementary to form a double-stranded structure, a single-stranded DNA and a single-stranded RNA complementary to form a DNA / RNA hybrid double strand, or two DNA / RNA chimeric nucleic acid single strands complementary to form a double strand.

[0010] As used herein, "treatment" includes alleviating or eliminating a medical condition or one or more symptoms or complications associated with the condition, and alleviating or eliminating one or more causes of the condition.

[0011] As used herein, "pharmaceutically acceptable" means that a substance is suitable for contact with the tissues and organs of a subject without undue irritation, allergic reaction, immunogenicity, and toxicity, and the substance has a reasonable benefit-risk ratio. A "pharmaceutically acceptable" carrier or excipient in a pharmaceutical composition or preparation is also compatible with the other components of the composition or preparation.

[0012] In this text, "optional" and "optionally" mean that the matter or event described thereafter may or may not occur, and the description includes the case where the event occurs and the case where the event does not occur.

[0013] Tetrahedral framework nucleic acid One aspect of the present invention provides a tetrahedral framework nucleic acid carrying miR-22.

[0014] The tetrahedral framework nucleic acid provided by the present invention is generally formed by four single-stranded oligonucleotides through base complementarity. Among them, the first, second, and third single-stranded oligonucleotides all have a basic structure in which an RNA segment - a turn segment - a DNA segment - a turn segment - a DNA segment - a turn segment - an RNA segment - a DNA segment are connected in sequence. Specifically: The first single-stranded oligonucleotide is composed of an RNA segment P, a turn segment, a DNA segment 1A, a turn segment, a DNA segment 1B, a turn segment, an RNA segment Q, and a DNA tail segment connected in sequence; The second single-stranded oligonucleotide is composed of an RNA segment P, a turn segment, a DNA segment 2A, a turn segment, a DNA segment 2B, a turn segment, an RNA segment Q, and a DNA tail segment connected in sequence; The third single-stranded oligonucleotide is composed of an RNA segment P, a turn segment, a DNA segment 3A, a turn segment, a DNA segment 3B, a turn segment, an RNA segment Q, and a DNA tail segment connected in sequence.

[0015] The DNA segment 1A in the first single-stranded oligonucleotide is reverse complementary to the DNA segment 3B in the third single-stranded oligonucleotide to form a DNA double strand. The DNA segment 1B in the first single-stranded oligonucleotide is reverse complementary to the DNA segment 2A in the second single-stranded oligonucleotide to form a DNA double strand. The DNA segment 2B in the second single-stranded oligonucleotide is reverse complementary to the DNA segment 3A in the third single-stranded oligonucleotide in sequence to form a DNA double strand, thereby building the central skeleton structure of the tetrahedral framework nucleic acid. The turn segments in the first, second, and third single-stranded oligonucleotides can be composed of one or more nucleotide residues, preferably one nucleotide residue (such as one adenine deoxynucleotide residue, or one cytosine deoxynucleotide residue, or one guanine deoxynucleotide residue, or one thymine deoxynucleotide residue), more preferably one adenine deoxynucleotide residue. When the first, second, and third single-stranded oligonucleotides form the tetrahedral central skeleton structure through base complementarity of the corresponding DNA segments, the turn segments are located at the inner vertices of the tetrahedral skeleton. The bases of adjacent two single-stranded oligonucleotides are not complementary at the corresponding turn segments, which helps the oligonucleotide chains to form a spatial bend at this site.

[0016] From the perspective of forming a tetrahedral framework nucleic acid with appropriate size, the lengths of the above DNA segment 1A, DNA segment 1B, DNA segment 2A, DNA segment 2B, DNA segment 3A, and DNA segment 3B can be 18-24 nucleotide residues (18-24 nt), preferably 18-22 nt, more preferably 19-20 nt, and further preferably 20 nt; the lengths of the above RNA segment P and RNA segment Q can be 7-9 nt, preferably 8 nt.

[0017] The fourth single-stranded oligonucleotide is an RNA strand, which is composed of four segments, that is, the fourth single-stranded oligonucleotide is successively connected by RNA segment 4A, RNA segment 4B, RNA segment 4C, and RNA tail segment. Among them, the lengths of RNA segment 4A and RNA segment 4C can be 7-9 nt, preferably 8 nt. The RNA strand formed by the successive connection of RNA segment 4A, RNA segment 4B, and RNA segment 4C is completely identical or highly similar to miR-22-3p (SEQ ID NO: 5) (that is, it has at least 95%, preferably 100% sequence identity with miR-22-3p). RNA segment 4A in the fourth single-stranded oligonucleotide is reverse complementary to RNA segment Q in the first, second, and third single-stranded oligonucleotides to form an RNA double strand, and RNA segment 4C in the fourth single-stranded oligonucleotide is reverse complementary to RNA segment P in the first, second, and third single-stranded oligonucleotides to form an RNA double strand, thereby loading the fourth single-stranded oligonucleotide onto the central framework structure formed by the first, second, and third single-stranded oligonucleotides.

[0018] The RNA tail segment in the fourth single-stranded oligonucleotide is reverse complementary to the DNA tail segments in the first, second, and third single-stranded oligonucleotides to form a DNA / RNA hybrid double strand that can be cleaved by RNase H. To meet the requirements for RNase H recognition and cleavage, generally, the length of the DNA / RNA hybrid double strand is at least 4 base pairs (bp), such as 4 bp, 5 bp, that is, the length of the DNA tail segments in the first, second, and third single-stranded oligonucleotides or the RNA tail segment in the fourth single-stranded oligonucleotide is at least 4 nucleotide residues (nt), such as 4 nt, 5 nt. The sequence of the DNA tail segments in the first, second, and third single-stranded oligonucleotides can be, for example, TAAG, and the sequence of the RNA tail segment in the fourth single-stranded oligonucleotide can be, for example, cuua.

[0019] Thus, the four single-stranded oligonucleotides form a tetrahedral framework nucleic acid through base complementarity of each segment, and miR-22 (or an RNA strand highly similar to miR-22) is loaded on the central framework of the tetrahedral framework nucleic acid, improving the stability of the RNA strand and preventing the RNA strand from being easily degraded during delivery. The framework nucleic acid formed by the four single-stranded oligonucleotides has a good tetrahedral overall spatial topology structure. In the case of loading miR-22 (or an RNA strand highly similar to miR-22), the tetrahedral framework nucleic acid still maintains structural stability and the property of passing through the cell membrane. Due to the existence of the DNA / RNA hybrid double-stranded region formed by the RNA tail segment of the fourth single-stranded oligonucleotide and the DNA tail segments of the first, second, and third single-stranded oligonucleotides, when the tetrahedral framework nucleic acid reaches the target action position, it can be cleaved by RNase H, and miR-22 (or an RNA strand highly similar to miR-22) is released to exert its biological activity.

[0020] In an exemplary instance of the tetrahedral framework nucleic acid provided by the present invention, the tetrahedral framework nucleic acid is formed by base complementarity of a first single-stranded oligonucleotide with a sequence as shown in SEQ ID NO:1, a second single-stranded oligonucleotide with a sequence as shown in SEQ ID NO:2, a third single-stranded oligonucleotide with a sequence as shown in SEQ ID NO:3, and a fourth single-stranded oligonucleotide with a sequence as shown in SEQ ID NO:2.

[0021] Nucleic acid composition Another aspect of the present invention provides a nucleic acid composition, which includes or consists of a first single-stranded oligonucleotide, a second single-stranded oligonucleotide, a third single-stranded oligonucleotide, and a fourth single-stranded oligonucleotide, wherein: The first single-stranded oligonucleotide is sequentially connected by an RNA segment P, a turn segment, a DNA segment 1A, a turn segment, a DNA segment 1B, a turn segment, an RNA segment Q, and a DNA tail segment; The second single-stranded oligonucleotide is sequentially connected by an RNA segment P, a turn segment, a DNA segment 2A, a turn segment, a DNA segment 2B, a turn segment, an RNA segment Q, and a DNA tail segment; The third single-stranded oligonucleotide is sequentially connected by an RNA segment P, a turn segment, a DNA segment 3A, a turn segment, a DNA segment 3B, a turn segment, an RNA segment Q, and a DNA tail segment; The fourth single-stranded oligonucleotide is sequentially connected by an RNA segment 4A, an RNA segment 4B, an RNA segment 4C, and an RNA tail segment; The sequence of the RNA segment P is reverse complementary to the sequence of the RNA segment 4C, the sequence of the RNA segment Q is reverse complementary to the sequence of the RNA segment 4A, the sequence of the DNA segment 1A is reverse complementary to the sequence of the DNA segment 3B, the sequence of the DNA segment 1B is reverse complementary to the sequence of the DNA segment 2A, the sequence of the DNA segment 2B is reverse complementary to the sequence of the DNA segment 3A, and the sequence of the DNA tail segment is reverse complementary to the sequence of the RNA tail segment; The sequence of the segment formed by sequentially connecting the RNA segment 4A, the RNA segment 4B, and the RNA segment 4C has at least 95%, preferably 100% identity with miR-22-3p.

[0022] In the nucleic acid composition provided by the present invention, the corner segment in the first, second, and third single-stranded oligonucleotides may be composed of one or more nucleotide residues, preferably one nucleotide residue (such as one deoxyadenosine nucleotide residue, or one deoxycytidine nucleotide residue, or one deoxyguanosine nucleotide residue, or one thymidine nucleotide residue), and more preferably one deoxyadenosine nucleotide residue.

[0023] In the nucleic acid composition provided by the present invention, the lengths of DNA segment 1A, DNA segment 1B, DNA segment 2A, DNA segment 2B, DNA segment 3A, and DNA segment 3B may be 18 - 24 nt, preferably 18 - 22 nt, more preferably 19 - 20 nt, and further preferably 20 nt; the lengths of RNA segment P and RNA segment Q may be 7 - 9 nt, preferably 8 nt.

[0024] In the nucleic acid composition provided by the present invention, the lengths of the DNA tails in the first, second, and third single-stranded oligonucleotides are at least 4 nt, such as 4 nt, 5 nt, and their sequences may be, for example, TAAG. The length of the RNA tail in the fourth single-stranded oligonucleotide is at least 4 nt, such as 4 nt, 5 nt, and its sequence may be, for example, cuua.

[0025] In the nucleic acid composition provided by the present invention, the molar ratio of the first, second, third, and fourth single-stranded oligonucleotides is preferably (0.9 - 1.1):(0.9 - 1.1):(0.9 - 1.1):(2.7 - 6.3), more preferably (0.9 - 1.1):(0.9 - 1.1):(0.9 - 1.1):(2.7 - 3.3), and further preferably 1:1:1:3.

[0026] In an exemplary example of the nucleic acid composition provided by the present invention, the sequences of the first, second, third, and fourth single-stranded oligonucleotides are shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively.

[0027] Use of tetrahedral framework nucleic acid The tetrahedral framework nucleic acid provided by the present invention can be used for treating eye diseases, or the tetrahedral framework nucleic acid provided by the present invention can be used for preparing a drug for treating eye diseases.

[0028] Furthermore, the eye diseases include eye diseases associated with abnormal angiogenesis.

[0029] Furthermore, the eye diseases include age-related macular degeneration, diabetic retinopathy, choroidal neovascularization secondary to pathologic myopia, neovascular glaucoma, retinopathy of prematurity, and presumed ocular histoplasmosis syndrome.

[0030] Furthermore, the aforementioned age-related macular degeneration is wet (neovascular) age-related macular degeneration, and the aforementioned diabetic retinopathy is proliferative diabetic retinopathy.

[0031] Pharmaceutical composition or pharmaceutical preparation The present invention also provides a pharmaceutical composition or a pharmaceutical preparation, which contains the tetrahedral framework nucleic acid described in the present invention, and optionally, a pharmaceutically acceptable carrier.

[0032] Examples of pharmaceutically acceptable carriers include solvents, cosolvents, preservatives, osmotic pressure regulators, buffers, stabilizers, pH regulators, wetting agents, emulsifiers, dispersants, excipients, thickeners, release promoters, absorption promoters, humectants, lubricants, semi-solid matrices, etc.

[0033] The pharmaceutical preparation provided by the present invention can be an ophthalmic preparation, such as ophthalmic liquid preparations (eye drops, eye washes, intraocular injections, etc.), ophthalmic semi-solid preparations (eye ointments, ophthalmic creams, ophthalmic gels, etc.), ophthalmic solid preparations (eye membranes, eye pills, intraocular inserts, etc.). The form of the ophthalmic preparation can also be a dry powder, which is packaged in a solid form and provided with a solvent, and is formulated into a solution or suspension before use.

[0034] Furthermore, the pharmaceutical preparation provided by the present invention can be an injection or a dry powder, especially an ophthalmic injection or a dry powder. Preferably, the injection contains the tetrahedral framework nucleic acid of the present invention at a concentration of 0.05 - 5 μM, or the liquid in the used state prepared from the dry powder contains the tetrahedral framework nucleic acid of the present invention at a concentration of 0.05 - 5 μM.

[0035] Furthermore, the pharmaceutical preparation provided by the present invention can be eye drops. Preferably, the eye drops contain the tetrahedral framework nucleic acid of the present invention at a concentration of 0.1 - 10 μM, preferably 0.5 - 5 μM.

[0036] Preparation method of tetrahedral framework nucleic acid The present invention also provides a preparation method of the tetrahedral framework nucleic acid described in the present invention, including the following steps: Prepare a mixed solution containing the first single-stranded oligonucleotide, the second single-stranded oligonucleotide, the third single-stranded oligonucleotide, and the fourth single-stranded oligonucleotide; The above mixed solution is maintained at a temperature sufficient to denature the first single-stranded oligonucleotide, the second single-stranded oligonucleotide, the third single-stranded oligonucleotide, and the fourth single-stranded oligonucleotide for a period of time (for example, maintained for 1 - 20 min, preferably 10 - 15 min) to denature each single-stranded oligonucleotide; Then the temperature is reduced to 2 - 40 °C (for example, 2 - 8 °C, for example, 4 °C) and maintained for a period of time (for example, maintained for more than 1 min, preferably 15 - 30 min) to assemble each single-stranded oligonucleotide into a tetrahedral framework nucleic acid.

[0037] The temperature sufficient to denature each single-stranded oligonucleotide is preferably above 70 °C, such as 75 °C, 80 °C, 85 °C, 90 °C, 95 °C.

[0038] Among them, when preparing the mixed solution, it can be prepared by mixing the solution containing the first single-stranded oligonucleotide, the solution containing the second single-stranded oligonucleotide, the solution containing the third single-stranded oligonucleotide, and the solution containing the fourth single-stranded oligonucleotide, or by mixing the dry powders of the first single-stranded oligonucleotide, the second single-stranded oligonucleotide, the third single-stranded oligonucleotide, and the fourth single-stranded oligonucleotide, and then adding a solvent to dissolve. In the prepared mixed solution, the molar ratio of the first single-stranded oligonucleotide, the second single-stranded oligonucleotide, the third single-stranded oligonucleotide, and the fourth single-stranded oligonucleotide is preferably (0.9 - 1.1):(0.9 - 1.1):(0.9 - 1.1):(2.7 - 6.3), more preferably (0.9 - 1.1):(0.9 - 1.1):(0.9 - 1.1):(2.7 - 3.3), and further preferably 1:1:1:3. In the prepared mixed solution, the concentration of the first, second, and third single-stranded oligonucleotides is preferably 0.25 μM - 10 μM, and the concentration of the fourth single-stranded oligonucleotide is preferably 0.75 μM - 30 μM.

[0039] When preparing the above mixed solution containing each single-stranded oligonucleotide, each single-stranded nucleotide can be dissolved in a buffer solution. Exemplary examples of the buffer solution include TM buffer solution (containing Tris-HCl, MgSO4 or MgCl2), HEPES-NaOH buffer solution, HEPES-KOH buffer solution, Tris-HCl buffer solution, sodium hydrogen phosphate - sodium dihydrogen phosphate buffer solution, etc. The pH of the buffer solution is preferably 7 - 8.

[0040] Effect of the invention The present invention uses tetrahedral framework nucleic acid as a carrier to carry miR-22 with the functions of protecting nerves and anti-angiogenesis, forming a novel nanocomplex. The tetrahedral framework nucleic acid carrying miR-22 has a reasonable spatial topological structure and can efficiently enter cells without the need for additional transfection reagents, solving the problem that miR-22 is difficult to enter cells autonomously. miR-22 is protected because it forms a hybrid chain with the backbone of the tetrahedral framework nucleic acid, can be protected from nuclease degradation during delivery, and can exert the biological activity of miR-22 in a timely manner after being enzymatically cleaved upon reaching the target site of action.

[0041] The tetrahedral framework nucleic acid carrying miR-22 of the present invention is a nucleic acid drug delivery system with high delivery efficiency, good structural stability, and low immunogenicity, providing a new highly efficient, precise, and low-toxicity solution for the treatment of eye diseases. The tetrahedral framework nucleic acid carrying miR-22 of the present invention can be prepared into an injection or eye drops for administration, providing a new practical and highly patient-compliant option for the treatment of eye diseases related to abnormal angiogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The present disclosure will be described in detail with reference to the following drawings according to one or more different embodiments. The provided drawings are for facilitating the understanding of the present disclosure and should not be considered as limiting the breadth, scope, size, or applicability of the present disclosure. For ease of illustration, the drawings are not necessarily drawn to scale.

[0043] Figure 1 It is a transmission electron microscope photograph of an exemplary tetrahedral framework nucleic acid YHK-803 of the present disclosure after successful assembly.

[0044] Figure 2 It is a schematic structural diagram of an exemplary tetrahedral framework nucleic acid YHK-803 of the present disclosure.

[0045] Figure 3 It is a bar graph showing the inhibitory effect of YHK-803 at various concentrations on cell proliferation under hypoxic conditions.

[0046] Figure 4 It is a bar graph showing the inhibitory effect of miR-22, YHK-803, and aflibercept on cell proliferation under hypoxic conditions.

[0047] Figure 5A It is a photograph of the wound healing of each group of cells in the cell scratch experiment, Figure 5B It is a bar graph showing the unhealed proportion of the cell scratches of each group in the cell scratch experiment.

[0048] Figure 6A It is a photograph of the tube structures formed by HUVEC cells of each group in the tube formation experiment in the gel, Figure 6B It is a bar graph showing the length and number of branches of the tubular structures formed by each group of cells in the tube formation experiment. Detailed implementation manners

[0049] The present invention will be further described below in conjunction with specific embodiments and the accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Any modifications, equivalent substitutions or improvements within the spirit and principles of the present invention shall be included within the protection scope of the present invention. The experimental methods in the following embodiments are all conventional methods for molecules, cells and animals unless otherwise specified. The experimental materials used are all purchased from conventional biochemical reagent manufacturers unless otherwise specified.

[0050] Example 1 Preparation of tetrahedral framework nucleic acid YHK-803 Dissolve four single-stranded oligonucleotides YHK-803-S1, YHK-803-S2, YHK-803-S3, miR22-YHK-803 (synthesized by Shanghai Sangon Biotech, and the sequences are shown in Table 1) in TM buffer (50 mM MgCl2·6H2O, 10 mM Tris-HCl, pH 8.0), and fully mix to make a mixed solution. The final concentration of YHK-803-S1 is 1000 nM, and the concentration ratio of YHK-803-S1, YHK-803-S2, YHK-803-S3, miR22-YHK-803 is 1:1:1:3.

[0051] Heat the mixed solution to 95°C and maintain it for 10 minutes to denature the single-stranded oligonucleotides, and then cool it to 4°C at the maximum speed and keep it for 30 minutes. The four single-stranded oligonucleotides are assembled into YHK-803. It is observed by transmission electron microscope (TEM) that the assembled YHK-803 is a tetrahedral particle (attached Figure 1 ). The structure of YHK-803 is as attached Figure 2 shown. Through the base complementarity of each segment, YHK-803-S1, YHK-803-S2, YHK-803-S3 form a tetrahedral central skeleton structure, and miR22-YHK-803 is carried on the central skeleton.

[0052]

[0053] Example 2 Effect of multiple concentrations of YHK-803 on cell proliferation under hypoxic conditions Adopt a hypoxia model of human umbilical vein endothelial cells (Human Umbilical Vein Endothelial Cells, HUVEC), and test the effect of tetrahedral framework nucleic acid YHK-803 at different concentrations on cell proliferation under hypoxic conditions by CCK-8 cell proliferation detection method.

[0054] Experimental method: HUVEC cells were divided into a non-modeling group (C), a hypoxia modeling group (HC), and treatment groups with different concentrations of YHK-803. Each group had 6 replicate wells. After inoculation, HUVEC cells were placed in an incubator and pre-cultured for 24 h at 37°C and 5% CO2. Subsequently, drug administration was carried out: YHK-803 was added to the treatment groups with different concentrations of YHK-803, and the final concentrations were 12.5 nM, 25 nM, 50 nM, 100 nM, 150 nM, 250 nM, 375 nM, and 500 nM respectively. The C and HC groups were added with PBS buffer. After drug administration, the C group maintained the original cell culture conditions, and the cells in the HC group and the treatment groups with different concentrations of YHK-803 were placed in a hypoxia incubator (the oxygen concentration in the incubator was 1.5%) and cultured for 24 h.

[0055] After the treatment of each group of cells was completed, CCK-8 reagent was added to each well at a ratio of 10% of the liquid volume in the well, and the incubation was continued for 2 h. Then, the absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Based on the C group, the relative viability of each group of cells was calculated according to the absorbance value.

[0056] Experimental results: As shown in the appendix Figure 3 The CCK-8 test results showed that the cell viability of the hypoxia modeling group (HC) was significantly increased compared with the non-modeling group (C) (P<0.0001), indicating that the model of promoting HUVEC cell proliferation under hypoxia conditions was successfully established. Compared with the HC group, YHK-803 at 150 nM, 250 nM, 375 nM, and 500 nM could all inhibit cell proliferation caused by hypoxia conditions, and the inhibitory effect became more significant with the increase in concentration (*: P<0.05; ***: P<0.001; ****: P<0.0001).

[0057] Example 3 Effects of miR-22, YHK-803 and aflibercept on cell proliferation under hypoxic conditions Using the HUVEC hypoxia model, the effects of miR-22 (i.e., miR-22-3p, the sequence is shown in Table 2), YHK-803, and aflibercept (AFL) on cell proliferation under hypoxia conditions were tested by the CCK-8 cell proliferation detection method.

[0058]

[0059] HUVEC cells were divided into a non-modeling group (C), a hypoxia modeling group (HC), a miR-22 treatment group, a YHK-803 treatment group, and an aflibercept treatment group, with 6 replicate wells in each group. After inoculation, the HUVEC cells were placed in an incubator and pre-cultured for 24 h under the conditions of 37 °C and 5% CO2. Subsequently, drug administration was carried out: the C group and the HC group were added with PBS buffer, the miR-22 treatment group was added with miR-22 (final concentration 375 nM), the YHK-803 treatment group was added with YHK-803 (final concentration 375 nM), and the aflibercept treatment group was added with aflibercept (Bayer Healthcare Co., Ltd., aflibercept intravitreal injection, stock solution 40 mg / mL, final concentration 100 ng / μl). After drug administration, the C group maintained the original cell culture conditions, and the cells in the HC group, miR-22 treatment group, YHK-803 treatment group, and aflibercept treatment group were placed in a hypoxic incubator (oxygen concentration in the incubator was 1.5%) and cultured for 24 h.

[0060] After the treatment of cells in each group was completed, CCK-8 reagent was added to each well at a ratio of 10% of the liquid volume in the well, and the incubation was continued for 2 h. Then, the absorbance was measured at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Based on the C group as the reference, the relative cell viability of each group was calculated according to the absorbance value.

[0061] Experimental results: As shown in the appendix Figure 4 The CCK-8 detection results showed that the cell viability of the modeling group (HC) was significantly increased compared with that of the non-modeling group (C), indicating that the model of promoting the proliferation of HUVEC cells under hypoxic conditions was successfully established. Compared with the HC group, miRNA-22 (P<0.05), YHK-803 (P<0.01), and aflibercept (P<0.01) could all significantly inhibit the cell proliferation caused by hypoxic conditions, and the cell viability of the YHK-803 treatment group was lower than that of the miRNA-22 group.

[0062] Example 4 Effects of miR-22, YHK-803 and aflibercept on cell migration under hypoxic conditions The scratch assay of HUVEC cells under hypoxic conditions was used to test the effects of miR-22, YHK-803, and aflibercept on cell migration by detecting cell migration.

[0063] Experimental method: HUVEC cells were divided into a non-modeling group (C), a hypoxia modeling group (HC), a miR-22 treatment group, a YHK-803 treatment group, and an aflibercept treatment group, with 2 replicate wells in each group. The cells were plated with a HUVEC cell suspension at a density of 3×10 5 cells / ml. After plating, the cells were placed in an incubator and pre-cultured for 24 h under the conditions of 37 °C and 5% CO2.

[0064] Subsequently, scratching and drug administration were carried out. Three scratches were made in each well, and a total of six scratches were made in each group. Photos were taken immediately after the scratches were made. Immediately afterwards, PBS buffer was added to Group C and Group HC, miR-22 (final concentration 375 nM) was added to the miR-22 treatment group, YHK-803 (final concentration 375 nM) was added to the YHK-803 treatment group, and aflibercept (final concentration 100 ng / μl) was added to the aflibercept treatment group. After drug administration, the cells in Group C were maintained under the original cell culture conditions, and the cells in Group HC, the miR-22 treatment group, the YHK-803 treatment group, and the aflibercept treatment group were placed in a hypoxic incubator (oxygen concentration in the incubator was 1.5%) and cultured for 24 h. Finally, photos were taken of each scratch to analyze the scratch healing situation.

[0065] Experimental results: As shown in Figure 5A and 5B , 24 h after scratching, the cell scratches in the hypoxic model group (HC) were basically healed, and the proportion of the unhealed area was significantly reduced compared with the non-model group (C), indicating that the model of promoting HUVEC cell migration under hypoxic conditions was successfully established. Compared with Group HC, the unhealed areas of the cell scratches in the miR-22 treatment group, the YHK-803 treatment group, and the aflibercept treatment group were significantly increased, indicating that the cell migration induced by hypoxic conditions was inhibited by the drugs (**: P < 0.01; ***: P < 0.001; ****: P < 0.0001).

[0066] Example 5 Effects of miR-22, YHK-803 and aflibercept on tube formation under hypoxic conditions The tube formation experiment under the HUVEC hypoxic model was used to test the effects of miR-22, YHK-803, and aflibercept on tube formation by detecting the tube formation situation.

[0067] Experimental method: HUVEC cells were divided into a non-model group (C), a hypoxic model group (HC), a miR-22 treatment group, a YHK-803 treatment group, and an aflibercept treatment group. HUVEC cells were seeded into 6-well plates at a plating density of 2×10 5 cells / ml. After plating, the cells were placed in an incubator and cultured with DMEM medium at 37 °C and 5% CO2 for 24 h.

[0068] Subsequently, drug administration was carried out for each group: PBS buffer was added to Group C and Group HC, miR-22 (final concentration 375 nM) was added to the miR-22 treatment group, YHK-803 (final concentration 375 nM) was added to the YHK-803 treatment group, and aflibercept (final concentration 100 ng / μl) was added to the aflibercept treatment group. After drug administration, the cells in Group C were maintained under the original cell culture conditions, and the cells in Group HC, the miR-22 treatment group, the YHK-803 treatment group, and the aflibercept treatment group were placed in a hypoxic incubator (oxygen concentration in the incubator was 1.5%) and cultured for 24 h.

[0069] Subsequently, the cells in each group were digested, cell counting was performed, and the cell suspensions of all groups were adjusted to 2×10 5 cells / ml. Then, the cells were seeded into a 96-well plate pre-coated with Matrigel, 100 μl of cell suspension was added to each well, and 8 replicate wells were set for each group. Then, the cells were placed in an incubator and cultured at 37°C and 5% CO2 for 4 h, after which photographs were taken to observe the formation of tubular structures by the cells in each group in Matrigel.

[0070] Among them, the method for pre-coating the 96-well plate with Matrigel was as follows: On ice, the Matrigel stock solution (Corning® Matrigel® Basement Membrane Matrix, high concentration (HC), free of LDEV) was diluted 1-fold with DMEM medium, mixed well, and 50 μl of the diluted Matrigel was added to each well of the 96-well plate. Subsequently, the 96-well plate was allowed to stand at 37°C to complete the coating.

[0071] Experimental results: As shown in Figure 6A , 6B , compared with the non-modeling group (C), the length of the tubular structures formed by HUVEC cells in the hypoxia modeling group (HC) and the number of tube branches both increased, indicating that the hypoxia-induced angiogenesis model of HUVEC cells was successfully established.

[0072] Compared with the HC group, the total length of the tubular structures formed by the miR-22 treatment group, YHK-803 treatment group, and aflibercept treatment group was significantly lower, that is, tube formation was inhibited, and the inhibitory effect of YHK-803 on tube formation was significantly better than that of miR-22 (P<0.01); compared with the HC group, the decrease in the number of tube branches in the miR-22 treatment group did not reach a significant level (P>0.05), the number of tube branches in the YHK-803 treatment group decreased significantly (P<0.001), the number of tube branches in the aflibercept treatment group decreased significantly (P<0.0001), and the inhibitory effect of YHK-803 on hypoxia-induced tube branch formation was significantly better than that of miR-22 (P<0.01).

[0073] Example 6 Effect of YHK-803 on choroidal neovascularization in rats Experimental method: Rats were fully dilated with Mydrin-P (Compound Tropicamide Eye Drops), anesthetized with sodium pentobarbital, the heads of the rats were fixed in front of an ophthalmic laser photocoagulator, and CNV modeling was performed by laser photocoagulation. Six laser spots were irradiated in each eye. The appearance of air bubbles in the fundus indicated that Bruch's membrane was ruptured, suggesting successful photocoagulation. Modeling and detection were both performed on the right eye. The rats were randomly divided into 6 groups, with 6 rats in each group, namely the PBS group, aflibercept group, low-dose YHK-803 group, medium-dose YHK-803 group, high-dose YHK-803 group, and YHK-803 eye drop group.

[0074] On the 7th day after establishing the rat CNV model, fundus fluorescein angiography (FFA) was performed first, and then administration started on the same day (recorded as administration day 0): In the PBS group, 4 μl of PBS buffer was injected intravitreally once. In the low-dose YHK-803 group, 4 μl of YHK-803 injection solution with a concentration of 0.1 μM was injected intravitreally once. In the medium-dose YHK-803 group, 4 μl of YHK-803 injection solution with a concentration of 0.6 μM was injected intravitreally once. In the high-dose YHK-803 group, 4 μl of YHK-803 injection solution with a concentration of 3.6 μM was injected intravitreally once. In the YHK-803 eye drop group, the eyes were instilled with YHK-803 eye drops at a concentration of 1 μM, 20 μL / eye each time, once a day, and instillation continued for 14 days. In the aflibercept group, 4 μl of aflibercept intravitreal injection solution (Bayer Healthcare Pharmaceuticals Inc.) at 40 mg / mL was injected intravitreally once.

[0075] Subsequently, FFA was performed again on the 14th day (i.e., 7 days after administration) and the 21st day (i.e., 14 days after administration) after model establishment.

[0076] Method for FFA detection: The operative eye of the rat was instilled with Mydrin-P (Compound Tropicamide Eye Drops) to dilate the pupil. After anesthesia with sodium pentobarbital, 10% sodium fluorescein injection solution was intraperitoneally injected at a dose of 0.5 mL / kg body weight. Subsequently, fundus angiography was immediately performed, and a retinal area containing 6 laser spots centered on the optic disc was photographed. The picture taken about 15 minutes after injecting sodium fluorescein was selected for fluorescence spot grading and scoring.

[0077] Method for fluorescence spot grading and scoring: Grade 1: No hyperfluorescence appears in the spot; Grade 2: The spot shows hyperfluorescence but no fluorescein leakage; Grade 3: The spot shows hyperfluorescence with mild fluorescein leakage, and the leakage does not exceed the edge of the spot; Grade 4: The spot shows hyperfluorescence with significant fluorescein leakage, and the leakage exceeds the edge of the spot.

[0078] The experimental results are shown in Table 3.

[0079] In the PBS group, obvious laser spots and sodium fluorescein leakage were visible on the 7th, 14th, and 21st days after model establishment, indicating successful establishment of the model.

[0080] Single intravitreal injection of medium or high dose of YHK-803 can effectively inhibit laser-induced choroidal neovascularization, and the drug effect can last for at least 2 weeks. Instillation of YHK-803 eye drops can also effectively inhibit laser-induced choroidal neovascularization.

[0081]

[0082] The experimental results of the above examples show that the tetrahedral framework nucleic acid of the present invention can significantly inhibit the excessive proliferation, migration and tube formation of vascular endothelial cells caused by hypoxia, and inhibit laser-induced choroidal neovascularization.

[0083] The tetrahedral framework nucleic acid of the present invention can complete the delivery of miR-22 without additional transfection reagents, protect miR-22 from degradation during delivery, and exert the biological activity of miR-22 at the target site. Compared with free miR-22, the inhibition effect of miR-22 carried by the tetrahedral framework nucleic acid structure on the proliferation and tube formation of HUVEC cells under hypoxia is significantly better. The tetrahedral framework nucleic acid carrying miR-22 of the present invention can effectively inhibit laser-induced choroidal neovascularization by intravitreal injection in an animal model, and the drug effect can last for at least 2 weeks, providing a good new option for the treatment of ocular diseases related to abnormal neovascularization. Instillation of the tetrahedral framework nucleic acid carrying miR-22 of the present invention can also effectively inhibit laser-induced choroidal neovascularization, providing a practical and effective option with high patient compliance for the treatment of ocular diseases related to abnormal neovascularization, filling the gap in this field.

[0084] Although the features of the present invention have been shown and described in detail with reference to the preferred embodiments, those skilled in the art will understand that other changes can be made therein without departing from the spirit of the scope of the present invention. Similarly, the various figures may depict exemplary architectures or other configurations for the present disclosure, which are used to understand the features and functions that may be included in the present disclosure. The present disclosure is not limited to the exemplary architectures or configurations shown, but can be implemented using various alternative architectures and configurations. Additionally, although the present disclosure has been described above according to various exemplary embodiments and implementations, it should be understood that the various features and functions described in one or more of the individual embodiments are not limited to their description of their applicability to the particular embodiments to which they belong. Instead, they can be applied individually or in some combination to one or more other embodiments of the present disclosure, whether or not such embodiments are described, and whether or not these features are presented as part of the described embodiments. Therefore, the breadth and scope of the present disclosure should not be limited by any of the above exemplary embodiments.

Claims

1. A tetrahedral framework nucleic acid carrying miR-22, which is formed by base complementarity of a first single-stranded oligonucleotide, a second single-stranded oligonucleotide, a third single-stranded oligonucleotide, and a fourth single-stranded oligonucleotide, wherein: The first single-stranded oligonucleotide is sequentially connected by an RNA segment P, a turn segment, a DNA segment 1A, a turn segment, a DNA segment 1B, a turn segment, an RNA segment Q, and a DNA tail segment; The second single-stranded oligonucleotide is sequentially connected by an RNA segment P, a turn segment, a DNA segment 2A, a turn segment, a DNA segment 2B, a turn segment, an RNA segment Q, and a DNA tail segment; The third single-stranded oligonucleotide is sequentially connected by an RNA segment P, a turn segment, a DNA segment 3A, a turn segment, a DNA segment 3B, a turn segment, an RNA segment Q, and a DNA tail segment; The fourth single-stranded oligonucleotide is sequentially connected by an RNA segment 4A, an RNA segment 4B, an RNA segment 4C, and an RNA tail segment; The sequence of the RNA segment P is reverse complementary to the sequence of the RNA segment 4C, the sequence of the RNA segment Q is reverse complementary to the sequence of the RNA segment 4A, the sequence of the DNA segment 1A is reverse complementary to the sequence of the DNA segment 3B, the sequence of the DNA segment 1B is reverse complementary to the sequence of the DNA segment 2A, the sequence of the DNA segment 2B is reverse complementary to the sequence of the DNA segment 3A, and the sequence of the DNA tail segment is reverse complementary to the sequence of the RNA tail segment; The sequence of the segment formed by sequentially connecting the RNA segment 4A, the RNA segment 4B, and the RNA segment 4C has at least 95% identity with miR-22-3p.

2. The tetrahedral framework nucleic acid according to claim 1, wherein the lengths of the DNA segment 1A, the DNA segment 1B, the DNA segment 2A, the DNA segment 2B, the DNA segment 3A, and the DNA segment 3B are 18-24 nucleotide residues; the lengths of the RNA segment P and the RNA segment Q are 7-9 nucleotide residues.

3. The tetrahedral framework nucleic acid according to claim 1 or 2, wherein the turn segment is 1 nucleotide residue.

4. The tetrahedral framework nucleic acid according to claim 1 or 2, wherein the DNA tail segment is a DNA strand with a length of 4 nucleotide residues.

5. The tetrahedral framework nucleic acid according to claim 4, wherein the sequence of the DNA tail segment is TAAG.

6. The tetrahedral framework nucleic acid according to claim 1, wherein the sequences of the first single-stranded oligonucleotide, the second single-stranded oligonucleotide, the third single-stranded oligonucleotide, and the fourth single-stranded oligonucleotide are shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively.

7. A nucleic acid composition, which comprises a first single-stranded oligonucleotide, a second single-stranded oligonucleotide, a third single-stranded oligonucleotide, and a fourth single-stranded oligonucleotide, wherein: The first single-stranded oligonucleotide is composed of an RNA segment P, a turn segment, a DNA segment 1A, a turn segment, a DNA segment 1B, a turn segment, an RNA segment Q, and a DNA tail segment connected in sequence; The second single-stranded oligonucleotide is composed of an RNA segment P, a turn segment, a DNA segment 2A, a turn segment, a DNA segment 2B, a turn segment, an RNA segment Q, and a DNA tail segment connected in sequence; The third single-stranded oligonucleotide is composed of an RNA segment P, a turn segment, a DNA segment 3A, a turn segment, a DNA segment 3B, a turn segment, an RNA segment Q, and a DNA tail segment connected in sequence; The fourth single-stranded oligonucleotide is composed of an RNA segment 4A, an RNA segment 4B, an RNA segment 4C, and an RNA tail segment connected in sequence; The sequence of the RNA segment P is reverse complementary to the sequence of the RNA segment 4C, the sequence of the RNA segment Q is reverse complementary to the sequence of the RNA segment 4A, the sequence of the DNA segment 1A is reverse complementary to the sequence of the DNA segment 3B, the sequence of the DNA segment 1B is reverse complementary to the sequence of the DNA segment 2A, the sequence of the DNA segment 2B is reverse complementary to the sequence of the DNA segment 3A, and the sequence of the DNA tail segment is reverse complementary to the sequence of the RNA tail segment; The sequence of the segment formed by connecting the RNA segment 4A, the RNA segment 4B, and the RNA segment 4C in sequence has at least 95% identity with miR-22-3p.

8. The nucleic acid composition according to claim 7, wherein the sequences of the first single-stranded oligonucleotide, the second single-stranded oligonucleotide, the third single-stranded oligonucleotide, and the fourth single-stranded oligonucleotide are shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively.

9. The nucleic acid composition according to claim 7 or 8, wherein The molar ratio of the first single-stranded oligonucleotide, the second single-stranded oligonucleotide, the third single-stranded oligonucleotide, and the fourth single-stranded oligonucleotide in the nucleic acid composition is (0.9 - 1.1):(0.9 - 1.1):(0.9 - 1.1):(2.7 - 6.3).

10. Use of the tetrahedral framework nucleic acid according to any one of claims 1 - 6 or the nucleic acid composition according to any one of claims 7 - 9 in the preparation of a drug for treating eye diseases.

11. The use according to claim 10, wherein the eye disease is an eye disease associated with abnormal angiogenesis.

12. The use according to claim 10, wherein the eye disease is selected from wet age-related macular degeneration, proliferative diabetic retinopathy, choroidal neovascularization secondary to pathologic myopia, neovascular glaucoma, retinopathy of prematurity, and ocular histoplasmosis syndrome.

13. A pharmaceutical preparation, which comprises the tetrahedral framework nucleic acid according to any one of claims 1 - 6, and optionally a pharmaceutically acceptable carrier.

14. The pharmaceutical preparation according to claim 13, wherein the pharmaceutical preparation is an ophthalmic preparation.

15. The pharmaceutical preparation according to claim 14, wherein the pharmaceutical preparation is an injection or a dry powder.

16. The pharmaceutical preparation according to claim 15, wherein the injection contains the tetrahedral framework nucleic acid at a concentration of 0.05 - 5 μM, or the liquid in the state of use prepared from the dry powder contains the tetrahedral framework nucleic acid at a concentration of 0.05 - 5 μM.

17. The pharmaceutical preparation according to claim 14, wherein the pharmaceutical preparation is an eye drop.

18. The pharmaceutical preparation according to claim 17, wherein the eye drop contains the tetrahedral framework nucleic acid at a concentration of 0.1 - 10 μM.

19. A method for preparing the tetrahedral framework nucleic acid according to any one of claims 1 - 6, comprising the following steps: Preparing a mixed solution containing the first single-stranded oligonucleotide, the second single-stranded oligonucleotide, the third single-stranded oligonucleotide, and the fourth single-stranded oligonucleotide; Maintaining the mixed solution at a temperature sufficient to denature the first single-stranded oligonucleotide, the second single-stranded oligonucleotide, the third single-stranded oligonucleotide, and the fourth single-stranded oligonucleotide for 1 - 20 min, and then lowering the temperature to 2 - 8°C and maintaining for more than 1 min.

20. The method according to claim 19, wherein, Dissolving the first single-stranded oligonucleotide, the second single-stranded oligonucleotide, the third single-stranded oligonucleotide, and the fourth single-stranded oligonucleotide in TM buffer to form a mixed solution, and the molar ratio of the first single-stranded oligonucleotide, the second single-stranded oligonucleotide, the third single-stranded oligonucleotide, and the fourth single-stranded oligonucleotide in the formed mixed solution is (0.9 - 1.1):(0.9 - 1.1):(0.9 - 1.1):(2.7 - 6.3); Maintaining the mixed solution at 95°C for 10 - 15 min, and then lowering the temperature to 4°C and maintaining for 15 - 30 min.

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