Tetrahedral framework nucleic acid related to mir-22, and preparation method therefor and use thereof

Abstract

Provided in the present invention are a tetrahedral framework nucleic acid related to miR-22, and a preparation method therefor and the use thereof. The tetrahedral framework nucleic acid is formed by means of base complementation of a first single-stranded oligonucleotide, a second single-stranded oligonucleotide, a third single-stranded oligonucleotide and a fourth single-stranded oligonucleotide. The tetrahedral framework nucleic acid of the present invention is a nucleic acid drug delivery system with high delivery efficiency, good structural stability and low immunogenicity, providing a new approach for the treatment of ocular diseases with high efficacy, precision, and low toxicity.

Description

A tetrahedral framework nucleic acid related to miR-22, its preparation method and uses

[0001] Cross-references to related applications

[0002] This application claims the benefit of Chinese Patent Application No. 202510349315.5, filed on March 24, 2025, the contents of which are incorporated herein by reference in their entirety.

[0003] Cross-references to related applications

[0004] The contents of the electronic sequence list (CYTB_002_01WO_SeqList_ST26.xml; size: 10,298 bytes; and creation date: March 16, 2026) are incorporated herein by reference in their entirety. Technical Field

[0005] This invention relates to the field of nucleic acid technology, and further to a tetrahedral framework nucleic acid related to miR-22, its preparation method, and its uses. Background Technology

[0006] Fundus diseases refer to a series of diseases occurring in the structures at the back of the eyeball, involving key areas such as the retina, optic nerve, and choroid. These diseases can severely affect visual function. Among them, age-related macular degeneration (AMD) is a blinding eye disease characterized by degenerative changes in the macular region, primarily affecting people over 50 years of age, and is the leading cause of irreversible vision loss in middle-aged and elderly people 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 drusen deposition; the disease progresses slowly but can develop into late-stage atrophy. Wet AMD is caused by abnormal proliferation of choroidal neovascularization (CNV), accompanied by hemorrhage, exudation, and scar formation, which can lead to a rapid decline in vision. Common symptoms include blurred central vision, distorted vision (such as distorted lines), and color vision abnormalities; in advanced stages, permanent loss of central vision may occur. The etiology of AMD is not fully understood, but it is currently believed to be the result of a combination of genetic, environmental, and metabolic factors. Oxidative stress, inflammatory response, and abnormal choroidal blood flow are the core pathological mechanisms of the disease. Currently, there is no specific treatment for dry AMD; treatment primarily involves supplementing with antioxidants (such as the AREDS2 formula) to slow progression. The first-line treatment for wet AMD is intravitreal injection of anti-vascular endothelial growth factor (anti-VEGF) drugs (such as ranibizumab and aflibercept), which can effectively inhibit angiogenesis and improve vision. However, treatment requires long-term, repeated injections, and some patients experience drug resistance or financial burden. Therefore, a new therapy is urgently needed to fill the current treatment gap.

[0007] MicroRNAs (miRNAs, miRs) are a class of endogenous non-coding RNA molecules approximately 20-24 nucleotides in length, playing a variety of important regulatory roles within cells. In recent years, miRNA therapy has attracted significant attention in the field of nucleic acid drug research. miRNA therapy involves using miRNA mimics for miRNA replacement therapy or using miRNA inhibitors to suppress miRNA function. miRNA molecules are easily degraded by nucleases in blood and tissues, and their negative charge makes it difficult for them to enter cells independently; therefore, the implementation of miRNA therapy relies on a suitable delivery system. Existing miRNA delivery systems, such as lipid nanoparticles, polymer nanoparticles, and viral vectors, suffer from several drawbacks, including insufficient delivery efficiency, immunogenicity or cytotoxicity, unclear metabolic pathways, poor stability, and complex manufacturing processes. These limitations restrict the application of miRNA therapy.

[0008] Tetrahedral framework nucleic acid (tFNA) is a novel nucleic acid nanomaterial that can freely penetrate cell membranes and enter cells, exhibiting stability and biocompatibility in the complex serum environment. tFNA enters cells via caveolin-mediated endocytosis, then undergoes microtubule-dependent intracellular transport, finally being transported in an orderly manner to lysosomes. Furthermore, tFNA can maintain a stable structure in the cytoplasm for up to 12 hours. Therefore, tFNA shows promise as a good drug delivery carrier. However, finding effective methods to mount other molecules onto tFNA to achieve satisfactory delivery results remains a challenge that researchers are still exploring. Summary of the Invention

[0009] definition

[0010] In this invention, the relevant terms have the following meanings:

[0011] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0012] As used herein, unless otherwise indicated, the term "or" can be a conjunction or a disjunctive conjunction. As used herein, unless otherwise indicated, any embodiment may be combined with any other embodiment.

[0013] As used herein, unless otherwise indicated, certain application embodiments herein envision a range of values. Where a range exists, the range includes the range endpoints. Furthermore, each subrange and value within that range exists as if explicitly written.

[0014] As used herein, the singular form (e.g., “a”, “an”) includes plural indicators unless the context clearly indicates otherwise.

[0015] As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized in,” and is inclusive or open-ended, and does not exclude additional unstated components, elements, or method steps, etc. As used herein, “consisting of” excludes any component, element, or method step not specified in the elements of the claim. As used herein, “consisting substantially of” does not exclude components, elements, or method steps that do not substantially affect the essential and novel features of the claim. Any statement of the term “comprising” herein, such as when describing compositional components or method steps, should be understood to include (i) those compositions and methods consisting of the stated components, elements, or method steps; and (ii) those compositions and methods substantially consisting of the stated components, elements, or method steps.

[0016] The term "oligonucleotide" as used in this article refers to a polymer of nucleotide monomers, specifically a linear polynucleotide fragment composed of multiple nucleotide residues linked by phosphodiester bonds. The oligonucleotides referred to in this article can be deoxyribonucleic acid (DNA) chains formed by the linkage of several deoxynucleotide residues, ribonucleic acid (RNA) chains formed by the linkage of several ribonucleotide residues, or chimeric nucleic acid chains (DNA / RNA chimeras) formed by the linkage of 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.

[0017] The oligonucleotides mentioned in this article typically range in length from 100 nucleotide residues (nt), for example, 20-70 nucleotide residues.

[0018] When oligonucleotides are represented by a letter sequence, it should be understood that the nucleotides are arranged in a 5'→3' direction from left to right. In this article, unless otherwise specified, "A" represents adenine deoxynucleotide, "C" represents cytosine deoxynucleotide, "G" represents guanine deoxynucleotide, "T" represents thymine deoxynucleotide, "a" represents adenine ribonucleotide, "c" represents cytosine ribonucleotide, "g" represents guanine ribonucleotide, and "u" represents uracil ribonucleotide.

[0019] The term "complementarity" as used in this article refers to the ability of two oligonucleotide chains to form base pairs. Base pairs are typically formed between nucleotides in antiparallel oligonucleotide chains via hydrogen bonds. Complementary oligonucleotide chains can form base pairs in a Watson-Crick manner, such as A / aT / u, C / cG / g.

[0020] When two oligonucleotides complement each other to form a double strand, it can be two single-stranded DNAs complementing each other to form a double-stranded structure, two single-stranded RNAs complementing each other to form a double-stranded structure, one single-stranded DNA and one single-stranded RNA complementing each other to form a DNA / RNA hybrid double strand, or two DNA / RNA chimeric nucleic acid single strands complementing each other to form a double strand.

[0021] The term "treatment" as used in this article includes the relief or elimination of a medical condition or one or more symptoms or complications associated with that condition, as well as the relief or elimination of one or more causes of that condition.

[0022] As used herein, "pharmaceutically acceptable" means that a substance is suitable for contact with the tissues and organs of a subject without excessive irritation, allergic reactions, immunogenicity, or toxicity, and that the substance has a reasonable benefit-risk ratio commensurate with this. "Pharmaceutically acceptable" carriers or excipients in pharmaceutical compositions or formulations are also compatible with other components of the composition or formulation.

[0023] In this article, "optional" or "optionally" means that the matter or event described thereafter may or may not occur, and the description includes both the circumstances under which the event occurs and the circumstances under which the event does not occur.

[0024] Synopsis

[0025] In one aspect, the present invention provides a tetrahedral framework nucleic acid carrying miR-22, wherein the tetrahedral framework nucleic acid 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. In some embodiments, a single tetrahedral framework nucleic acid carrying miR-22 comprises one first single-stranded oligonucleotide, one second single-stranded oligonucleotide, one third single-stranded oligonucleotide, and three fourth single-stranded oligonucleotides. In some embodiments, the three fourth single-stranded oligonucleotides have the same nucleotide sequence.

[0026] In another aspect, the present invention provides a nucleic acid composition, wherein the tetrahedral framework nucleic acid 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.

[0027] In some embodiments, the first single-stranded oligonucleotide is formed by sequentially connecting RNA segment P, a turn segment, DNA segment 1A, a turn segment, DNA segment 1B, a turn segment, RNA segment Q, and a DNA tail segment; the second single-stranded oligonucleotide is formed by sequentially connecting the RNA segment P, a turn segment, DNA segment 2A, a turn segment, DNA segment 2B, a turn segment, RNA segment Q, and the DNA tail segment; the third single-stranded oligonucleotide is formed by sequentially connecting the RNA segment P, a turn segment, DNA segment 3A, a turn segment, DNA segment 3B, a turn segment, RNA segment Q, and the DNA tail segment; and the fourth single-stranded oligonucleotide is formed by sequentially connecting RNA segment 4A, RNA segment 4B, RNA segment 4C, and an RNA tail segment. In some embodiments, the RNA segment P is anticomplementary to the RNA segment 4C, the RNA segment Q is anticomplementary to the RNA segment 4A, the DNA segment 1A is anticomplementary to the DNA segment 3B, the DNA segment 1B is anticomplementary to the DNA segment 2A, the DNA segment 2B is anticomplementary to the DNA segment 3A, and the DNA tail is anticomplementary to the RNA tail. In some embodiments, the sequence of the segment formed by sequentially connecting the RNA segments 4A, 4B, and 4C has at least 95% identity with miR-22-3p.

[0028] In some embodiments, the DNA segments 1A, 1B, 2A, 2B, 3A, and 3B are 18-24 nucleotide residues in length, preferably 19 or 20 nucleotide residues. In some embodiments, the RNA segments P and Q are 7-9 nucleotide residues in length, preferably 8 nucleotide residues.

[0029] In some embodiments, the DNA tail is a DNA strand of 4 nucleotide residues in length. In some embodiments, the sequence of the DNA tail is TAAG.

[0030] The corner segments may be the same or different. In some embodiments, each corner segment is DNA. In some embodiments, each corner segment is 1 nucleotide residue in length. In some embodiments, each corner segment has the sequence A.

[0031] In some embodiments, the sequences of the first single-chain oligonucleotide, the second single-chain oligonucleotide, the third single-chain oligonucleotide, and the fourth single-chain oligonucleotide are shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively.

[0032] In some embodiments, the sequence formed by sequentially linking RNA segment 4A, RNA segment 4B, and RNA segment 4C has 100% identity with SEQ ID NO:5. In some embodiments, the sequence of RNA segment 4A is aagcugcc, the sequence of RNA segment 4B is aguuga, and the sequence of RNA segment 4C is agaacugu.

[0033] In some embodiments, the sequence of the segment formed by sequentially connecting RNA segment 4A, RNA segment 4B, and RNA segment 4C has a mutation (substitution, deletion, or addition) of up to one nucleotide compared to SEQ ID NO:5. In some embodiments, the sequence of RNA segment 4A is aagcugcc. In some embodiments, RNA segment 4B is aguuga. In some embodiments, the sequence of RNA segment 4C is agaacugu.

[0034] In some embodiments, the sequence of the RNA tail segment is cuua.

[0035] In some embodiments, the molar ratio of the first single-chain oligonucleotide, the second single-chain oligonucleotide, the third single-chain oligonucleotide, and the fourth single-chain oligonucleotide in the nucleic acid composition is (0.9-1.1):(0.9-1.1):(0.9-1.1):(2.7-6.3).

[0036] In another aspect, the present invention provides a pharmaceutical formulation comprising the tetrahedral framework nucleic acid or nucleic acid composition described herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation is an ophthalmic formulation. In some embodiments, the pharmaceutical formulation is an injection or a dry powder. In some embodiments, the injection contains 0.05-5 μM of the tetrahedral framework nucleic acid. In some embodiments, the dry powder, when formulated into a liquid in its usable state, contains 0.05-5 μM of the tetrahedral framework nucleic acid. In some embodiments, the pharmaceutical formulation is an eye drop. In some embodiments, the eye drop contains 0.1-10 μM of the tetrahedral framework nucleic acid. In some embodiments, the pharmaceutical formulation does not contain a transfection reagent.

[0037] In another aspect, the present invention provides the use of the tetrahedral framework nucleic acid or nucleic acid composition described herein in the preparation of a medicament for treating ocular lesions.

[0038] In another aspect, the present invention provides a method for treating ocular lesions, comprising administering to a patient in need the tetrahedral framework nucleic acid, nucleic acid composition, or pharmaceutical preparation described herein.

[0039] In some embodiments, the ocular lesion is an ocular lesion associated with abnormal angiogenesis. In some embodiments, the ocular lesion is selected from wet age-related macular degeneration, proliferative diabetic retinopathy, choroidal neovascularization secondary to pathological myopia, neovascular glaucoma, retinopathy of prematurity, and ocular pseudohistoplasmosis syndrome.

[0040] In some embodiments, the administration method comprises treatment by eye drops. In some embodiments, the administration method comprises intravitreal injection. In some embodiments, the administration method comprises a single intravitreal injection.

[0041] In another aspect, the present invention provides a method for preparing the tetrahedral framework nucleic acid described herein, comprising the following steps: preparing a mixed solution containing a first single-stranded oligonucleotide, a second single-stranded oligonucleotide, a third single-stranded oligonucleotide, and a fourth single-stranded oligonucleotide; placing 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 it for at least 1 min. In some embodiments, the method comprises dissolving the first single-stranded oligonucleotide, the second single-stranded oligonucleotide, the third single-stranded oligonucleotide, and the fourth single-stranded oligonucleotide in a buffer solution to prepare a mixed solution. In some embodiments, the buffer solution is a TM buffer (containing Tris-HCl, MgSO4, or MgCl2). In some embodiments, 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 prepared mixed solution is (0.9-1.1):(0.9-1.1):(0.9-1.1):(2.7-6.3). In some embodiments, the method includes placing the mixed solution at approximately 95°C for 10-15 minutes, and then lowering the temperature to approximately 4°C for 15-30 minutes.

[0042] Tetrahedral framework nucleic acid

[0043] One aspect of the present invention provides a tetrahedral framework nucleic acid carrying miR-22.

[0044] The tetrahedral framework nucleic acid provided by this invention is typically formed by four single-stranded oligonucleotides through base complementarity. Specifically, the first, second, and third single-stranded oligonucleotides all have a basic structure consisting of a sequentially linked RNA segment-corner segment-DNA segment-corner segment-DNA segment-corner segment-RNA segment-DNA segment.

[0045] The first single-stranded oligonucleotide is composed of RNA segment P, corner segment, DNA segment 1A, corner segment, DNA segment 1B, corner segment, RNA segment Q, and DNA tail segment linked together in sequence.

[0046] The second single-stranded oligonucleotide is composed of RNA segment P, turn segment, DNA segment 2A, turn segment, DNA segment 2B, turn segment, RNA segment Q, and DNA tail segment linked together in sequence.

[0047] The third single-stranded oligonucleotide is composed of RNA segment P, turn segment, DNA segment 3A, turn segment, DNA segment 3B, turn segment, RNA segment Q, and DNA tail segment linked together in sequence.

[0048] DNA segment 1A in the first single-stranded oligonucleotide and DNA segment 3B in the third single-stranded oligonucleotide are anticomplementary to form a DNA double strand. DNA segment 1B in the first single-stranded oligonucleotide and DNA segment 2A in the second single-stranded oligonucleotide are anticomplementary to form a DNA double strand. The sequences of DNA segment 2B in the second single-stranded oligonucleotide and DNA segment 3A in the third single-stranded oligonucleotide are anticomplementary to form a DNA double strand, thereby constructing the central backbone structure of the tetrahedral framework nucleic acid. The corner segments in the first, second, and third single-stranded oligonucleotides can be composed of one or more nucleotide residues, preferably one nucleotide residue, preferably a deoxynucleotide residue (e.g., one adenine deoxynucleotide residue, or one cytosine deoxynucleotide residue, or one guanine deoxynucleotide residue, or one thymine deoxynucleotide residue), and more preferably one adenine deoxynucleotide residue. When the first, second, and third single-stranded oligonucleotides form the tetrahedral central backbone structure through base complementarity of the corresponding DNA segments, the corner segments are located at the inner vertices of the tetrahedral backbone. The bases of two adjacent oligonucleotide single strands are not complementary at the corresponding turn segments, which helps the oligonucleotide chain to form a spatial bend at that location.

[0049] From the perspective of forming a tetrahedral framework nucleic acid of suitable size, the lengths of the aforementioned DNA segments 1A, 1B, 2A, 2B, 3A, and 3B can be 18-24 nucleotide residues (18-24 nt), preferably 18-22 nt, more preferably 19-20 nt, and even more preferably 20 nt; the lengths of the aforementioned RNA segments P and Q can be 7-9 nt, preferably 8 nt. In some embodiments, the lengths of the first, second, and third single-stranded oligonucleotides are all 57-69 nt, preferably 60-66 nt, more preferably 61-65 nt, more preferably 62-64 nt, and even more preferably 63 nt.

[0050] The fourth single-stranded oligonucleotide is an RNA chain composed of four segments: RNA segment 4A, RNA segment 4B, RNA segment 4C, and an RNA tail segment, linked sequentially. The lengths of RNA segments 4A and 4C can be 7-9 nt, preferably 8 nt. The RNA chain formed by the sequential linking of RNA segments 4A, 4B, and 4C is completely identical to or highly similar to miR-22-3p (SEQ ID NO:5) (i.e., possessing at least 95%, preferably 100%, sequence identity with miR-22-3p). RNA segment 4A in the fourth single-stranded oligonucleotide is anticomplementary to RNA segment Q in the first, second, and third single-stranded oligonucleotides, forming an RNA double strand. Similarly, RNA segment 4C in the fourth single-stranded oligonucleotide is anticomplementary to RNA segment P in the first, second, and third single-stranded oligonucleotides, forming an RNA double strand. This allows the fourth single-stranded oligonucleotide to be mounted on the central backbone structure formed by the first, second, and third single-stranded oligonucleotides.

[0051] The RNA tail in the fourth single-stranded oligonucleotide is anticomplementary to the DNA tails in the first, second, and third single-stranded oligonucleotides, forming a DNA / RNA hybrid double strand that can be cleaved by RNase H. To meet the requirements for RNase H recognition and cleavage, the length of the DNA / RNA hybrid double strand is typically at least 4 base pairs (bp), for example, 4bp or 5bp; that is, the length of the DNA tails in the first, second, and third single-stranded oligonucleotides or the RNA tail in the fourth single-stranded oligonucleotide is at least 4 nucleotide residues (nt), for example, 4nt or 5nt. The sequences of the DNA tails in the first, second, and third single-stranded oligonucleotides can be, for example, TAAG, and the sequence of the RNA tail in the fourth single-stranded oligonucleotide can be, for example, cuua. In some embodiments, the total length of RNA segment 4A, RNA segment 4B, and RNA segment 4C is 22nt. In some embodiments, the length of the fourth single-stranded oligonucleotide is 26nt.

[0052] Thus, four single-stranded oligonucleotides form a tetrahedral framework nucleic acid through base complementarity at each segment. miR-22 (or an RNA strand highly similar to miR-22) is mounted on the central backbone of the tetrahedral framework nucleic acid, improving the stability of the RNA strand and preventing its easy degradation during delivery. The framework nucleic acid formed by the four single-stranded oligonucleotides has a good tetrahedral overall spatial topology. Even with miR-22 (or an RNA strand highly similar to miR-22) mounted, the tetrahedral framework nucleic acid still maintains structural stability and the ability to pass through the cell membrane. Due to the presence of the DNA / RNA hybrid double-stranded region formed by the RNA tail of the fourth single-stranded oligonucleotide and the DNA tails of the first, second, and third single-stranded oligonucleotides, the tetrahedral framework nucleic acid can be cleaved by RNase H upon reaching the target site, releasing miR-22 (or an RNA strand highly similar to miR-22) to exert its biological activity.

[0053] In some embodiments, the RNA segments P, Q, and DNA tails of the first, second, and third single-stranded oligonucleotides and the RNA segments 4A, 4C, and RNA tails of the fourth single-stranded oligonucleotide are anticomplementary to form three edges of a triangular face of a tetrahedral framework, and the DNA segments of the first, second, and third single-stranded oligonucleotides are anticomplementary to form the remaining three edges of the tetrahedral framework.

[0054] In some embodiments, the RNA segment 4B of the fourth single-stranded oligonucleotide, and / or the turn segments of the first, second, and third single-stranded oligonucleotides, do not participate in the complementary pairing of the tetrahedral framework nucleic acids.

[0055] In some embodiments, the 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 tetrahedral framework nucleic acid is 1:1:1:3.

[0056] In an exemplary example 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 the sequence shown in SEQ ID NO:1, a second single-stranded oligonucleotide with the sequence shown in SEQ ID NO:2, a third single-stranded oligonucleotide with the sequence shown in SEQ ID NO:3, and a fourth single-stranded oligonucleotide with the sequence shown in SEQ ID NO:4.

[0057] Nucleic acid composition

[0058] Another aspect of the present invention provides a nucleic acid composition comprising a first single-stranded oligonucleotide, a second single-stranded oligonucleotide, a third single-stranded oligonucleotide, and a fourth single-stranded oligonucleotide, or composed of the first single-stranded oligonucleotide, the second single-stranded oligonucleotide, the third single-stranded oligonucleotide, and the fourth single-stranded oligonucleotide, wherein:

[0059] The first single-stranded oligonucleotide is composed of RNA segment P, corner segment, DNA segment 1A, corner segment, DNA segment 1B, corner segment, RNA segment Q, and DNA tail segment linked together in sequence.

[0060] The second single-stranded oligonucleotide is composed of RNA segment P, turn segment, DNA segment 2A, turn segment, DNA segment 2B, turn segment, RNA segment Q, and DNA tail segment linked together in sequence.

[0061] The third single-stranded oligonucleotide is composed of RNA segment P, turn segment, DNA segment 3A, turn segment, DNA segment 3B, turn segment, RNA segment Q, and DNA tail segment linked together in sequence;

[0062] The fourth single-stranded oligonucleotide is composed of RNA segment 4A, RNA segment 4B, RNA segment 4C, and RNA tail segment linked together in sequence;

[0063] The RNA segment P is inversely complementary to the RNA segment 4C; the RNA segment Q is inversely complementary to the RNA segment 4A; the DNA segment 1A is inversely complementary to the DNA segment 3B; the DNA segment 1B is inversely complementary to the DNA segment 2A; the DNA segment 2B is inversely complementary to the DNA segment 3A; and the DNA tail is inversely complementary to the RNA tail.

[0064] 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.

[0065] In some embodiments, the lengths of the first single-stranded oligonucleotide, the second single-stranded oligonucleotide, and the third single-stranded oligonucleotide are all 57-69 nt, preferably 60-66 nt, more preferably 61-65 nt, more preferably 62-64 nt, and even more preferably 63 nt.

[0066] In some embodiments, the total length of RNA segment 4A, RNA segment 4B, and RNA segment 4C is 22 nt. In some embodiments, the length of the fourth single-stranded oligonucleotide is 26 nt.

[0067] In the nucleic acid composition provided by the present invention, the turn segments in the first, second, and third single-stranded oligonucleotides may be composed of one or more nucleotide residues, preferably one nucleotide residue, more preferably a deoxynucleotide residue (e.g., one adenine deoxynucleotide residue, or one cytosine deoxynucleotide residue, or one guanine deoxynucleotide residue, or one thymine deoxynucleotide residue), and more preferably one adenine deoxynucleotide residue.

[0068] 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 can be 18-24 nt, preferably 18-22 nt, more preferably 19-20 nt, and even more preferably 20 nt; the lengths of RNA segment P and RNA segment Q can be 7-9 nt, preferably 8 nt.

[0069] In the nucleic acid composition provided by this invention, the DNA tail segments in the first, second, and third single-stranded oligonucleotides are at least 4 nt in length, for example, 4 nt or 5 nt, and their sequences may be, for example, TAAG. The RNA tail segment in the fourth single-stranded oligonucleotide is at least 4 nt in length, for example, 4 nt or 5 nt, and its sequence may be, for example, cuua.

[0070] In the nucleic acid composition provided by the present invention, 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 even more preferably 1:1:1:3.

[0071] In an exemplary example of the nucleic acid composition provided by the present invention, 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 as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively.

[0072] Applications of tetrahedral framework nucleic acids

[0073] The tetrahedral framework nucleic acid or nucleic acid composition provided by the present invention can be used to treat eye diseases, or the tetrahedral framework nucleic acid or nucleic acid composition provided by the present invention can be used to prepare drugs for treating eye diseases.

[0074] Furthermore, ocular lesions include those associated with abnormal angiogenesis.

[0075] Furthermore, ocular lesions include age-related macular degeneration, diabetic retinopathy, choroidal neovascularization secondary to pathological myopia, neovascular glaucoma, retinopathy of prematurity, and ocular pseudohistomycosis syndrome.

[0076] Furthermore, the aforementioned age-related macular degeneration is wet (neovascular) age-related macular degeneration, and the aforementioned diabetic retinopathy is proliferative diabetic retinopathy.

[0077] Pharmaceutical composition or pharmaceutical preparation

[0078] The present invention also provides a pharmaceutical composition or pharmaceutical formulation comprising the tetrahedral framework nucleic acid or nucleic acid composition described herein, and optionally, a pharmaceutically acceptable carrier.

[0079] Examples of pharmaceutically acceptable carriers include solvents, cosolvents, preservatives, osmotic pressure regulators, buffers, stabilizers, pH adjusters, wetting agents, emulsifiers, dispersants, excipients, thickeners, release promoters, absorption enhancers, humectants, lubricants, and semi-solid matrices.

[0080] The pharmaceutical preparations provided by this invention can be ophthalmic preparations, such as ophthalmic liquid preparations (eye drops, eye washes, intraocular injections, etc.), ophthalmic semi-solid preparations (ophthalmic ointments, ophthalmic creams, ophthalmic gels, etc.), and ophthalmic solid preparations (ophthalmic films, ophthalmic pills, intraocular inserts, etc.). The ophthalmic preparations can also be in the form of dry powder, packaged in a solid state, with a separate solvent to prepare a solution or suspension before use.

[0081] Furthermore, the pharmaceutical formulation provided by the present invention can be an injection solution or a dry powder, particularly an ophthalmic injection solution or dry powder. Preferably, the injection solution contains 0.05-5 μM of the tetrahedral framework nucleic acid of the present invention, or the liquid in use prepared from the dry powder contains 0.05-5 μM of the tetrahedral framework nucleic acid of the present invention.

[0082] Furthermore, the pharmaceutical preparation provided by the present invention can be an eye drop. Preferably, the eye drop contains 0.1-10 μM, more preferably 0.5-5 μM, of the tetrahedral framework nucleic acid of the present invention.

[0083] Preparation methods of tetrahedral framework nucleic acids

[0084] This invention also provides a method for preparing the tetrahedral framework nucleic acid described herein, comprising the following steps:

[0085] Prepare a mixed solution containing the first single-chain oligonucleotide, the second single-chain oligonucleotide, the third single-chain oligonucleotide, and the fourth single-chain oligonucleotide;

[0086] The above mixed solution is placed 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 (e.g., 1-20 min, preferably 10-15 min) to denature each single-stranded oligonucleotide.

[0087] Then the temperature is lowered to 2-40℃ (e.g., 2-8℃, e.g., 4℃) and maintained for a period of time (e.g., for more than 1 minute, preferably 15-30 minutes) to allow the single-stranded oligonucleotides to assemble into a tetrahedral framework nucleic acid.

[0088] The temperature at which the single-stranded oligonucleotides are denatured is preferably above 70°C, for example 75°C, 80°C, 85°C, 90°C, or 95°C.

[0089] The mixed solution can be prepared by mixing solutions containing a first single-chain oligonucleotide, a second single-chain oligonucleotide, a third single-chain oligonucleotide, and a fourth single-chain oligonucleotide, or by mixing dry powders of the first, second, third, and fourth single-chain oligonucleotides and then dissolving them in a solvent. In the prepared mixed solution, the molar ratio of the first, second, third, and fourth single-chain 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 even more preferably 1:1:1:3. In the prepared mixed solution, the concentrations of the first, second, and third single-stranded oligonucleotides are preferably 0.25 μM-10 μM, and the concentration of the fourth single-stranded oligonucleotide is preferably 0.75 μM-30 μM.

[0090] When preparing the above-mentioned mixed solution containing each single-stranded oligonucleotide, each single-stranded oligonucleotide can be dissolved in a buffer solution. Exemplary examples of buffer solutions include TM buffer (containing Tris-HCl, MgSO4, or MgCl2), HEPES-NaOH buffer, HEPES-KOH buffer, Tris-HCl buffer, sodium monohydrogen phosphate-sodium dihydrogen phosphate buffer, etc. The pH of the buffer solution is preferably 7-8.

[0091] The effects of the invention

[0092] This invention utilizes tetrahedral framework nucleic acids as carriers, incorporating miR-22, which possesses neuroprotective and anti-angiogenic properties, to form a novel nanocomposite. The tetrahedral framework nucleic acid carrying miR-22 has a rational spatial topology, enabling efficient cell entry without additional transfection reagents, thus solving the problem of miR-22's difficulty in autonomous cell entry. miR-22 is protected by forming a hybrid strand with the tetrahedral framework nucleic acid backbone, preventing degradation by nucleases during delivery. Upon reaching the target site, it can be cleaved by enzymes to exert its biological activity as appropriate.

[0093] This invention presents a tetrahedral framework nucleic acid system incorporating miR-22, which offers a highly efficient, structurally stable, and low-immunogenic nucleic acid drug delivery system, providing a novel, efficient, precise, and low-toxicity approach for the treatment of ocular diseases. The miR-22-incorporated tetrahedral framework nucleic acid can be formulated into injections or eye drops for administration, providing a practical, effective, and highly patient-compliant new option for treating ocular diseases associated with abnormal angiogenesis.

[0094] In some embodiments, the tetrahedral framework nucleic acid of the present invention can encapsulate and protect miRNA, enabling efficient delivery and controlled release of miRNA in response to RNase H. In the presence of RNase H, the RNA portion of the DNA-RNA hybridization region is hydrolyzed, resulting in the dissociation of the tetrahedral structure and the intracellular release of miR-22. Experiments have shown that this tetrahedral framework nucleic acid structure exhibits high stability and good biocompatibility, and can cross the blood-retinal barrier, making it suitable for use as eye drops for the treatment of ocular diseases. This method is suitable for long-term, frequent administration, solving the problem of adverse reactions caused by invasive treatments in ophthalmic diseases, including pain, bleeding, inflammation, infection, and even choroidal / retinal detachment and visual impairment, thus improving patient compliance and quality of life.

[0095] All references throughout this application, such as patent documents containing published or granted patents or their equivalents, patent application publications, and non-patent literature or other sources, are incorporated herein by reference in their entirety, as if individually incorporated by reference. All patents and publications referenced in the specification indicate the level of skill of a person skilled in the art to which this invention relates. References cited herein are incorporated in their entirety by reference to indicate the state of the art in the art (in some cases) at their filing date, and this information may be used in the invention, excluding (e.g., abandoning) specific embodiments in the prior art if necessary. For example, when claiming a compound, it is understood that compounds known in the art that include a particular compound disclosed in the references disclosed herein (particularly in the referenced patent documents) are not included in the claims. Attached Figure Description

[0096] The present disclosure will be described in detail with reference to the following accompanying drawings, based on one or more different embodiments. The drawings are provided to facilitate understanding of the disclosure and should not be construed as limiting its breadth, scope, size, or applicability. For ease of illustration, the drawings are not necessarily drawn to scale.

[0097] Figure 1 is a transmission electron microscope image of an exemplary tetrahedral framework nucleic acid YHK-803 after successful assembly according to this disclosure.

[0098] Figure 2 is a schematic diagram of the structure of an exemplary tetrahedral framework nucleic acid YHK-803 of this disclosure.

[0099] Figure 3 is a bar chart showing the inhibitory effect of different concentrations of YHK-803 on cell proliferation under hypoxic conditions.

[0100] Figure 4 is a bar chart showing the inhibitory effects of miR-22, YHK-803, and aflibercept on cell proliferation under hypoxic conditions.

[0101] Figure 5A shows photographs of cell scratch healing in each group during the cell scratch experiment, and Figure 5B is a bar chart showing the proportion of cell scratches that did not heal in each group during the cell scratch experiment.

[0102] Figure 6A shows photographs of HUVEC cells forming tube structures in gel during the tube formation experiment, and Figure 6B is a bar chart showing the length and number of branches of the tube structures formed by cells in each group during the tube formation experiment.

[0103] Figure 7 shows the percentage (%) of grade 4 fluorescent spots before administration and on days 14 and 28 of the administration period. Differences were statistically analyzed using the Kruskal-Wallis H rank-sum test (KW test) and the Dunn rank-sum test. The sample size for the negative control group and the YHK803 injection group was n = 4. **P ≤ 0.01: Negative control group VS YHK803 injection group.

[0104] Figure 8A is a bar chart of fluorescence leakage area (mm2) before administration and on days 14 and 28 of the administration period. Differences between groups were statistically analyzed using independent samples t-tests. The sample size for the negative control group and the YHK803 injection group was n=4. *P≤0.05, ***P≤0.001: Negative control group vs. YHK803 injection group. Figure 8B is a bar chart of the improvement rate (%) of fluorescence leakage area on days 14 and 28 of the administration period. Differences between groups were statistically analyzed using independent samples t-tests. The sample size for the negative control group and the YHK803 injection group was n=4. **P≤0.01, ***P≤0.001: Negative control group vs. YHK803 injection group.

[0105] Figure 9 shows bar charts of retinal thickness (μm) before modeling, before drug administration, and on days 14 and 28 of the drug administration period. Differences between groups were statistically analyzed using independent samples t-tests (t-tests) or approximate t-tests (t'-tests). The sample size for the negative control group and the YHK803 injection group was n=4. Detailed Implementation

[0106] The present invention will be further described below with reference to specific embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications, equivalent substitutions, or improvements within the spirit and principles of the invention should be included within the scope of protection of the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional molecular, cellular, and animal methods. Unless otherwise specified, the experimental materials used were purchased from conventional biochemical reagent manufacturers.

[0107] Example 1: Preparation of tetrahedral framework nucleic acid YHK-803

[0108] Four single-stranded oligonucleotides, YHK-803-S1, YHK-803-S2, YHK-803-S3, and miR22-YHK-803 (synthesized by Shanghai Sangon Biotech, sequences shown in Table 1), were dissolved in TM buffer (50 mM MgCl2·6H2O, 10 mM Tris-HCl, pH 8.0) and thoroughly mixed to prepare a mixed solution. The final concentration of YHK-803-S1 was 1000 nM, and the concentration ratio of YHK-803-S1, YHK-803-S2, YHK-803-S3, and miR22-YHK-803 was 1:1:1:3.

[0109] The mixed solution was heated to 95°C and held for 10 minutes to denature the single-stranded oligonucleotides. It was then cooled to 4°C at maximum rate and held for 30 minutes, allowing the four single-stranded oligonucleotides to assemble into YHK-803. Transmission electron microscopy (TEM) revealed that the assembled YHK-803 was a tetrahedral particle (Figure 1). The structure of YHK-803 is shown in Figure 2. Through base complementarity of each segment, YHK-803-S1, YHK-803-S2, and YHK-803-S3 form a tetrahedral central backbone structure, with miR22-YHK-803 mounted on the central backbone.

[0110] Table 1. Sequences of single-stranded oligonucleotides used to prepare YHK-803

[0111] In the nucleotide sequences in the table, the uppercase letters represent deoxyribonucleotides, and the lowercase letters represent ribonucleotides.

[0112] Example 2: Effects of multiple concentrations of YHK-803 on cell proliferation under hypoxic conditions

[0113] Using a human umbilical vein endothelial cell (HUVEC) hypoxia model, the effects of different concentrations of tetrahedral framework nucleic acid YHK-803 on cell proliferation under hypoxic conditions were tested using the CCK-8 cell proliferation assay.

[0114] Experimental Methods: HUVEC cells were divided into three groups: a non-model group (C), a hypoxia-model group (HC), and various concentrations of YHK-803 treatment groups, with six replicate wells in each group. After seeding, HUVEC cells were pre-cultured at 37°C and 5% CO2 for 24 hours. Subsequently, YHK-803 was administered: the YHK-803 treatment groups were treated with YHK-803 at final concentrations of 12.5 nM, 25 nM, 50 nM, 100 nM, 150 nM, 250 nM, 375 nM, and 500 nM, respectively, while the C and HC groups were treated with PBS buffer. After administration, the C group maintained its original cell culture conditions, while the HC group and the YHK-803 treatment groups were placed in a hypoxia incubator (oxygen concentration 1.5%) for 24 hours.

[0115] After each group of cells was treated, CCK-8 reagent was added to each well at a ratio of 10% of the liquid volume in the well, and incubation was continued for 2 hours. Then, the absorbance was measured at 450 nm using a microplate reader. The relative viability of cells in each group was calculated based on the absorbance value, with group C as the baseline.

[0116] Experimental Results: As shown in Figure 3, CCK-8 assay results showed that the cell viability of the hypoxic model group (HC) was significantly increased compared with the non-model group (C) (P<0.0001), indicating that the model of hypoxia promoting HUVEC cell proliferation was successfully established. Compared with the HC group, YHK-803 at concentrations of 150 nM, 250 nM, 375 nM, and 500 nM all inhibited cell proliferation induced by hypoxia, and the inhibitory effect became more significant with increasing concentration (*: P<0.05; ***: P<0.001; ****: P<0.0001).

[0117] Example 3: Effects of miR-22, YHK-803, and aflibercept on cell proliferation under hypoxic conditions

[0118] Using the HUVEC hypoxia model, the effects of miR-22 (i.e. miR-22-3p, sequence shown in Table 2), YHK-803, and aflibercept (AFL) on cell proliferation under hypoxic conditions were tested using the CCK-8 cell proliferation assay.

[0119] Table 2 shows the sequence of miR-22-3p.

[0120] HUVEC cells were divided into four groups: no model group (C), hypoxia model group (HC), miR-22 treatment group, YHK-803 treatment group, and aflibercept treatment group, with 6 replicate wells in each group. After seeding, HUVEC cells were pre-cultured at 37°C and 5% CO2 for 24 hours. Subsequently, the following drug administrations were performed: PBS buffer was added to groups C and 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 (Bayer Healthcare Co., Ltd., aflibercept intraocular injection, 40 mg / mL stock solution, final concentration 100 ng / μl) was added to the aflibercept treatment group. After drug administration, the original cell culture conditions were maintained in group C. Cells from 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.

[0121] After each group of cells was treated, CCK-8 reagent was added to each well at a ratio of 10% of the liquid volume in the well, and incubation was continued for 2 hours. Then, the absorbance was measured at 450 nm using a microplate reader. The relative viability of cells in each group was calculated based on the absorbance value, with group C as the baseline.

[0122] Experimental Results: As shown in Figure 4, CCK-8 assay results showed that the cell viability of the model group (HC) was significantly increased compared to the non-model group (C), indicating that the model of hypoxia-induced HUVEC cell proliferation was successfully established. Compared with the HC group, miRNA-22 (P<0.05), YHK-803 (P<0.01), and aflibercept (P<0.01) all significantly inhibited hypoxia-induced cell proliferation, and the cell viability of the YHK-803 treatment group was lower than that of the miRNA-22 group.

[0123] Example 4: Effects of miR-22, YHK-803, and aflibercept on cell migration under hypoxic conditions

[0124] The effects of miR-22, YHK-803, and aflibercept on cell migration under hypoxic conditions were tested using a cell scratch assay in a HUVEC hypoxia model and cell migration was detected.

[0125] Experimental methods: HUVEC cells were divided into four groups: no model group (C), hypoxia model group (HC), miR-22 treatment group, YHK-803 treatment group, and aflibercept treatment group. Each group had two replicate wells. 3 × 10⁻⁶ cells were used. 5 HUVEC cell suspension was plated at 1 cell / ml and then placed in an incubator for pre-culture at 37°C and 5% CO2 for 24 hours.

[0126] Following this, scratching and drug administration were performed. Three scratches were made in each well, for a total of six scratches in each group, and photographs were taken immediately after scratching. Then, PBS buffer was added to group C and 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, group C maintained its original cell culture conditions, while cells from the HC, miR-22, YHK-803, and aflibercept treatment groups were placed in a hypoxic incubator (oxygen concentration 1.5%) for 24 hours. Finally, each scratch was photographed to analyze the scratch healing process.

[0127] Experimental Results: As shown in Figures 5A and 5B, after 24 hours of scratch treatment, the cell scratches in the hypoxia model group (HC) were basically healed, and the proportion of unhealed area was significantly smaller compared to the non-model group (C), indicating that the model of hypoxia promoting HUVEC cell migration was successfully established. Compared with the HC group, the unhealed area of ​​cell scratches in the miR-22 treatment group, YHK-803 treatment group, and aflibercept treatment group was significantly increased, indicating that cell migration induced by hypoxia was inhibited by drugs (**: P < 0.01; ***: P < 0.001; ****: P < 0.0001).

[0128] Example 5: Effects of miR-22, YHK-803, and aflibercept on tube formation under hypoxic conditions

[0129] A tube formation experiment was conducted using the HUVEC hypoxia model. By detecting tube formation, the effects of miR-22, YHK-803, and aflibercept on tube formation were tested.

[0130] Experimental methods: HUVEC cells were divided into four groups: no model group (C), hypoxia model group (HC), miR-22 treatment group, YHK-803 treatment group, and aflibercept treatment group. HUVEC cells were cultured at a rate of 2 × 10⁻⁶ cells / cells. 5 Cells were seeded into 6-well plates at a density of cells / ml. After seeding, the cells were placed in an incubator and cultured in DMEM medium at 37°C and 5% CO2 for 24 hours.

[0131] Subsequently, the following drugs were administered to each group: PBS buffer was added to group C and 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 original cell culture conditions were maintained in group C, while the cells from the HC, miR-22, YHK-803, and aflibercept treatment groups were placed in a hypoxic incubator (oxygen concentration 1.5%) and cultured for 24 h.

[0132] The cells in each group were then digested, and cell counts were performed. The cell suspensions of all groups were adjusted to 2 × 10⁻⁶. 5 Cells were seeded at a density of 100 μl / ml and then seeded into 96-well plates pre-coated with Matrigel. Each well contained 100 μl of cell suspension, with 8 replicates per group. The cells were then incubated at 37°C and 5% CO2 for 4 hours. Images were then taken to observe the formation of tubular structures in the Matrigel for each group of cells.

[0133] The method for pre-coating the 96-well plate with matrix gel is as follows: The matrix gel stock solution (Corning) is placed on ice in DMEM medium. The basement membrane matrix (high concentration (HC), LDEV-free) was diluted 1-fold and thoroughly mixed. 50 μl of the diluted matrix gel was added to each well of the 96-well plate, and the plate was then incubated at 37°C to complete the coating.

[0134] Experimental results: As shown in Figures 6A and 6B, compared with the unmodeled group (C), the length of the tubular structures formed by HUVEC cells in the hypoxia modeled group (HC) and the number of tubular branches were increased, indicating that the hypoxia condition promoted the successful establishment of the HUVEC cell tubular model.

[0135] Compared with the HC group, the total length of tubular structures formed in the miR-22 treatment group, YHK-803 treatment group, and aflibercept treatment group was significantly lower, indicating that tube formation was inhibited. YHK-803 was significantly more effective than miR-22 in inhibiting tube formation (P<0.01). Compared with the HC group, the reduction in the number of tube branches in the miR-22 treatment group was not significant (P>0.05), while the number of tube branches in the YHK-803 treatment group and the aflibercept treatment group was significantly reduced (P<0.001). YHK-803 was significantly more effective than miR-22 in inhibiting hypoxia-induced tube branch formation (P<0.01).

[0136] Example 6: Effect of YHK-803 on choroidal angiogenesis in rats

[0137] Experimental Methods: Rats were instilled with Typicalide (compound tropicamide eye drops) to fully dilate their pupils, and anesthetized with sodium pentobarbital. The rats' heads were fixed in front of an ophthalmic laser photocoagulation instrument, and CNV modeling was performed using laser photocoagulation. Six laser points were irradiated in each eye. The presence of bubbles in the fundus indicated that Bruch's membrane had been ruptured, indicating successful photocoagulation. Modeling and testing were performed in the right eye. Rats were randomly divided into 6 groups, with 6 rats in each group: 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.

[0138] On day 7 after CNV modeling in rats, fundus fluorescein angiography (FFA) was performed first, followed by drug administration on the same day (day 0 of administration).

[0139] The PBS group received a single intravitreal injection of 4 μl of PBS buffer.

[0140] The low-dose group of YHK-803 received a single intravitreal injection of 4 μl of 0.1 μM YHK-803 solution.

[0141] The medium-dose group of YHK-803 received a single intravitreal injection of 4 μl of 0.6 μM YHK-803 solution.

[0142] The high-dose group of YHK-803 received a single intravitreal injection of 4 μl of 3.6 μM YHK-803 solution.

[0143] The YHK-803 eye drop group used 1μM YHK-803 eye drops, 20μL / eye each time, once a day, for 14 consecutive days.

[0144] The aflibercept group received a single intravitreal injection of 40 mg / mL aflibercept intravitreal injection solution (Bayer Healthcare Co., Ltd.) 4 μl.

[0145] Subsequently, FFA testing was performed again at 14 days after modeling (i.e., 7 days after drug administration) and 21 days after modeling (i.e., 14 days after drug administration).

[0146] Methods for FFA detection: The rat eye was instilled with methylparaben (compound tropicamide eye drops) to dilate the pupil. After anesthesia with sodium pentobarbital, 10% sodium fluorescein injection was injected intraperitoneally at a dose of 0.5 mL / kg body weight. Immediately afterwards, fundus angiography was performed, and images of the retinal region containing 6 laser points centered on the optic disc were taken. Images taken about 15 minutes after the injection of sodium fluorescein were selected for fluorescence spot grading and scoring.

[0147] Fluorescent spot grading method: Grade 1: No high fluorescence in the spot; Grade 2: High fluorescence in the spot but no fluorescein leakage; Grade 3: High fluorescence in the spot, slight fluorescein leakage, leakage does not exceed the edge of the spot; Grade 4: High fluorescence in the spot, significant fluorescein leakage, leakage exceeds the edge of the spot.

[0148] The experimental results are shown in Table 3.

[0149] In the PBS group, obvious laser spots and sodium fluorescein leakage were observed at 7, 14, and 21 days after modeling, indicating that the model was successfully established.

[0150] A single intravitreal injection of a high dose of YHK-803 can effectively inhibit laser photocoagulation-induced choroidal angiogenesis, with the effect lasting for at least two weeks. YHK-803 eye drops can also effectively inhibit laser photocoagulation-induced choroidal angiogenesis.

[0151] Table 3 Fluorescence spot grading data ( n = 6 / group)

[0152] * Compared with the PBS group, P < 0.05.

[0153] The experimental results of the above embodiments 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 choroidal angiogenesis induced by laser photocoagulation.

[0154] Example 7: Preclinical In vivo pharmacodynamic study - Pharmacodynamic test of the effect of a single intravitreal injection of YHK803 solution on laser-induced choroidal neovascularization in rhesus monkeys.

[0155] This experiment used laser-induced damage to the outer Bruch's membrane of the rhesus monkey retina to establish a choroidal neovascularization model. Fluorescence angiography was performed two weeks after modeling, and four monkeys that successfully developed the model were selected for the study. The experiment consisted of two groups, two monkeys per group, with half male and half female. The negative control group and the YHK803 injection group received a single intravitreal injection in both eyes of the corresponding phosphate buffer or YHK803 injection solution.

[0156] During the experiment, the general condition and near-death / death status of the monkeys were observed daily; body weight was measured once each during the adaptation and model periods, and once every two weeks during the drug administration period; slit-lamp and fundus examinations were performed on both eyes on day 5 of the adaptation period, day 14 of the model period, and days 7, 14, and 28 of the drug administration period; fundus photography, fluorescein angiography (FFA), and optical coherence tomography (OCT) of the retina were performed on day 5 of the adaptation period, day 14 of the model period, and days 14 and 28 of the drug administration period.

[0157] The main results are as follows:

[0158] Fundus fluorescein angiography

[0159] On days 14 and 28 of the dosing period, the percentage of grade IV fluorescent spots in the YHK803 injection group showed a continuous decrease, and on day 28 of the dosing period, there was a statistically significant difference compared with the negative control group (P≤0.01). (See Figure 7) On days 14 and 28 of the dosing period, the fluorescence leakage area in the negative control group increased, and the leakage continued to worsen over time, while the fluorescence leakage area in the YHK803 injection group decreased, and the leakage continued to improve over time, showing a statistically significant difference compared with the negative control group (see Figures 8A and 8B).

[0160] According to optical coherence tomography (OCT) scans of the retina, there were no statistically significant differences in retinal thickness between the two groups before modeling, before the first dose, and on days 14 and 28 of the treatment period (P > 0.05). (See Figure 9)

[0161] In summary, under the conditions of this experiment, a single intravitreal injection of YHK803 solution had an inhibitory effect on choroidal neovascularization induced by laser in rhesus monkeys (detailed data are shown in Table 4).

[0162] Table 4-1 Effect of a single intravitreal injection of YHK803 solution on the percentage of grade 4 fluorescein spots in rhesus monkeys (%) Note: *The difference in means compared with the negative control group was statistically significant (P≤0.05).

[0163] Table 4-2 Statistical tests on the effect of a single intravitreal injection of YHK803 on the percentage of grade 4 fluorescein spots in rhesus monkeys

[0164] Table 4-3 Effect of a single intravitreal injection of YHK803 solution on the fluorescein leakage area in rhesus monkeys (mm) 2 ) Note: *The difference in means compared with the negative control group was statistically significant (P≤0.05).

[0165] Table 4-4 Statistical tests on the effect of a single intravitreal injection of YHK803 solution on the fluorescein leakage area in rhesus monkeys

[0166] Table 4-5 Effect of a single intravitreal injection of YHK803 solution on the improvement rate of fluorescein leakage area in rhesus monkeys (%) Note: *The difference in means compared with the negative control was statistically significant (P≤0.05).

[0167] Table 4-6 Statistical tests on the effect of a single intravitreal injection of YHK803 solution on the improvement rate of fluorescein leakage area in rhesus monkeys

[0168] Table 4-7 Effect of a single intravitreal injection of YHK803 solution on retinal thickness in rhesus monkeys (μm)

[0169] Table 4-8 Statistical analysis of the effect of a single intravitreal injection of YHK803 on retinal thickness in rhesus monkeys.

[0170] Example 8: Preliminary toxicity test of YHK803 administered via single intravitreal / intravenous injection in cynomolgus monkeys.

[0171] ●Introduction to Experimental Design

[0172] ●Objective: To observe the nature, extent, and reversibility of potential toxic reactions caused by the test product in cynomolgus monkeys via single intravitreal injection in both eyes and single intravenous injection within 14 days, to identify the target organs or tissues for toxicity, and to provide a reference for subsequent studies.

[0173] ● Animals and Grouping: Crab-eating macaques, 8 in total, half male and half female; 4 dosage groups: intravitreal injection of YHK803 at 12.6 and 37.8 μg / eye, and intravenous injection of YHK803 at 168 and 1512 μg / kg.

[0174] ●Route and frequency of administration: Monkeys in the intravitreal injection group received a single intravitreal injection of YHK803 at concentrations of 252 and 756 μg / mL, administered at a volume of 50 μL / eye; monkeys in the intravenous injection group received a single intravitreal injection of YHK803 at concentrations of 84 and 756 μg / mL, administered at a volume of 2 mL / kg.

[0175] ●Observation indicators: The day of administration was defined as day 1 of the administration period / day 1 of the experiment. During the experiment, the general condition of the monkeys in each group was observed daily, and their weight was measured once a week. Slit-lamp and fundus examinations were performed on days 3, 7, and 14 of the experimental period for monkeys in the intravitreal injection group, and on day 14 of the experimental period for monkeys in the intravenous injection group. Fundus color photography, fluorescein angiography (FFA), and optical coherence tomography (OCT) were performed on days 7 and 14 of the experimental period for monkeys in the intravitreal injection group, and on days 3, 7, and 14 of the experimental period for monkeys in the intravitreal injection group. On day 15 of the experimental period, after anesthesia with sodium pentobarbital and euthanasia by exsanguination from the abdominal aorta, gross anatomical observation and histopathological examination were performed on the monkeys in each group.

[0176] ●Experimental Results and Conclusions

[0177] Under the experimental conditions, cynomolgus monkeys in the intravitreal injection group received a single intravitreal injection of 12.6 and 37.8 μg / eye of YHK803 in both eyes. After 14 days of observation, slit-lamp and fundus examinations mainly revealed aqueous humor cells and anterior vitreous cells. These signs significantly lessened or recovered with prolonged observation. No abnormalities were found in fundus color photography, fluorescein angiography, optical coherence tomography, or ocular histopathological examination. Blood biochemistry also showed no abnormalities related to the test product. In the intravenous injection group, cynomolgus monkeys received a single intravenous injection of 168 and 1512 μg / kg of YHK803. After 14 days of observation, no abnormalities related to the test product were found in slit-lamp and fundus examinations, blood biochemistry tests, or histopathological examinations of the eye, liver, and kidneys. (Detailed data are shown in Table 5.)

[0178] Table 5-1 Summary of Observation Results of General Condition of Male Monkeys (Days 1-15 of the Experiment) Note: a represents the number of animals showing symptoms; b represents the number of days since symptoms appeared.

[0179] Table 5-2 Summary of Observation Results of General Condition of Female Monkeys (Days 1-15 of the Experiment) Note: a represents the number of animals showing symptoms; b represents the number of days since symptoms appeared.

[0180] Table 5-3 Effects of a single intravitreal / intravenous injection of YHK803 on the body weight of cynomolgus monkeys (kg)

[0181] Table 5-4 Summary of ophthalmic observation results in male monkeys (day 2 of the adaptation period) Note: a represents the number of animals showing symptoms; b represents the number of days since symptoms appeared.

[0182] Appendix Table 5-5 Summary of ophthalmic observation results in female monkeys (day 2 of the acclimatization period) Note: a represents the number of animals showing symptoms; b represents the number of days since symptoms appeared.

[0183] In summary, YHK803 demonstrated good local and systemic safety within the tested dose range under both administration routes. Only the intravitreal injection group experienced transient and reversible ocular inflammation, which may be related to the injection procedure rather than the drug's inherent toxicity. In cynomolgus monkeys, a single intravitreal injection of 12.6 and 37.8 μg / eye of YHK803 resulted in a no-adverse-effect level (NOAEL) of 37.8 μg / eye. Similarly, a single intravenous injection of 168 and 1512 μg / kg of YHK803 in cynomolgus monkeys resulted in a NOAEL of 1512 μg / kg.

[0184] Example 9: Toxicity Evaluation of a Single Intravitreal Injection of YHK803 in New Zealand Rabbits (High Dose)

[0185] ●Introduction to Experimental Design

[0186] ●Objective: To observe the nature, extent, and reversibility of potential toxic reactions caused by a single intravitreal injection of YHK803 into New Zealand rabbits within 14 days, and to determine the target organs or tissues for toxicity, so as to provide a reference for subsequent studies.

[0187] ● Animals and grouping: 3 New Zealand rabbits, female; 1 dosage group: 37.8 μg / eye (756 μg / mL, 50 μL / eye)

[0188] ●Route and frequency of administration: Single intravitreal injection in both eyes

[0189] ●Observation indicators: The day of administration is defined as day 1 of the administration period / day 1 of the experiment. During the observation period after administration, the general condition of the New Zealand rabbits was observed daily, and their weight was measured weekly; hematological and blood biochemical tests were performed on day 14 of the experiment; slit-lamp and fundus examinations were performed on days 3, 7, and 14 of the experiment; retinal optical coherence tomography, fundus color photography, and fluorescein angiography were performed on day 14 of the experiment; on day 15 of the experiment, the rabbits were anesthetized by intravenous injection of sodium pentobarbital and euthanized by exsanguination from the abdominal aorta, followed by gross anatomical observation and histopathological examination.

[0190] ●Experimental Results and Conclusions

[0191] During the experiment, the New Zealand rabbits were generally in good condition, with normal spontaneous activity, clean skin and fur, normal feces and urine, and no other toxic reactions or abnormal weight changes. On day 14 of the experiment, slit-lamp examination, fundus examination, fundus color photography and fluorescein angiography, and optical coherence tomography showed no abnormalities in any rabbit. No significant abnormalities were observed in the hematological and blood biochemical indicators of the New Zealand rabbits. On day 15 of the experiment, gross anatomical examination of the major organs, including the eyeball, brain, heart, liver, spleen, lungs, kidneys, gastrointestinal tract, and reproductive system, revealed no significant abnormalities in morphology, color, or texture. On day 15 of the experiment, in the YHK803 group, 4 / 6 eyes showed vitreous mononuclear cell infiltration, which could not be ruled out as being related to YHK803. Apart from this, no histopathological changes were observed in other subtissues of the eyeball (conjunctiva, cornea, limbus, sclera, anterior chamber, posterior chamber, iris, ciliary body, lens, and choroid). (Detailed data are shown in Table 6)

[0192] Table 6-1 Summary of Observation Results of General Condition of Female Rabbits (Days 1-15 of the Experiment) Note: a represents the number of animals showing symptoms; b represents the number of days since symptoms appeared.

[0193] Table 6-2 Effect of a single intravitreal injection of high-dose YHK803 on the body weight (kg) of female rabbits.

[0194] Table 6-3 Changes in mean values ​​(%) of female rabbit blood biochemical samples at each time point after drug administration compared to the environmental adaptation period. Note: 1) Mean change rate (%) = (mean at each time point after drug administration - mean during environmental adaptation period) / |mean during environmental adaptation period| × 100.

[0195] 2) ▲ The differences in mean time points compared to the environmental adaptation period were statistically significant (P≤0.05).

[0196] In summary, under the conditions of this experiment, a single intravitreal injection of 50 μL / eye of YHK803 (756 μg / mL) into New Zealand rabbits, followed by a 14-day observation period, revealed no significant abnormalities in the general condition, weight, slit-lamp and fundus examinations, fundus color photography and fluorescein angiography, optical coherence tomography, hematological parameters, blood biochemical parameters, and gross anatomical observation of the rabbits. Histopathological examination of the ocular tissue mainly revealed mild vitreous mononuclear cell infiltration in some eyes, which could not be ruled out as being related to YHK803. Overall assessment indicates that YHK803 exhibits good intraocular safety characteristics at the experimental dose of 37.8 μg / eye, and did not induce systemic toxicity.

[0197] Example 10: Preliminary toxicity test of intravitreal injection of YHK803 in New Zealand rabbits over 4 weeks.

[0198] ●Introduction to Experimental Design

[0199] ●Objective: To administer YHK803 via intravitreal injection into both eyes of New Zealand rabbits once every 2 weeks for 4 consecutive weeks (3 administrations), and observe for 14 days after the last administration to observe the nature, extent, dose-response relationship and reversibility of the possible toxic reactions caused by the test product, to determine the target organs or tissues of toxicity, and to provide a reference for subsequent studies.

[0200] ● Animals and Grouping: Six female New Zealand rabbits; three dosage groups: YHK803 4.2, 12.6, and 37.8 μg / eye.

[0201] ●Route and frequency of administration: Administer YHK803 via intravitreal injection in both eyes at a volume of 50 μL / eye, at concentrations of 84, 252, and 756 μg / mL, respectively, once every 2 weeks for 4 consecutive weeks (3 administrations).

[0202] ●Observation indicators: The day of administration was defined as day 1 of the administration period / day 1 of the experiment, and the day after the last administration was defined as day 1 of the recovery period. During the administration and recovery periods, the general condition of rabbits in each group was observed daily, and their weight was measured weekly. Slit-lamp and fundus examinations were performed on days 1, 3, 7, and 13 after the first and second administrations, and on days 1, 3, 7, and 14 after the third administration. Retinal optical coherence tomography, fundus color photography, and fluorescein angiography were performed in weeks 1 and 4 of the administration period and week 2 of the recovery period. Hematological and blood biochemical tests were performed on day 14 of the administration and recovery periods. On day 15 of the recovery period, rabbits in each group were anesthetized by intravenous injection of sodium pentobarbital and euthanized by exsanguination from the abdominal aorta, followed by gross anatomical observation and histopathological examination.

[0203] ●Experimental Results and Conclusions

[0204] During the administration and recovery periods, the rabbits in all groups were in good general condition, with normal spontaneous activity, clean skin and fur, normal feces and urine, and no other toxic reactions or abnormal weight changes. At each time point during the administration and recovery periods, slit-lamp and fundus examinations of the eyelids, conjunctiva, cornea, anterior chamber, iris, lens, vitreous body, optic disc, retina, and retinal vessels in all YHK803 groups showed no abnormalities related to the test substance. At weeks 1 and 4 of the administration period and week 2 of the recovery period, the retinal structures of all layers in the YHK803 groups were continuous, of moderate thickness, and showed normal signal intensity (neurofibrillary and pigment epithelial layers showed high reflectivity; ganglion cell layer, inner and outer plexiform layers, and photoreceptor ganglion junction layer showed medium reflectivity; inner and outer granular layers and photoreceptor ganglion layers showed low reflectivity). No abnormalities related to the test substance were observed. No abnormal changes were observed. During weeks 1 and 4 of the drug administration period and week 2 of the recovery period, the optic discs of rabbits in all YHK803 groups showed normal color and clear boundaries. The retinal branches of arteries and veins showed normal thickness and course, and the retina was normal in color. The retinal arteries and veins were well-filled, with normal vessel course and diameter. The choroidal background fluorescence was uniform, and no abnormal changes related to the test product were observed. On day 14 of the drug administration period and day 14 of the recovery period, no abnormal changes related to the test product were observed in the hematological and blood biochemical indicators of rabbits in all YHK803 groups. On day 15 of the recovery period, gross anatomical observation of the major organs of the New Zealand rabbits, including the eyeball, brain, heart, liver, spleen, lungs, kidneys, gastrointestinal tract, and reproductive system, showed no abnormal changes in morphology, color, or texture. Microscopic observation did not reveal any lesions that might be related to YHK803. (Detailed data are shown in Table 7) Table 7-1 Summary of general condition observation results of New Zealand rabbits (days 1-29 of drug administration period) Note: a represents the number of animals showing symptoms; b represents the number of days since symptoms appeared.

[0205] Table 7-2 Summary of General Condition Observation Results of New Zealand Rabbits (Recovery Period Days 1-15) Note: a represents the number of animals showing symptoms; b represents the number of days since symptoms appeared.

[0206] Table 7-3 Effects of intravitreal injection of YHK803 on body weight (kg) in New Zealand rabbits

[0207] Table 7-4 Summary of Ophthalmic Observation Results in New Zealand Rabbits (Drug Administration Period) Note: a represents the number of animals showing symptoms; b represents the number of days since symptoms appeared.

[0208] Table 7-5 Summary of Ophthalmic Observation Results in New Zealand Rabbits (Recovery Period) Note: a represents the number of animals showing symptoms; b represents the number of days since symptoms appeared.

[0209] In summary, under the conditions of this experiment, New Zealand rabbits were administered YHK803 intravitreal injections of 4.2, 12.6, and 37.8 μg / eye every two weeks for four consecutive weeks (three administrations). After the last administration, rabbits were observed for 14 days. No abnormalities related to the test product were found in the general condition, body weight, slit-lamp and fundus examinations, fundus color photography and fluorescein angiography, retinal optical coherence tomography, hematological parameters, blood biochemical parameters, gross anatomical observation, and histopathological examination of the rabbits in each YHK803 group. No systemic toxicity was observed in any of the dosage groups, indicating that YHK803 has good safety within the experimental dosage range.

[0210] The tetrahedral framework nucleic acid of this invention can deliver miR-22 without additional transfection reagents, preventing miR-22 degradation during delivery and allowing it to exert its biological activity at the target site. Compared to free miR-22, the tetrahedral framework nucleic acid structure carrying miR-22 exhibits significantly better inhibitory effects on HUVEC cell proliferation and tube formation under hypoxic conditions. In animal models, the tetrahedral framework nucleic acid carrying miR-22, administered intravitreally, effectively inhibits laser photocoagulation-induced choroidal angiogenesis, with the effect lasting at least two weeks, providing a promising new option for treating ocular lesions related to abnormal angiogenesis. Furthermore, the tetrahedral framework nucleic acid carrying miR-22, when formulated as eye drops, also effectively inhibits laser photocoagulation-induced choroidal angiogenesis, providing a practical and highly patient-compliant treatment option for ocular lesions related to abnormal angiogenesis, filling a gap in this field.

[0211] While features of the invention have been shown and described in detail with reference to preferred embodiments, those skilled in the art will understand that other changes may be made therein without departing from the spirit and scope of the invention. Similarly, various figures may depict exemplary architectures or other configurations for use in this disclosure, serving to illustrate the features and functions that may be included in this disclosure. This disclosure is not limited to the example architectures or configurations shown, but may be implemented using various alternative architectures and configurations. Furthermore, although this disclosure has been described above with reference to various exemplary embodiments and implementations, it should be understood that the various features and functions described in one or more individual embodiments are not limited to descriptions of their suitability for the particular embodiment to which they belong. Rather, they may be applied individually or in some combination to one or more other embodiments of this disclosure, whether or not such embodiments are described, and whether these features are presented as part of the described embodiments. Therefore, the breadth and scope of this disclosure should not be limited to any of the exemplary embodiments described above.

Claims

1. A tetrahedral framework nucleic acid carrying miR-22, wherein the tetrahedral framework nucleic acid 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 composed of RNA segment P, corner segment 1, DNA segment 1A, corner segment 2, DNA segment 1B, corner segment 3, RNA segment Q, and DNA tail segment connected sequentially. The second single-stranded oligonucleotide is composed of the RNA segment P, the corner segment 4, the DNA segment 2A, the corner segment 5, the DNA segment 2B, the corner segment 6, the RNA segment Q, and the DNA tail segment connected sequentially. The third single-stranded oligonucleotide is formed by sequentially connecting the RNA segment P, the corner segment 7, the DNA segment 3A, the corner segment 8, the DNA segment 3B, the corner segment 9, the RNA segment Q, and the DNA tail segment. The fourth single-stranded oligonucleotide is composed of RNA segment 4A, RNA segment 4B, RNA segment 4C, and RNA tail segment linked together in sequence; The RNA segment P is inversely complementary to the RNA segment 4C; the RNA segment Q is inversely complementary to the RNA segment 4A; the DNA segment 1A is inversely complementary to the DNA segment 3B; the DNA segment 1B is inversely complementary to the DNA segment 2A; the DNA segment 2B is inversely complementary to the DNA segment 3A; and the DNA tail is inversely complementary to the RNA tail. 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 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 in length, preferably 19 or 20 nucleotide residues; and the RNA segment P and the RNA segment Q are 7-9 nucleotide residues in length, preferably 8 nucleotide residues.

3. The tetrahedral framework nucleic acid according to claim 1 or 2, wherein the corner segments 1-9 are all DNA; optionally, the length of each of the corner segments 1-9 is 1 nucleotide residue; optionally, the sequence of each of the corner segments 1-9 is A.

4. The tetrahedral framework nucleic acid according to claim 1 or 2, wherein the DNA tail 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 as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively.

7. The tetrahedral framework nucleic acid according to any one of claims 1-6, wherein, Each of the aforementioned tetrahedral framework nucleic acids carrying miR-22 comprises one first single-stranded oligonucleotide, one second single-stranded oligonucleotide, one third single-stranded oligonucleotide, and three fourth single-stranded oligonucleotides; Optionally, the three fourth single-stranded oligonucleotides have the same nucleotide sequence.

8. The tetrahedral framework nucleic acid of any one of claims 1-7, wherein, The sequence formed by sequentially linking RNA segment 4A, RNA segment 4B, and RNA segment 4C has 100% identity with SEQ ID NO:5; Optionally, the sequence of RNA segment 4A is aagcugcc, the sequence of RNA segment 4B is aguuga, and the sequence of RNA segment 4C is agaacugu.

9. The tetrahedral framework nucleic acid according to any one of claims 1-7, wherein, The sequence of the segment formed by sequentially connecting the RNA segment 4A, the RNA segment 4B, and the RNA segment 4C has a mutation (substitution, deletion, or addition) of up to one nucleotide compared with SEQ ID NO:5; Optionally, the sequence of RNA segment 4A is aagcugcc, the sequence of RNA segment 4B is aguuga, and / or the sequence of RNA segment 4C is agaacugu.

10. A nucleic acid composition comprising 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 RNA segment P, corner segment 1, DNA segment 1A, corner segment 2, DNA segment 1B, corner segment 3, RNA segment Q, and DNA tail segment connected sequentially. The second single-stranded oligonucleotide is composed of the RNA segment P, the corner segment 4, the DNA segment 2A, the corner segment 5, the DNA segment 2B, the corner segment 6, the RNA segment Q, and the DNA tail segment connected sequentially. The third single-stranded oligonucleotide is formed by sequentially connecting the RNA segment P, the corner segment 7, the DNA segment 3A, the corner segment 8, the DNA segment 3B, the corner segment 9, the RNA segment Q, and the DNA tail segment. The fourth single-stranded oligonucleotide is composed of RNA segment 4A, RNA segment 4B, RNA segment 4C, and RNA tail segment linked together in sequence; The RNA segment P is inversely complementary to the RNA segment 4C; the RNA segment Q is inversely complementary to the RNA segment 4A; the DNA segment 1A is inversely complementary to the DNA segment 3B; the DNA segment 1B is inversely complementary to the DNA segment 2A; the DNA segment 2B is inversely complementary to the DNA segment 3A; and the DNA tail is inversely complementary to the RNA tail. 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.

11. The nucleic acid composition according to claim 10, 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 as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively.

12. The nucleic acid composition according to claim 10 or 11, 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).

13. Use of the tetrahedral framework nucleic acid of any one of claims 1-9 or the nucleic acid composition of any one of claims 10-12 in the preparation of a medicament for treating ocular diseases.

14. The use according to claim 13, wherein the ocular lesion is an ocular lesion associated with abnormal angiogenesis.

15. The use according to claim 13 or 14, wherein the ocular lesion is selected from wet age-related macular degeneration, proliferative diabetic retinopathy, choroidal neovascularization secondary to pathological myopia, neovascular glaucoma, retinopathy of prematurity, and ocular pseudohistomycosis syndrome.

16. A pharmaceutical formulation comprising a tetrahedral framework nucleic acid according to any one of claims 1-9 or a nucleic acid composition according to any one of claims 10-12, and optionally a pharmaceutically acceptable carrier.

17. The pharmaceutical preparation according to claim 16, wherein the pharmaceutical preparation is an ophthalmic preparation.

18. The pharmaceutical preparation according to claim 17, wherein the pharmaceutical preparation is an injection solution or a dry powder.

19. The pharmaceutical preparation according to claim 18, wherein the injection solution contains 0.05-5 μM of the tetrahedral framework nucleic acid, or the liquid in use prepared from the dry powder contains 0.05-5 μM of the tetrahedral framework nucleic acid.

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

21. The pharmaceutical formulation according to claim 20, wherein the eye drops contain the tetrahedral framework nucleic acid at a concentration of 0.1-10 μM.

22. The pharmaceutical preparation according to any one of claims 16-21, wherein, The pharmaceutical preparation does not contain transfection reagents.

23. A method of treating an ocular lesion, comprising administering to a patient in need a tetrahedral framework nucleic acid according to any one of claims 1-9, a nucleic acid composition according to any one of claims 10-12, or a pharmaceutical preparation according to any one of claims 16-21.

24. The method of claim 23, wherein, The method of application includes treatment in the form of eye drops.

25. The method according to claim 23, wherein, The method of application includes intravitreal injection.

26. The method of any one of claims 22-25, wherein, The ocular lesions mentioned are ocular lesions associated with abnormal angiogenesis.

27. The method according to any one of claims 22-26, wherein the ocular lesion is selected from wet age-related macular degeneration, proliferative diabetic retinopathy, choroidal neovascularization secondary to pathological myopia, neovascular glaucoma, retinopathy of prematurity, and ocular pseudohistomycosis syndrome.

28. The method according to any one of claims 22-27, wherein the ocular lesion is a choroidal neovascularization-related lesion.

29. A method for preparing a tetrahedral framework nucleic acid according to any one of claims 1-9, comprising the following steps: Prepare a mixed solution containing the first single-chain oligonucleotide, the second single-chain oligonucleotide, the third single-chain oligonucleotide, and the fourth single-chain oligonucleotide; The mixture is kept 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 minutes, and then the temperature is lowered to 2-8°C and kept for more than 1 minute.

30. The method of claim 29, wherein, The first single-chain oligonucleotide, the second single-chain oligonucleotide, the third single-chain oligonucleotide, and the fourth single-chain oligonucleotide are dissolved in TM buffer (containing Tris-HCl and MgSO4 or MgCl2) to prepare a mixed solution. The molar ratio of the first single-chain oligonucleotide, the second single-chain oligonucleotide, the third single-chain oligonucleotide, and the fourth single-chain oligonucleotide in the prepared mixed solution is (0.9-1.1):(0.9-1.1):(0.9-1.1):(2.7-6.3). The mixture was kept at approximately 95°C for 10-15 minutes, and then the temperature was lowered to approximately 4°C and kept for 15-30 minutes.

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