Preparation method of nucleic acid medicine taking non-coding RNA (Ribonucleic Acid) of brain central neuron as target spot

Abstract

The invention discloses a preparation method of a nucleic acid medicine taking brain central neuron non-coding RNA as a target spot, the nucleic acid medicine comprises an annular or linear DNA coding sequence for expressing an ASO sequence, a promoter sequence and a transcription termination signal, the ASO sequence is a reverse complementary sequence of non-coding RNA such as snRNA, circRNA, lncRNA and miRNA or an RNA sequence with homology greater than 80% with the reverse complementary sequence of the ASO sequence, the invention also discloses a brain-targeted delivery system containing the DNA coding sequence. According to the DNA coding sequence and delivery system provided by the preparation method of the nucleic acid medicine, after peripheral administration, coding DNA can be directionally delivered to brain central neurons in animal bodies, ASO sequences are transcribed, effective intervention on expression of non-coding RNA related to the Alzheimer's disease is achieved, the stability and bioavailability of the nucleic acid medicine can be effectively improved, and the application prospect is wide. Meanwhile, a nucleic acid chain can be accurately conveyed to target tissue cells, so that the stability of a nucleic acid drug for treatment is greatly improved, and the influence of the nucleic acid drug on other tissue cells is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to a method for preparing a nucleic acid drug targeting non-coding RNA of central neurons of the brain. Background Art

[0002] Nucleic acid drugs are the frontier of current biomedical development, including deoxyribonucleic acid (DNA), ribonucleic acid (RNA), antisense nucleic acid (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), aptamer, ribozyme, antibody nucleic acid conjugate (ARC), etc. It is a form of gene therapy and a new generation of pharmaceutical technology after small molecule drugs, protein drugs, and antibody drugs, and has received more and more attention. Since its invention, nucleic acid therapy has been considered a promising strategy for the treatment of difficult human diseases, but many problems have been encountered in clinical practice, making the development of this therapy far behind expectations. For example: Alzheimer's disease (AD) is a central nervous system degenerative disease with cognitive impairment as the main clinical symptom that occurs in the elderly and seriously endangers the physical and mental health of patients. The current AD intervention strategy mainly focuses on its main pathological components: accumulation of β-amyloid protein (Aβ), hyperphosphorylation of tau protein, and cholinergic dysfunction. However, AD interventions targeting these classic pathologies have only mildly improved clinical symptoms to a certain extent and cannot reverse AD. Currently, there are many existing technologies related to RNA nucleic acid drugs, which mainly focus on the following aspects: 1. Designing RNA nucleic acid drugs with medical effects; 2. Chemically modifying RNA chains to improve the stability of nucleic acid drugs in the body and increase their effective utilization rate; 3. Designing and improving various artificial carriers (such as lipid nanoparticles, cationic polymers and viruses) to improve the efficiency of nucleic acid drug delivery in the body.

[0003] The second and third aspects of the prior art are the most widely used, but the final working effect is still limited. The fundamental reason is that there is still a lack of suitable nucleic acid drug modification and delivery systems to safely, accurately and efficiently deliver nucleic acid chains to target tissue cells. The above defects have become the core issues restricting the development of RNA nucleic acid therapy, and there is still room for improvement in the current RNA nucleic acid therapy. Summary of the invention

[0004] In order to overcome the above-mentioned defects of the prior art, an embodiment of the present invention provides a method for preparing a nucleic acid drug targeting non-coding RNA of central neurons in the brain. By using a new type of AD nucleic acid drug that can be transcribed intracellularly and targets non-coding RNA of central neurons in the brain, a drug preparation scheme is provided while providing a use, providing a new idea for AD intervention to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for preparing a nucleic acid drug targeting non-coding RNA in central neurons of the brain, comprising the following steps:

[0006] S1. First, nanosized DGL and NHS-PEG-NHS were dissolved in phosphate buffer (PBS, pH=7.0) at a molar ratio of 1:2.5 to 1:18, and stirred at room temperature in the dark for 12 h to covalently link the active group -NHS to DGL to obtain NHS-PEG-NHS-modified DGL nanoparticles;

[0007] S2, centrifuging the product of the previous step at 8000-12000g for 15 min, discarding the supernatant and washing with PBS (pH=7.0), chromatographically detecting until there is no free NHS-PEG-NHS, and freeze-drying;

[0008] S3, dissolving the product of the previous step and Tet1 in PBS (pH=7.0) at a molar ratio of 1:1.5 to 1:10, and reacting at room temperature in the dark for 12 h;

[0009] S4, dissolving the product of the previous step and KLVFFAED in PBS (pH=7.0) at a molar ratio of 1:1.5 to 1:10, and reacting at room temperature in the dark for 12 hours;

[0010] S5, centrifuge the product of the previous step at 8000-12000g for 15 min, discard the supernatant and wash with PBS (pH=7.0), perform chromatography detection until there is no free polypeptide, and freeze-dry;

[0011] S6. Dissolve the product of the previous step and the prepared circular or linear DNA in DNase-free water at a molar ratio of 1:0.5 to 1:2. Mix well and react in the dark for 30 minutes. Centrifuge at 8000-12000g for 15 minutes. Discard the supernatant and wash with DNase-free water. Perform chromatographic detection until there is no free DNA and freeze-dry.

[0012] In a preferred embodiment, the molar ratio of DGL to NHS-PEG-NHS is 1:5; the molar ratio of PEG-modified DGL to KLVFFAED and Tet1 is 1:5; the molar ratio of NHS-PEG-NHS, KLVFFAED and Tet1-modified DGL to circular or linear DNA is 1:2.

[0013] In a preferred embodiment, the administration of the nucleic acid drug includes one or more combinations of oral administration, transdermal absorption, respiratory inhalation, subcutaneous injection, intramuscular injection, and intravenous injection.

[0014] In a preferred embodiment, the particle size of the nucleic acid drug is 15-100 nm, and the dosage of the circular DNA is 0.01-20 mg / kg.

[0015] In a preferred embodiment, the in vitro preparation process of circular or linear DNA used for expressing ASO sequence in the nucleic acid drug comprises the following steps:

[0016] S1. Use PCR technology to clone promoter and poly(A) signal sequences in vitro. If circularization is required, the promoter upstream primer and poly(A) signal downstream primer retain the same restriction site;

[0017] S2, synthesize the DNA coding sequence of the specific ASO sequence by in vitro gene synthesis;

[0018] S3, connect the above fragments into a "5'-promoter-ASO sequence-poly(A) signal-3'" linear DNA sequence by overlap PCR;

[0019] S4. When circularization is required, the restriction sites upstream of the promoter and downstream of the poly(A) signal can be cut with endonucleases, and after purification, T4 DNA ligase can be used to complete DNA circularization.

[0020] In a preferred embodiment, the circular or linear DNA promoter is the neuron-specific promoter hSyn promoter, and the termination signal is the hGH poly(A) signal.

[0021] In a preferred embodiment, the ASO sequence is the reverse complementary sequence of circRNA or an RNA sequence with a homology greater than 80% to its reverse complementary sequence, and the AD intervention target is hsa_circ_0111650.

[0022] In a preferred embodiment, the ASO sequence is the reverse complementary sequence of snRNA or an RNA sequence having a homology greater than 80% to the reverse complementary sequence, and the AD intervention target is U1 snRNA.

[0023] In a preferred embodiment, the transcribed ASO sequence can achieve functional blocking of non-coding RNA by complementing the target gene nucleic acid sequence.

[0024] In a preferred embodiment, the poly(A) is selected from Synthetic poly(A) signal, SV40 late poly(A) signal, SV40 early poly(A) signal or bGH poly(A) signal; the promoter is selected from Ubi promoter, CMV promoter, CAG promoter, EF1α promoter, EFS promoter or GFAP promoter.

[0025] Beneficial effects of the present invention:

[0026] 1. The present invention designs a circular or linear DNA coding sequence, a neuron-specific promoter hSyn promoter sequence and an hGH poly(A) signal sequence that can self-transcribe the ASO sequence in the target cells, which greatly improves the stability of the nucleic acid drugs used for treatment and reduces the impact on other tissue cells. The nucleic acid chain can be safely, accurately and efficiently delivered to the target tissue cells, thereby improving the effect of the nucleic acid drugs.

[0027] 2. The circular or linear DNA prepared by the present invention and its delivery system can be enriched in the central neurons of the mammalian brain after peripheral administration, and the ASO sequence can be synthesized with the help of the enzyme system of the host cell to knock down the expression level of the target non-coding RNA in the cell.

[0028] 3. The present invention provides a new idea for AD intervention, targeting AD-related non-coding RNA. The carrier materials and nucleic acid sequences used as drugs are biodegradable in the body and will not form drug emboli. Drug administration can be achieved in a variety of ways. Through the targeting system, the drug can be selectively enriched in the central neurons of the brain to achieve more significant efficacy, while improving the safety of nucleic acid drugs in treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram of the particle size of the self-transcribed nucleic acid drug targeting the central neuron hsa_circ_0111650 of the brain according to Example 2 of the present invention;

[0030] Figure 2This is a schematic diagram of the brain targeting ability of the self-transcribed nucleic acid drug targeting the brain central neuron hsa_circ_0111650 and the in vivo metabolic cycle of the vector according to Example 3 of the present invention;

[0031] Figure 3 This is a schematic diagram of detecting the transcriptional ability and metabolic cycle of the self-transcribed nucleic acid drug targeting the central neurons of the brain hsa_circ_0111650 in Example 4 of the present invention;

[0032] Figure 4 This is a schematic diagram of the effect and safety of the self-transcribed nucleic acid drug targeting the central neuron hsa_circ_0111650 in the brain for treating AD according to Example 5 of the present invention;

[0033] Figure 5 This is a schematic diagram of the ability of the self-transcribed nucleic acid drug targeting hsa_circ_0111650 in the central neurons of the brain to inhibit the expression of human hsa_circ_0111650 homologous circRNA in the central neurons of the brain of experimental mice in Example 5 of the present invention;

[0034] Figure 6 This is a schematic diagram of the effect of the self-transcribed nucleic acid drug targeting the central neuron hsa_circ_0111650 in alleviating the blood-brain barrier damage of AD according to Example 6 of the present invention;

[0035] Figure 7 This is a schematic diagram of the preparation process of the self-transcribed nucleic acid drug targeting U1snRNA in central neurons of the brain, the brain targeting efficiency, and the improvement of cognitive impairment in PS1 / APP mice according to Example 7 of the present invention. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Example 1

[0038] A method for preparing a nucleic acid drug targeting non-coding RNA in central neurons of the brain, comprising the following steps:

[0039] S1. First, nanosized DGL and NHS-PEG-NHS were dissolved in phosphate buffer (PBS, pH=7.0) at a molar ratio of 1:2.5 to 1:18, and stirred at room temperature in the dark for 12 h to covalently link the active group -NHS to DGL to obtain NHS-PEG-NHS-modified DGL nanoparticles;

[0040] S2, centrifuging the product of the previous step at 8000-12000g for 15 min, discarding the supernatant and washing with PBS (pH=7.0), chromatographically detecting until there is no free NHS-PEG-NHS, and freeze-drying;

[0041] S3, dissolving the product of the previous step and Tet1 in PBS (pH=7.0) at a molar ratio of 1:1.5 to 1:10, and reacting at room temperature in the dark for 12 h;

[0042] S4, dissolving the product of the previous step and KLVFFAED in PBS (pH=7.0) at a molar ratio of 1:1.5 to 1:10, and reacting at room temperature in the dark for 12 hours;

[0043] S5, centrifuge the product of the previous step at 8000-12000g for 15 min, discard the supernatant and wash with PBS (pH=7.0), perform chromatography detection until there is no free polypeptide, and freeze-dry;

[0044] S6. Dissolve the product of the previous step and the prepared circular or linear DNA in DNase-free water at a molar ratio of 1:0.5 to 1:2. Mix well and react in the dark for 30 minutes. Centrifuge at 8000-12000g for 15 minutes. Discard the supernatant and wash with DNase-free water. Perform chromatographic detection until there is no free DNA and freeze-dry.

[0045] The molar ratio of DGL to NHS-PEG-NHS is 1:5; the molar ratio of PEG-modified DGL to KLVFFAED and Tet1 is 1:5; the molar ratio of NHS-PEG-NHS, KLVFFAED and Tet1-modified DGL to circular or linear DNA is 1:2. The administration method of the nucleic acid drug includes one or more of oral administration, transdermal absorption, respiratory inhalation, subcutaneous injection, intramuscular injection and intravenous injection. The particle size of the nucleic acid drug is 15-100 nm, and the dosage of DNA is 0.01-20 mg / kg.

[0046] The in vitro preparation process of circular or linear DNA used for expressing ASO sequence in nucleic acid drugs includes the following steps:

[0047] S1. Use PCR technology to clone promoter and poly(A) signal sequences in vitro. If circularization is required, the promoter upstream primer and poly(A) signal downstream primer retain the same restriction site;

[0048] S2, synthesize the DNA coding sequence of the specific ASO sequence by in vitro gene synthesis;

[0049] S3, connect the above fragments into a "5'-promoter-ASO sequence-poly(A) signal-3'" linear DNA sequence by overlap PCR;

[0050] S4. When circularization is required, the restriction sites upstream of the promoter and downstream of the poly(A) signal can be cut with endonucleases, and after purification, T4 DNA ligase can be used to complete DNA circularization.

[0051] Wherein, the circular or linear DNA promoter is a neuron-specific promoter hSyn promoter, and the termination signal is hGH poly (A) signal.

[0052] The ASO sequence is a reverse complementary sequence of a non-coding RNA such as snRNA, circRNA, lncRNA, miRNA, or an RNA sequence with a homology greater than 80% to its reverse complementary sequence. The AD intervention target is preferably hsa_circ_0111650 or U1 snRNA. The transcribed ASO sequence can achieve functional blocking of the non-coding RNA by complementary pairing with the target gene nucleic acid sequence. The poly(A) is selected from Synthetic poly(A) signal, SV40 late poly(A) signal, SV40 earlypoly(A) signal, or bGH poly(A) signal; the promoter is selected from Ubi promoter, CMV promoter, CAG promoter, EF1α promoter, EFS promoter, or GFAP promoter.

[0053] Among them, a delivery system involving a linear or circular DNA component can be enriched in central neurons of the brain and endogenously synthesize RNA sequences in the cells of the administration object; the RNA sequence is an ASO sequence of a target non-coding RNA with medical significance or a sequence with a homology greater than 80% thereto, the delivery system is a non-viral vector, including liposome vectors, cationic polymer vectors, nanoparticle vectors, multifunctional envelope nanocarriers, exosomes, lysosomes, Golgi bodies, artificial nanovesicles, etc., the delivery vector is preferably a polylysine (DGL) nanoparticle, the delivery system also has a brain targeting structure for locating and binding to central neurons of the brain, and delivering the carried DNA coding sequence into cells, the targeting tag of the targeting structure of the delivery system is selected from a targeting polypeptide or protein with a brain targeting function, the targeting peptide is an Aβ-derived blood-brain barrier-crossing peptide KLVFFAED targeting the RAGE receptor and a central neuron targeting peptide tetanus toxin mimetic peptide Tet1, and the delivery system is a delivery system for mammals.

[0054] The nucleic acid drug provided above uses nano-DGL as the basic carrier, and uses the small molecule polypeptide KLVFFAED derived from the natural human Aβ sequence and the tetanus toxin mimetic peptide Tet1 as ligands for achieving cross-blood-brain barrier and brain central neuron targeting. The surface of DGL is modified with succinimide double-modified bifunctional polyethylene glycol (NHS-PEG-NHS), which is beneficial for the subsequent modification of the targeting polypeptide and avoids DGL being engulfed by the reticuloendothelial system, and is also beneficial for increasing the in vivo circulation time and reducing the cytotoxicity of the carrier. The modified DGL nanoparticles are combined with the circular or linear DNA used to express the ASO sequence through electrostatic adsorption, which reduces the immunogenicity of the DNA and increases its in vivo circulation time and bioavailability.

[0055] Preparation of circular DNA expressing the hsa_circ_0111650ASO sequence.

[0056] The detailed implementation steps are as follows:

[0057] (1) Synthesize hSyn promoter upstream primer (SEQ ID NO.1) carrying EcoR I restriction site, hSyn promoter downstream primer (SEQ ID NO.3) carrying upstream homologous sequence of hsa_circ_0111650ASO coding sequence (SEQ ID NO.2), hsa_circ_0111650ASO sequence upstream primer (SEQ ID NO.4) carrying downstream homologous sequence of hSyn promoter, hsa_circ_0111650ASO sequence upstream primer (SEQ ID NO.5) carrying upstream homologous sequence of hGH poly(A) signal, hGH poly(A) signal upstream primer (SEQ ID NO.6) carrying downstream homologous sequence of hsa_circ_0111650ASO sequence, and hGH poly(A) signal downstream primer (SEQ ID NO.7) carrying EcoR I restriction site, and use Primer The Max DNA Polymerase Kit is used to prepare the PCR reaction solution according to the following system:

[0058] Reagents Usage Final concentration PrimeSTAR Max Premix (2X) 25μl 1X Primer 1 10~15pmol 0.2~0.3μM Primer 2 10~15pmol 0.2~0.3μM Template <200ng Sterilized water Up to 50μl

[0059] Carry out PCR reaction according to the following reaction steps:

[0060]

[0061] Obtain the DNA fragments of each component of the hSyn promoter with an EcoR I restriction site upstream and the hGH poly(A) signal with an EcoR I restriction site downstream.

[0062] (2) Purify the PCR product using the TaKaRa MiniBEST DNA Fragment Purification Kit, and then prepare the overlap PCR reaction solution according to the following system:

[0063] PrimeSTAR Max Premix (2×) 25μL PCR product 1-hSynpromoter 500ng PCR product 2-hGH poly(A)signal 500ng ASO sequence DNA template 500ng Sterilized water up to 50μL

[0064] Place the prepared reaction solution in a PCR instrument, incubate at 98°C for 60 seconds, incubate at 72°C for 60 seconds, and repeat three times.

[0065] (3) The above reaction product was subjected to agarose gel electrophoresis, the target fragment was isolated by cutting the gel, and the linear DNA fragment "5'-hSyn promoter-ASO sequence-hGH poly(A) signal-3'" with EcoR I restriction sites upstream and downstream was recovered using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit.

[0066] (4) The DNA fragments were digested with EcoR I endonuclease, subjected to agarose gel electrophoresis, and the target fragments were separated by gel excision. The nucleic acid fragments were recovered using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit, and then DNA chain circularization was completed using T4 DNA ligase.

[0067] Using Plasmid Safe TM ATP Dependent DNase was used to remove residual linear DNA, and then the circular DNA fragments were purified using TaKaRaMiniBEST DNA Fragment Purification Kit. Sanger sequencing confirmed that the base sequence of the connected nucleic acid chain was correct.

[0068] Example 2

[0069] Preparation of self-transcribed nucleic acid drugs targeting hsa_circ_0111650 in central neurons of the brain.

[0070] The detailed implementation steps are as follows:

[0071] (1) Nanosized DGL particles and NHS-PEG-NHS were dissolved in PBS (pH = 7.0) at a molar ratio of 1:5, and incubated with stirring at room temperature in the dark for 12 h to covalently link the active group -NHS with DGL to obtain NHS-PEG-NHS-modified DGL nanoparticles.

[0072] (2) The NHS-PEG-NHS modified DGL nanoparticles were centrifuged at 8000 g for 15 min at room temperature, the supernatant was discarded and washed with PBS (pH = 7.0), chromatographically detected until there was no free PEG, and then freeze-dried.

[0073] (3) The dried NHS-PEG-NHS-modified DGL nanoparticles and Tet1 were dissolved in PBS (pH = 7.0) at a molar ratio of 1:5, and reacted at room temperature in the dark for 12 h; then KLVFFAED was added at a molar ratio of 1:5, and reacted at room temperature in the dark for 12 h to obtain NHS-PEG-NHS, KLVFFAED and Tet1-modified DGL nanoparticles.

[0074] (4) The reaction solution was centrifuged at 8000 g for 15 min at room temperature, the supernatant was discarded and washed with PBS (pH = 7.0), and chromatographic detection was performed until no free polypeptide was found, and then freeze-dried.

[0075] (5) The DGL nanoparticles modified with PEG, KLVFFAED and Tet1 and the circular DNA prepared in Example 1 were dissolved in DNase-free water at a molar ratio of 1:2, mixed and reacted in the dark for 30 minutes, centrifuged at 8000 g for 15 minutes at room temperature, the supernatant was discarded and washed with DNase-free water, and the chromatographic detection was performed until there was no free circular DNA, and freeze-dried to obtain a targeted nucleic acid drug targeting the central neuron hsa_circ_0111650 in the brain. The particle size of the prepared nucleic acid drug was determined by dynamic light scattering to be in the range of 15-100 nm ( Figure 1 ).

[0076] Example 3

[0077] The brain targeting ability of the self-transcribed nucleic acid drug targeting hsa_circ_0111650 in the central neurons of the brain and the in vivo metabolism time of the vector.

[0078] Twelve-month-old PS1 / APP mice with a C57BL / 6J genetic background were depilated and injected with the fluorescently labeled nucleic acid drug once via the tail vein at a dose of 5 mg / kg / d. The in vivo distribution of DGL nanoparticles was observed by in vitro imaging before injection and 4h, 12h, and 24h after injection to evaluate the brain targeting efficiency and in vivo metabolism time.

[0079] Test results: The nucleic acid drug constructed by the present invention with DGL as a carrier showed good brain tissue tropism distribution and the ability to be completely metabolized. After 24 hours, the DGL nanocarrier was completely metabolized in the body ( Figure 2 ). It indicates that the fluorescently labeled nucleic acid drug of the vector injected through the tail vein can be enriched in the brain of the AD model mouse with PS1 / APP mutation in the C57BL / 6J gene background, and the vector nanoparticles are completely metabolized after 24 hours.

[0080] Example 4

[0081] Detection of the level of ASO sequence expressed in the central neurons of the brain of PS1 / APP mice by self-transcribed nucleic acid drugs targeting hsa_circ_0111650 in the central neurons of the brain.

[0082] 12-month-old PS1 / APP mice with C57BL / 6J gene background were injected with nucleic acid drugs once through the tail vein at a dose of 5 mg / kg / d. After the injection, the experimental mice were killed by cervical dislocation at 7 days, 14 days, 1 month, 3 months, 6 months, 9 months and 12 months, and hippocampal neurons were isolated. The detailed operation steps for hippocampal neuron isolation are as follows: After killing the mice, the brain tissue was removed, the temporal cortex of the brain was peeled off from the midline of the brain to expose the hippocampal tissue, the hippocampal tissue was removed and placed in pre-cooled PBS, the microvessels were carefully removed and the tissue was fully chopped with ophthalmic scissors; then an equal volume of 0.125% trypsin was added and digested at 37°C for 30 minutes, shaking it gently every 5 minutes; then a few drops of fetal bovine serum were added to terminate the digestion, and the liquid was gently blown with a pipette until it became a rice paste, and then the cell suspension was collected by filtering with a 200-mesh cell sieve. The cells were resuspended in DMEM / F12 complete medium and the cell density was adjusted to 10 5 / mL; after 6 hours of culture at 37°C, the DMEM / F12 complete medium was replaced with a neuron-specific medium. After 48 hours, cytarabine was added to inhibit the growth of non-neuronal cells, and then half the medium was replaced every 3 days. After 7 days of culture, the medium was discarded, the cells were washed twice with PBS, total RNA was extracted with Trizol, and cDNA was synthesized using Random 6-mer primers. Premix Ex Taq TM II was used to detect the expression of target circRNA. Total DNA extraction was performed using TaKaRa MiniBESTUniversal Genomic DNA Extraction Kit, and RT-qPCR was performed using TB Premix ExTaq TM II is carried out.

[0083] Test results: After a single injection of nucleic acid drugs, the expression of ASO sequences can be detected in the hippocampal central neurons of 12-month-old PS1 / APP mice. The expression level is stable in the first 6 months, and the expression level decreases at 9 months. No obvious ASO expression can be detected at 12 months ( Figure 3 A). DNA RT-qPCR results showed that the level of circular DNA in the central neurons of the hippocampus remained stable at a high level in the first 6 months, then decreased at 9 months and was completely metabolized at 12 months ( Figure 3 B). It shows that the nucleic acid drug prepared by the present invention has high stability, strong brain targeting and can be completely metabolized after drug withdrawal. After a single injection of PS1 / APP mice via the tail vein, the circular DNA can stably exist in the central neurons of the brain and continue to stably express the ASO sequence for more than 6 months. After 12 months, the circular DNA is completely metabolized and stops expressing the ASO sequence.

[0084] Example 5

[0085] The efficacy and safety of self-transcribed nucleic acid drugs targeting hsa_circ_0111650 in central neurons of the brain in alleviating cognitive impairment in AD.

[0086] The 12-month-old wild-type (WT) and PS1 / APP mice with C57BL / 6J genetic background were divided into four groups: the wild-type control group, which was injected with PBS through the tail vein every day, defined as the WT-PBS group; the AD model group, which was injected with PBS through the tail vein every day, defined as the PS1 / APP-PBS group; the AD sham intervention group, which was injected with NHS-PEG-NHS, KLVFFAED and Tet1-modified DGL nanoparticles without circular DNA through the tail vein at a dose of 0.5 mg / kg / d every day, defined as the PS1 / APP-DGL group; the AD intervention group, which was injected with NHS-PEG-NHS, KLVFFAED and Tet1-modified DGL nanoparticles containing circular DNA through the tail vein every day at a dose of 0.5 mg / kg / d, defined as the PS1 / APP-DGL circDNA group, for a total of 12 injections. Three months after the injection, the learning and memory ability of the experimental mice was tested by Morris water maze. Morris water maze is an experimental method that forces experimental animals (usually rats or mice) to swim in water and trains them to learn to find a platform hidden in the water to avoid swimming. It is mainly used to test the spatial position sense and spatial positioning learning and memory ability of experimental animals. The water maze experimental process is shown in the following table:

[0087]

[0088] After the water maze experiment, HE staining was used to detect the damage of the main organs of the experimental mice to confirm the toxicity of the self-transcribed nucleic acid drug targeting the central neurons of the brain hsa_circ_0111650 on the main organs. After the mice were anesthetized and killed, the perfused heart, liver, spleen, lung, and kidney were removed, soaked in 4% PFA overnight, soaked in 20% and 30% sucrose solutions for 1 day, and soaked in 40% sucrose solution for 3 days. Tissue sections with a thickness of 15 μm were cut by frozen section. Staining steps: Wash the sections in water for 5 minutes, then stain with hematoxylin for 3 minutes, and then wash with water 3 times, each time for 1 minute; differentiate the sections in 1% hydrochloric acid alcohol for 1 minute, then place in 0.5% ammonia water for 10 minutes, wash with water 3 times, each time for 1 minute; stain in eosin for 3 minutes, wash with water 3 times, each time for 1 minute; dehydrate in gradient ethanol (75%, 85%, 95%, 100%, 100%) for 1 minute each, and finally place in xylene to clarify for 5 minutes, seal with 50% glycerol, and observe the image under an upright microscope.

[0089] Detection of the expression level of human hsa_circ_0111650 homologous circRNA in mouse hippocampal neurons: After anesthetizing and killing the mice, the brain tissue was removed, the hippocampal neurons were separated, and the total RNA was extracted. Random 6-mer random primers were used to synthesize cDNA, and RT-qPCR was used to detect the expression level of human hsa_circ_0111650 homologous circRNA in the hippocampal neurons of PS1 / APP mice.

[0090] Test results: The self-transcribed nucleic acid drug prepared by the present invention can effectively alleviate the cognitive impairment of PS1 / APP mice after being injected into the tail vein. Figure 4 A) and no obvious organic damage to major organs ( Figure 4 B). The expression of circRNA homologous to human hsa_circ_0111650 in hippocampal neurons of experimental mice was effectively inhibited ( Figure 5 ), is an AD intervention strategy with great application potential. After administration, the cognitive impairment of PS1 / APP mice was improved and there was no obvious organic damage to the main organs of the experimental mice. The nanoparticles had good biosafety, and the self-transcribed nucleic acid drug could effectively inhibit the expression of target circRNA in the central neurons of the brain.

[0091] Example 6

[0092] The effectiveness of self-transcribed nucleic acid drugs targeting hsa_circ_0111650 in central neurons of the brain in alleviating blood-brain barrier damage in AD.

[0093] 12-month-old WT and PS1 / APP mice with C57BL / 6J genetic background were divided into three groups, namely WT-PBS, WT-DGL circDNA and PS1 / APP-DGL circDNA. The WT-DGL circDNA and PS1 / APP-DGL circDNA groups were injected with 0.5 mg / kg / d of self-transcribed nucleic acid drugs targeting hsa_circ_0111650 in central neurons of the brain through the tail vein 12 times daily, and the WT-PBS group was injected with the same dose of PBS 12 times. The mice were killed before injection and 2, 4, 6, and 8 months after the injection, and brain tissues were obtained. The hemoglobin level in the brain was detected using the Drabkin kit to confirm the reverse osmosis of blood through the blood-brain barrier.

[0094] Test results: After the injection of nucleic acid drugs, the hemoglobin reverse osmosis of the experimental mouse brain tissue gradually decreased ( Figure 6 ), indicating that it has the ability to repair blood-brain barrier damage caused by AD.

[0095] Example 7

[0096] The ability of self-transcribed nucleic acid drugs targeting U1 snRNA in central neurons of the brain to alleviate cognitive impairment in AD.

[0097] The methods described in Examples 1 and 2 were used to prepare brain neuron-targeted nanoparticles carrying the U1 snRNA ASO linear DNA coding sequence (SEQ ID NO. 8), and the methods described in Examples 3 and 5 were used to detect the brain targeting ability of the drug and its ability to alleviate AD cognitive impairment.

[0098] Test results: After the preparation of the self-transcribed nucleic acid drug targeting U1 snRNA in the central neurons of the brain, it was injected into 12-month-old PS1 / APP mice via the tail vein. In vitro imaging showed good brain targeting ability and effectively alleviated the cognitive impairment of PS1 / APP mice ( Figure 7 ).

[0099] Finally, a few points should be explained: First, in the description of this application, it should be noted that, unless otherwise specified and limited, the term "connection" should be understood in a broad sense, which can be a mechanical connection or an electrical connection, or the internal communication of two components, or a direct connection; the terms "upstream" and "downstream" are only used to indicate a relative position relationship, and when the absolute position of the object being described changes, the relative position relationship may change;

[0100] Secondly: In the drawings of the embodiments disclosed in the present invention, only the structures related to the embodiments disclosed in the present invention are involved, and other structures can refer to the general design. In the absence of conflict, the same embodiment and different embodiments of the present invention can be combined with each other;

[0101] Finally: The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a nucleic acid drug targeting non-coding RNA in central neurons of the brain, characterized in that: The steps include: S1. First, nanosized DGL and NHS-PEG-NHS were dissolved in phosphate buffer (PBS, pH=7.0) at a molar ratio of 1:2.5 to 1:18, and stirred at room temperature in the dark for 12 h to covalently link the active group -NHS to DGL to obtain NHS-PEG-NHS-modified DGL nanoparticles; S2, centrifuging the product of the previous step at 8000-12000g for 15 min, discarding the supernatant and washing with PBS (pH=7.0), chromatographically detecting until there is no free NHS-PEG-NHS, and freeze-drying; S3, dissolving the product of the previous step and Tet1 in PBS (pH=7.0) at a molar ratio of 1:1.5 to 1:10, and reacting at room temperature in the dark for 12 h; S4, dissolving the product of the previous step and KLVFFAED in PBS (pH=7.0) at a molar ratio of 1:1.5 to 1:10, and reacting at room temperature in the dark for 12 hours; S5, centrifuge the product of the previous step at 8000-12000g for 15 min, discard the supernatant and wash with PBS (pH=7.0), perform chromatography detection until there is no free polypeptide, and freeze-dry; S6. Dissolve the product of the previous step and the prepared circular or linear DNA in DNase-free water at a molar ratio of 1:0.5 to 1:

2. Mix well and react in the dark for 30 minutes. Centrifuge at 8000-12000g for 15 minutes. Discard the supernatant and wash with DNase-free water. Perform chromatographic detection until there is no free DNA and freeze-dry.

2. The method for preparing a nucleic acid drug targeting non-coding RNA in central neurons of the brain according to claim 1, characterized in that: The molar ratio of DGL to NHS-PEG-NHS is 1:5; the molar ratio of PEG-modified DGL to KLVFFAED and Tet1 is 1:5; the molar ratio of NHS-PEG-NHS, KLVFFAED and Tet1-modified DGL to circular or linear DNA is 1:

2.

3. The method for preparing a nucleic acid drug targeting non-coding RNA in central neurons of the brain according to claim 1, characterized in that: The administration method of the nucleic acid drug includes one or more combinations of oral administration, transdermal absorption, respiratory inhalation, subcutaneous injection, intramuscular injection, and intravenous injection.

4. The method for preparing a nucleic acid drug targeting non-coding RNA in central neurons of the brain according to claim 1, characterized in that: The particle size of the nucleic acid drug is 15-100 nm, and the dosage of DNA is 0.01-20 mg / kg.

5. The method for preparing a nucleic acid drug targeting non-coding RNA in central neurons of the brain according to claim 1, characterized in that: The in vitro preparation process of circular or linear DNA used for expressing ASO sequence by nucleic acid drugs includes the following steps: S1. Use PCR technology to clone promoter and poly(A) signal sequences in vitro. If circularization is required, the promoter upstream primer and poly(A) signal downstream primer retain the same restriction site; S2, synthesize the DNA coding sequence of the specific ASO sequence by in vitro gene synthesis; S3, connect the above fragments into a "5'-promoter-ASO sequence-poly(A) signal-3'" linear DNA sequence by overlap PCR; S4. When circularization is required, the restriction sites upstream of the promoter and downstream of the poly(A) signal can be cut with endonucleases, and after purification, T4 DNA ligase can be used to complete DNA circularization.

6. The method for preparing a nucleic acid drug targeting non-coding RNA in central neurons of the brain according to claim 5, characterized in that: The circular or linear DNA promoter is a neuron-specific promoter hSyn promoter, and the termination signal is hGH poly (A) signal.

7. The method for preparing a nucleic acid drug targeting non-coding RNA in central neurons of the brain according to claim 5, characterized in that: The ASO sequence is the reverse complementary sequence of snRNA, circRNA, lncRNA, miRNA non-coding RNA or an RNA sequence with a homology greater than 80% to its reverse complementary sequence, and the AD intervention targets are preferably hsa_circ_0111650 and U1snRNA.

8. The method for preparing a nucleic acid drug targeting non-coding RNA in central neurons of the brain according to claim 5, characterized in that: The transcribed ASO sequence can achieve functional blocking of non-coding RNA by complementary pairing with the target gene nucleic acid sequence.

9. The method for preparing a nucleic acid drug targeting non-coding RNA in central neurons of the brain according to claim 5, characterized in that: The poly(A) is selected from Synthetic poly(A) signal, SV40late poly(A) signal, SV40early poly(A) signal or bGH poly(A) signal; the promoter is selected from Ubi promoter, CMV promoter, CAG promoter, EF1α promoter, EFS promoter or GFAP promoter.

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