CRISPR / dCas9-SunTag mediated RBM25 controllable activation system and application thereof in ischemic heart failure myocardial repair

Abstract

The invention relates to the field of gene therapy, in particular to an RBM25 gene epigenetics activation technology based on a CRISPR / dCas9-SunTag system and application of the RBM25 gene epigenetics activation technology in ischemic heart failure treatment, the RBM25 gene epigenetics activation technology comprises the following steps: (a) protein containing inactivated Cas9 (dCas9) fused with a plurality of GCN4 peptide repetitive sequences, the number of the GCN4 peptide repetitive sequences is 8-12, and the dCas9 contains D10A and H840A double mutations; (b) a protein containing fusion of an anti-GCN4 single-chain antibody fragment (scFv) and a TET1 catalytic structural domain; (c) at least one gRNA of a target RBM25 gene promoter region, wherein a target sequence of the gRNA is selected from a sequence as shown in SEQ ID NO: 1-3; and (d) a lipid nanoparticle (LNP) system for delivery of said (a), (b), and (c).

Description

Technical Field

[0001] The present invention relates to the field of gene therapy, and particularly to an epigenetic activation technology of RBM25 gene based on the CRISPR / dCas9-SunTag system and its application in the treatment of ischemic heart failure. Background Art

[0002] Ischemic heart failure is caused by myocardial cell damage and dysfunction due to coronary artery disease, and is one of the main causes of death in cardiovascular diseases. Traditional treatment methods include drug therapy, interventional therapy and surgery, but these methods cannot achieve the regeneration and reconstruction of myocardial tissue.

[0003] Gene therapy provides a new treatment idea for ischemic heart failure, especially the development of epigenetic regulation technology makes it possible to precisely regulate gene expression. Among them, the CRISPR / Cas9 system has been widely used in the field of gene editing due to its high efficiency and specificity. And dCas9 (inactivated Cas9) can be used to target specific gene loci without causing DNA breakage, and combined with other functional domains, it can achieve precise regulation of gene expression.

[0004] However, the main challenges faced by current gene therapy include low delivery efficiency, poor targeting and insufficient controllability. Especially for myocardial tissue, due to its special anatomical and physiological characteristics, efficient targeted delivery is still an urgent problem to be solved.

[0005] Recent studies have found that the RNA-binding protein RBM25 may play an important role in myocardial cell survival and angiogenesis, but its potential in myocardial repair has not been fully developed. Therefore, developing a treatment system that can precisely target and controllably activate the RBM25 gene is of great significance for the gene therapy of ischemic heart failure. Summary of the Invention

[0006] The object of the present invention is to provide a controllable activation technology of RBM25 gene based on the CRISPR / dCas9-SunTag system and its delivery system for the treatment of ischemic heart failure.

[0007] The first aspect of the present invention provides a composition for activating the expression of the RBM25 gene in cardiomyocytes, comprising: (a) a protein comprising inactivated Cas9 (dCas9) fused with a plurality of GCN4 peptide repeats, the number of GCN4 peptide repeats being 8 - 12, and the dCas9 containing D10A and H840A double mutations; (b) a protein comprising an anti-GCN4 single-chain antibody fragment (scFv) fused with the TET1 catalytic domain; (c) at least one gRNA targeting the promoter region of the RBM25 gene, wherein the targeting sequence of the gRNA is selected from the sequences shown in SEQ ID NO: 1 - 3; and (d) a lipid nanoparticle (LNP) system for delivering (a), (b), and (c), the LNP system comprising an ionizable lipid, a helper lipid, cholesterol, and a PEGylated lipid, wherein the mass ratio of the ionizable lipid, the helper lipid, cholesterol, and the PEGylated lipid is 45 - 55:35 - 42:8 - 12:1 - 2.

[0008] In some embodiments of the present invention, the ionizable lipid is SM-102, the helper lipid is DSPC, and the PEGylated lipid is DMG-PEG2000.

[0009] In some embodiments of the present invention, the LNP system further comprises CRPPR cardiomyocyte-targeting peptide modification, and the CRPPR cardiomyocyte-targeting peptide is conjugated with the PEGylated lipid at a molar ratio of 0.1 - 0.3%.

[0010] In some embodiments of the present invention, (a) and (b) are provided in the form of mRNA, the mRNA comprising a 5' cap structure, an optimized 5' UTR, a coding sequence, an optimized 3' UTR, and a poly-A tail, and the uridine in the mRNA is completely replaced by 5-methoxyuridine.

[0011] In some embodiments of the present invention, the composition further comprises ultrasound microbubbles, the ultrasound microbubbles comprising: (i) a lipid shell layer formed by 60 - 65% (w / w) of DPPC, 4 - 6% (w / w) of DSPE-PEG2000, and 30 - 35% (w / w) of cholesterol; and (ii) a core of sulfur hexafluoride gas; wherein the particle size of the ultrasound microbubbles is 1.0 - 2.0 μm.

[0012] The second aspect of the present invention provides a method for preparing the above-mentioned composition, comprising the following steps: (a) cloning the coding sequences of dCas9-SunTag, scFv-TET1 and gRNA into expression vectors respectively; (b) preparing dCas9-SunTag mRNA and scFv-TET1 mRNA by in vitro transcription, including: (i) linearizing the expression vector using a restriction endonuclease; (ii) performing a transcription reaction using an in vitro transcription kit, and the reaction system contains an ARCA cap analogue, ATP, CTP, GTP and 5-methoxy UTP; (iii) treating with DNase I to remove template DNA; and (iv) purifying and quantifying the obtained mRNA; (c) preparing lipid nanoparticles, including: (i) dissolving an ionic lipid, a co-lipid, cholesterol and a PEGylated lipid in ethanol; (ii) dissolving dCas9-SunTag mRNA, scFv-TET1 mRNA and gRNA in sodium acetate buffer at pH 4.0 according to a mass ratio of 1:1:3; (iii) using a microfluidic mixing device to mix at a flow rate ratio of 1:3 (lipid:RNA); and (iv) dialyzing to remove the organic solvent and adjusting the pH to 7.4; and (d) when ultrasonic microbubbles are included, further comprising the following steps: (i) preparing a lipid film composed of DPPC, DSPE-PEG2000 and cholesterol; (ii) hydrating the lipid film and performing ultrasonic treatment; (iii) replacing the air with sulfur hexafluoride gas; and (iv) oscillating to prepare microbubbles and performing size screening.

[0013] The third aspect of the present invention provides a method for treating ischemic heart failure, comprising administering the above-mentioned composition to a patient, wherein: (a) the composition is administered by intravenous injection; and (b) ultrasonic waves are applied to the cardiac region of the patient within 5 minutes after the injection, and the ultrasonic parameters are: frequency 0.8 - 1.2 MHz, intensity 1.0 - 2.0 W / cm 2 , duty cycle 40 - 60%, duration 3 - 7 minutes.

[0014] In some embodiments of the present invention, the method further comprises administering a tetracycline compound to the patient to induce the expression of dCas9-SunTag, and the tetracycline compound is doxycycline, which is administered at a dose of 2 - 10 mg / kg / day.

[0015] In some embodiments of the present invention, the composition is administered within 3 - 7 days after myocardial infarction in the patient.

[0016] The fourth aspect of the present invention provides a system for in vitro activation of RBM25 gene expression, comprising: (a) a first expression vector containing the dCas9-SunTag-2A-mCherry coding sequence controlled by the TRE3G promoter; (b) a second expression vector containing the scFv-TET1-P2A-Hygro coding sequence controlled by the EF1α promoter; and (c) a third expression vector containing the gRNA coding sequence targeting the RBM25 promoter region controlled by the U6 promoter; wherein the expression of the dCas9-SunTag-2A-mCherry is induced and controlled by tetracycline or its derivatives. Detailed implementation manners

[0017] The present invention will be described in detail below through specific embodiments, but the protection scope of the present invention is not limited thereto.

[0018] Example 1: Construction of the dCas9-SunTag-mCherry expression vector

[0019] This example relates to a method for constructing a dCas9-SunTag-mCherry expression vector containing the TRE3G inducible promoter.

[0020] First, based on Addgene #61425 (dCas9) and #60903 (SunTag) as templates, the dCas9-SunTag fusion gene was obtained by gene synthesis. Adapter primers were designed, the forward primer was 5'-AAGCTTGCCACCATGGACAAGAAGTACAGCATCGGCCTGG-3' (containing HindIII site and Kozak sequence),

[0021] The reverse primer was:

[0022] 5'-GGATCCCTTGTACAGCTCGTCCATGCCGCCGGTGGAGTGG-3' (containing BamHI site). The PCR reaction conditions were: pre-denaturation at 98°C for 30 seconds; followed by 25 cycles of denaturation at 98°C for 10 seconds, annealing at 65°C for 30 seconds, and extension at 72°C for 5 minutes; finally, final extension at 72°C for 10 minutes. Amplification was carried out using Phusion High-Fidelity DNA polymerase (NEB, M0530).

[0023] Secondly, the P2A-mCherry fragment was constructed using the mCherry vector (Addgene #54631) as a template. Adapter primers were designed, the forward primer was:

[0024] 5'-GGATCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCCATGGTGAGCAAGGGCGAGGAG-3' (containing BamHI site and P2A sequence),

[0025] The reverse primer is 5'-GCGGCCGCTTACTTGTACAGCTCGTCCATGCC-3' (containing NotI site). PCR conditions are as follows: pre-denaturation at 98°C for 30 seconds; then 30 cycles of denaturation at 98°C for 10 seconds, annealing at 62°C for 30 seconds, and extension at 72°C for 1 minute; finally, final extension at 72°C for 5 minutes.

[0026] Then, the dCas9-SunTag and P2A-mCherry fragments were digested with HindIII / BamHI and BamHI / NotI restriction endonucleases respectively. The two fragments were ligated with the HindIII / NotI-digested pAAV-TRE3G backbone vector in a three-fragment ligation reaction, which was carried out at 16°C for 12 hours using T4 DNA ligase (NEB, M0202).

[0027] Finally, correct construction was verified by restriction enzyme digestion and Sanger sequencing, and the plasmid was purified using the EndoFree Plasmid MaxiKit (Qiagen, 12362). This expression vector was named pAAV-TRE3G-dCas9-SunTag-2A-mCherry, in which the expression of dCas9-SunTag is under the inducible control of doxycycline (Dox), and the expression can be monitored by the mCherry fluorescence signal.

[0028] In the constructed dCas9-SunTag protein, dCas9 contains double mutations of D10A and H840A, which makes it lose nuclease activity but retain DNA binding ability; SunTag contains 10 GCN4 peptide repeats, and each GCN4 peptide sequence is EELLSKNYHLENEVARLKK, and the peptide segments are connected by (GGGGS)3 flexible linker peptides. This design enables each dCas9 molecule to recruit up to 10 effector molecules, thus significantly amplifying the activation effect of target genes.

[0029] Example 2: Construction of scFv-TET1 expression vector

[0030] This example relates to a method for constructing an scFv-TET1 expression vector containing an EF1α promoter.

[0031] First, using Addgene #82561 as a template, adapter primers were designed. The forward primer was 5'-AAGCTTGCCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACA-3' (containing the HindIII site and Kozak sequence), and the reverse primer was 5'-GGATCCTTTCTTCTTCTTGTACACCTCCACTTTTGCCTCAGG-3' (containing the BamHI site). The PCR conditions were the same as those for the amplification of the dCas9-SunTag fragment in Example 1.

[0032] Second, a vector containing the hygromycin resistance gene was used as a template to construct the P2A-Hygro fragment. When designing the adapter primers, the P2A sequence and appropriate restriction sites were added. The PCR conditions were the same as those for the amplification of the P2A-mCherry fragment in Example 1.

[0033] Then, a three-fragment ligation strategy was adopted to ligate the scFv-TET1 and P2A-Hygro fragments to the pAAV-EF1α backbone vector. The ligation conditions were 16 °C for 12 hours, using T4 DNA ligase.

[0034] Finally, correct construction was verified by restriction enzyme digestion and Sanger sequencing, and the plasmid was purified using the EndoFree Plasmid Maxi Kit. This expression vector was named pAAV-EF1α-scFv-TET1-P2A-Hygro, in which scFv-TET1 was continuously expressed under the drive of the EF1α promoter.

[0035] In the constructed scFv-TET1 fusion protein, the scFv was derived from an anti-GCN4 single-chain antibody fragment and could specifically recognize and bind to the GCN4 peptide in the SunTag system; the TET1 catalytic domain was derived from the catalytic domain of human Ten-eleven translocation 1 protein (amino acids 1418 - 2136) and was capable of catalyzing the oxidation of DNA methylation sites to promote gene expression. The scFv and TET1 were connected by a (GGGGS)2 flexible linker peptide to ensure that the two functional domains could function independently.

[0036] Example 3: Construction of the gRNA Expression Vector Targeting the Promoter Region of RBM25

[0037] This example relates to a method for constructing a gRNA expression vector targeting the promoter region of the RBM25 gene.

[0038] First, analyze the promoter region of RBM25 (chr14:73,526,841 - 73,542,190) using the CRISPR Design Tool (https: / / crispr.mit.edu), and screen candidate gRNA sequences with an off-target score < 0.1. Select the top 3 candidate sequences for experimental verification.

[0039] Sequence 1 (SEQ ID NO:1): 5'-GCACTTGCAGGAACGTCCGA-3'

[0040] Sequence 2 (SEQ ID NO:2): 5'-GTACGCATGCCTGAGTCACC-3'

[0041] Sequence 3 (SEQ ID NO:3): 5'-GCTAGCTTGCAGCAATCCGT-3'

[0042] Secondly, design oligonucleotide pairs containing the target sequences. Taking Sequence 1 as an example, the forward oligonucleotide is 5'-CACCGCACTTGCAGGAACGTCCGA-3', and the reverse oligonucleotide is 5'-AAACTCGGACGTTCCTGCAAGTGC-3'. Anneal the oligonucleotide pairs at 95°C for 5 minutes, and then cool them to 25°C at a rate of 0.1°C / second.

[0043] Then, ligate the annealed oligonucleotide pairs with the BbsI-digested pAAV-U6-gRNA backbone vector. The ligation reaction is carried out at 16°C for 4 hours using T4 DNA ligase.

[0044] Finally, verify the correct insertion of the gRNA sequence by Sanger sequencing, and purify the plasmid using the Plasmid Mini Kit. The three constructed gRNA expression vectors are named pAAV-U6-gRNA-RBM25-1, pAAV-U6-gRNA-RBM25-2, and pAAV-U6-gRNA-RBM25-3 respectively.

[0045] These gRNAs target different positions in the promoter region of the RBM25 gene, and can specifically guide the binding of the dCas9-SunTag complex to the target site, thereby recruiting scFv-TET1 to catalyze the removal of DNA methylation and promoting the activation of RBM25 gene expression. Through experimental verification, gRNA-RBM25-1 (SEQ ID NO:1) shows the highest targeting efficiency and activation effect, with an off-target rate < 0.05.

[0046] Example 4: In vitro transcription and synthesis of dCas9-SunTag and scFv-TET1 mRNA

[0047] This example relates to an in vitro transcription and synthesis method for dCas9-SunTag and scFv-TET1 mRNA.

[0048] First, use the NotI restriction endonuclease to linearize the pAAV-TRE3G-dCas9-SunTag-2A-mCherry plasmid. The linearization reaction is carried out at 37 °C for 2 hours, and contains 5 μg plasmid DNA, 5 μL 10× buffer, 2 μL NotI enzyme (10 U / μL), and nuclease-free water up to 50 μL. After the reaction is completed, use Phenol:Chloroform (1:1) extraction and ethanol precipitation to purify the linearized template. The precipitated DNA is dissolved in RNase-free water to 1 μg / μL.

[0049] Second, use HiScribe TM T7 ARCA mRNA Kit (NEB, E2065) for in vitro transcription. The reaction system (100 μL) includes: 1 μg linear template DNA, ARCA capping analog (final concentration 4 mM), ATP, CTP, GTP each (final concentration 3 mM), 5-methoxy UTP (final concentration 3 mM), 10 μL 10× reaction buffer, 10 μL T7 RNA polymerase mixture, and nuclease-free water up to 100 μL. The reaction is carried out at 37 °C for 2 hours. Subsequently, add 2 μL of DNase I and incubate at 37 °C for 15 minutes to remove the template DNA.

[0050] Then, use the RNeasy Mini Kit (Qiagen, 74104) to purify the transcribed mRNA. Verify the integrity of the mRNA by agarose gel electrophoresis and Bioanalyzer, and use a UV spectrophotometer to measure the concentration and purity (A260 / A280 ratio > 2.0). Finally, aliquot and store the purified mRNA at -80 °C to avoid repeated freezing and thawing.

[0051] Using the same method, use the linearized pAAV-EF1α-scFv-TET1-P2A-Hygro plasmid as the template to synthesize scFv-TET1 mRNA.

[0052] Both the synthesized dCas9-SunTag mRNA and scFv-TET1 mRNA contain an ARCA cap structure, an optimized β-globin 5' UTR, the corresponding coding sequence, an optimized β-globin 3' UTR, and a poly A tail of about 120 A nucleotides. All uridines in the mRNA are completely replaced by 5-methoxyuridine, and this modification significantly improves the stability and translation efficiency of the mRNA, while reducing the immunogenicity.

[0053] Example 5: In vitro synthesis of gRNA

[0054] This example relates to a method for in vitro synthesis of gRNA targeting the promoter region of RBM25.

[0055] First, a DNA fragment containing the T7 promoter and the gRNA sequence was amplified by PCR. Using pAAV-U6-gRNA-RBM25-1 as a template, the following primers were used: forward primer 5'-TAATACGACTCACTATAGGGCACTTGCAGGAACGTCCGAGTTT-3' and reverse primer 5'-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTG-3'. The PCR conditions were: pre-denaturation at 98°C for 30 seconds; [98°C for 10 seconds, 60°C for 30 seconds, 72°C for 30 seconds] × 35 cycles; final extension at 72°C for 5 minutes.

[0056] Second, in vitro transcription was performed using the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB, E2050). The reaction system (20 μL) included: 0.5 μg of PCR product template DNA, 2 μL of NTP mixture, 2 μL of 10× reaction buffer, 2 μL of T7 RNA polymerase mixture, and nuclease-free water to 20 μL. The reaction was carried out at 37°C for 4 hours.

[0057] Then, 1 μL of DNase I was added and incubated at 37°C for 15 minutes to remove the template DNA. The gRNA was purified using the RNA Clean & Concentrator-25 Kit (ZymoResearch, R1017). The integrity of the gRNA was verified by denaturing agarose gel electrophoresis, and the concentration was measured using a spectrophotometer.

[0058] Finally, the purified gRNA was aliquoted and stored at -80°C. The same method was used for the synthesis of gRNA-RBM25-2 and gRNA-RBM25-3.

[0059] Example 6: Preparation of Cardiac-Targeting Lipid Nanoparticles (LNP) - Standard Formulation

[0060] This example relates to a method for preparing cardiac-targeting lipid nanoparticles for delivering components of the dCas9-SunTag system.

[0061] First, prepare the lipid mixture. Dissolve 50 mg of SM-102 (50% w / w), 38.5 mg of DSPC (38.5% w / w), 10 mg of cholesterol (10% w / w), and 1.5 mg of DMG-PEG2000 (1.5% w / w) in 4 mL of ethanol. Heat the mixture in a 60 °C water bath to dissolve it, and assist with 5 minutes of sonication to ensure complete dissolution.

[0062] Second, prepare the mRNA mixture. Dissolve 20 μg of dCas9-SunTag mRNA, 20 μg of scFv-TET1 mRNA, and 60 μg of gRNA-RBM25-1 (total mass ratio 1:1:3) in 4 mL of 50 mM sodium acetate buffer at pH 4.0.

[0063] Then, use a NanoAssemblr Benchtop (Precision NanoSystems) microfluidic mixing device to assemble the LNPs. Set the flow rate ratio of the lipid solution to the RNA solution to 1:3, and the total flow rate to 12 mL / min. The mixing is carried out at room temperature (20 - 25 °C).

[0064] Next, use an Amicon Ultra-15 centrifugal filter with a molecular weight cut-off of 100 kDa for purification and concentration. Replace with PBS buffer at pH 7.4 three times to ensure complete removal of ethanol and acidic buffer. Finally, adjust the LNP concentration so that the total mRNA concentration is 0.5 mg / mL.

[0065] Finally, measure the particle size and polydispersity of the LNPs by dynamic light scattering (DLS), with a target particle size of 80 - 100 nm and a polydispersity index (PDI) < 0.2. Use the RiboGreen fluorescent dye method to determine the mRNA encapsulation efficiency, with a target encapsulation efficiency > 95%. Observe the morphological characteristics of the LNPs by transmission electron microscopy (TEM).

[0066] The prepared standard LNPs have an average particle size of approximately 90 nm, a PDI of approximately 0.15, a Zeta potential of approximately +2 mV, and an mRNA encapsulation efficiency > 98%. This formulation provides good stability and high transfection efficiency, but lacks tissue-specific targeting ability.

[0067] Example 7: Preparation of Cardiomyocyte-Targeted Lipid Nanoparticles (LNPs) - Modification with CRPPR Peptide

[0068] This example relates to a method for preparing LNPs modified with the CRPPR cardiomyocyte-targeting peptide to improve the targeting to cardiomyocyte tissue.

[0069] First, synthesize CRPPR-PEG-DSPE. React 5 mg of the cyclized form of the CRPPR peptide (sequence CRPPR-GGGSC) with 20 mg of maleimide-PEG2000-DSPE in 5 mL of HEPES buffer at pH 7.0. The reaction is carried out at room temperature for 24 hours under nitrogen protection throughout the process. After the reaction is completed, dialyze for 48 hours using a dialysis membrane with a molecular weight cut-off of 10 kDa to remove the unreacted peptide.

[0070] Second, prepare the modified lipid mixture. In addition to the standard lipid components in Example 6, add CRPPR-PEG-DSPE additionally, so that its molar ratio is 0.2% (relative to the total lipids). The specific components are: 48 mg of SM-102 (48% w / w), 38.5 mg of DSPC (38.5% w / w), 11 mg of cholesterol (11% w / w), 1.5 mg of DMG-PEG2000 (1.5% w / w), and 1 mg of CRPPR-PEG-DSPE (1% w / w), dissolved in 4 mL of ethanol.

[0071] Then, prepare the mRNA mixture in the same method as in Example 6, and use a microfluidic mixing device for LNP assembly, purification, and characterization.

[0072] The prepared CRPPR peptide-modified LNP has an average particle size of about 95 nm, a PDI of about 0.18, a Zeta potential of about -1 mV, and an mRNA encapsulation efficiency > 96%. In the in vivo distribution study, the accumulation of CRPPR-modified LNP in the myocardial tissue is 3.5 times that of the unmodified LNP, significantly improving the myocardial targeting of the delivery system. This enhanced targeting is due to the ability of the CRPPR peptide to specifically bind to the receptors on the surface of cardiomyocytes, thereby promoting the endocytosis of LNP.

[0073] Example 8: Preparation of ultrasound microbubbles

[0074] This example relates to a method for preparing ultrasound microbubbles for enhancing the myocardial delivery of LNP.

[0075] First, prepare a lipid film. Mix 13 mg of DPPC (65% w / w), 1 mg of DSPE-PEG2000 (5% w / w), and 6 mg of cholesterol (30% w / w) in a 25 mL round-bottom flask, and dissolve in 5 mL of a chloroform:methanol (9:1) mixed solvent. Use a rotary evaporator to evaporate the solvent under reduced pressure at 40 °C to form a uniform lipid film. Place the flask in a high-vacuum drying system to degas for 4 hours to remove the residual solvent.

[0076] Secondly, prepare microbubbles. Add 5 mL of sterile PBS (pH 7.4) to the lipid film, and heat it in a 60 °C water bath for 10 minutes to hydrate the lipids. Perform intermittent sonication using a probe-type sonicator (power set at 20%) for a total of 10 minutes. Replace the air in the sealed container containing the lipid suspension with sulfur hexafluoride (SF6) gas. Use a Vialmix mechanical oscillator to oscillate at high speed for 45 seconds to form microbubbles.

[0077] Then, perform microbubble sorting and characterization. Remove large particles using differential centrifugation (300×g, 3 minutes). Measure the particle size distribution of the microbubbles using a Multisizer or Coulter Counter, and adjust the concentration to 2×10^9 microbubbles / mL. Further characterize the microbubbles using a fluorescence microscope and a flow cytometer.

[0078] The prepared microbubbles have an average particle size of 1.5 μm, and 90% of the microbubbles have a particle size distribution in the range of 1.0 - 2.0 μm. The microbubbles can be stably stored for 6 hours at 4 °C under SF6 gas protection. These microbubbles can produce a cavitation effect under the action of an ultrasonic field, temporarily increase vascular permeability, and significantly improve the efficiency of LNP penetrating from the vascular endothelium into the myocardial tissue.

[0079] Example 9: In Vivo Evaluation of the LNP and Ultrasound Microbubble Combined Delivery System

[0080] This example involves the evaluation of the delivery efficiency of the LNP and ultrasound microbubble combined delivery system in a mouse myocardial infarction model.

[0081] First, construct a mouse myocardial infarction model. Use 8 - 10-week-old male C57BL / 6J mice, weighing 22 - 25 g.

[0082] Example 9: In Vivo Evaluation of the LNP and Ultrasound Microbubble Combined Delivery System (continued)

[0083] First, construct a mouse myocardial infarction model. Use 8 - 10-week-old male C57BL / 6J mice, weighing 22 - 25 g. Anesthetize the mice by inhaling isoflurane (1.5 - 2%), and perform ligation of the left anterior descending coronary artery. After the operation, administer ibuprofen (10 mg / kg) for analgesia every 6 hours for 48 hours.

[0084] Secondly, implement the treatment protocol. Three days after myocardial infarction, randomly divide the mice into 4 groups (n = 10 in each group): (1) PBS control group; (2) pure LNP group (without ultrasound); (3) unmodified LNP + ultrasound group; (4) CRPPR-modified LNP + ultrasound group. Administer LNP by tail vein injection at a dose of 0.5 mg / kg (calculated based on mRNA), and simultaneously inject ultrasound microbubbles at a dose of 1×10^8 microbubbles / kg body weight.

[0085] Then, sonication was performed. It was carried out immediately after the injection of LNP and microbubbles, using a commercial diagnostic ultrasound system equipped with a linear array probe, with the frequency set at 1 MHz and the intensity at 1.5 W / cm 2 , duty cycle 50%, for 5 minutes. The ultrasound probe was aligned with the anterior chest wall of the mouse and focused on the left ventricular region.

[0086] Finally, the delivery efficiency was evaluated. At 48 hours after administration, the mice were sacrificed and heart tissues were taken. The expression of mCherry was observed by fluorescence microscopy, the expression level of RBM25 mRNA was detected by qRT-PCR, and the level of RBM25 protein was analyzed by Western Blot.

[0087] The results showed that the transfection efficiency of the CRPPR-modified LNP + ultrasound group was the highest in the myocardial tissue, reaching 70%, while that of the simple LNP group was only 20%, and that of the unmodified LNP + ultrasound group was 45%. This indicates that the combination of CRPPR peptide modification and ultrasound microbubble technology produced a significant synergistic effect, greatly improving the delivery efficiency of LNP in the myocardial tissue. The levels of RBM25 mRNA and protein also showed the same trend, and the expression level of RBM25 in the CRPPR-modified LNP + ultrasound group was 4 times that of the PBS control group.

[0088] Example 10: Study on the dose-time relationship of Dox-induced RBM25 expression

[0089] This example involves a study on the dose-time relationship of doxycycline (Dox)-induced dCas9-SunTag expression system, aiming to determine the optimal induction protocol.

[0090] First, in vitro cell experiments were carried out. HEK293T cells were transfected with CRPPR-modified LNP, and after 48 hours, different concentrations of Dox (0, 10, 100, 500, 1000 ng / mL) were added and incubated for 24, 48, and 72 hours respectively. The expression of mCherry was monitored by real-time fluorescence imaging, the level of RBM25 mRNA was detected by qRT-PCR, and the level of RBM25 protein was analyzed by Western Blot.

[0091] Secondly, in vivo mouse experiments were carried out. In mice with myocardial infarction models, CRPPR-modified LNP + ultrasound treatment was given according to the method of Example 9, and then different doses of Dox were given to the groups: (1) no Dox; (2) low-dose group (2 mg / kg / day); (3) medium-dose group (5 mg / kg / day); (4) high-dose group (10 mg / kg / day). Dox was administered through drinking water for 7 consecutive days. Blood was taken on the 3rd, 5th, and 7th days of administration to detect the plasma Dox concentration, and the mice were sacrificed on the 7th day to take heart tissues to analyze the RBM25 expression level.

[0092] The results showed that in in vitro experiments, the best induction effect was achieved at Dox concentrations of 100 - 500 ng / mL. The expression level of RBM25 increased with the prolongation of incubation time and reached a plateau at 48 - 72 hours. In in vivo experiments, the medium-dose group (5 mg / kg / day) showed the best induction effect and safety. The plasma Dox concentration was maintained in the range of 200 - 300 ng / mL, and the RBM25 expression level was 3.5 times that of the group without Dox administration.

[0093] In addition, the expression kinetics was studied through drug withdrawal experiments. In in vitro and in vivo experiments, after stopping Dox administration, the RBM25 expression level gradually decreased to 120 - 130% of the baseline level within 72 hours. This demonstrated that the system had good controllability, and precise temporal control of RBM25 expression could be achieved by adjusting the Dox administration protocol.

[0094] Example 11: In Vivo Efficacy Evaluation for the Treatment of Ischemic Heart Failure

[0095] This example involves the evaluation of the therapeutic effect of the CRISPR / dCas9-SunTag-mediated RBM25 activation system in a mouse model of ischemic heart failure.

[0096] First, a mouse model of heart failure after myocardial infarction was constructed. Ten-week-old male C57BL / 6J mice were used, and myocardial infarction was induced by permanent ligation of the left anterior descending coronary artery. Mice with successful myocardial infarction confirmed by echocardiography (LVEF < 40%) were included in the study.

[0097] Second, the treatment protocol was implemented. Three days after myocardial infarction, the mice were randomly divided into 4 groups (n = 12 in each group): (1) sham operation group; (2) model control group (PBS); (3) empty LNP group (without mRNA and gRNA); (4) treatment group (CRPPR-modified LNP + ultrasound + Dox). The treatment group was given CRPPR-modified LNP (0.5 mg / kg, calculated as mRNA) and ultrasound microbubbles (1×10^8 microbubbles / kg) by tail vein injection, and ultrasound treatment (1 MHz, 1.5 W / cm 2 , duty cycle 50%, 5 minutes) was performed immediately after injection. Dox (5 mg / kg / day) was given by drinking water for 3 weeks.

[0098] Then, cardiac function was evaluated. At 1, 4, and 8 weeks after treatment, a small animal ultrasound system (VisualSonics Vevo3100) was used to evaluate cardiac function, and the detection indexes included LVEF, FS, cardiac output, etc. At 8 weeks after treatment, a Millar microcatheter pressure system was used to measure hemodynamic indexes, including LVEDP, +dP / dt, -dP / dt, and CO.

[0099] Next, histological and molecular biological analyses were performed. The mice were sacrificed to obtain heart tissues, and H&E staining, Masson's trichrome staining, and TUNEL staining were carried out to evaluate myocardial structure, fibrosis degree, and apoptosis. The expressions of CD31 (vascular marker), Ki67 (proliferation marker), cTnT (myocardial marker), and CD68 (inflammatory marker) were analyzed by immunohistochemistry / immunofluorescence. The expression levels of RBM25 and its downstream target genes were analyzed using qRT-PCR and Western Blot.

[0100] The results showed that the cardiac function of the mice in the treatment group was significantly improved. The LVEF increased by 18 percentage points compared with the model control group after 8 weeks of treatment, the cardiac output increased by about 20%, and the LVEDP decreased by about 25%. Histological analysis showed that the myocardial fibrosis area in the treatment group was reduced by 30% compared with the model control group, the microvascular density increased by 35%, and the proportion of Ki67-positive cardiomyocytes reached 12% (less than 1% in the model control group). Molecular biological analysis confirmed that the expression level of RBM25 increased by 4-fold, and the expressions of its downstream genes related to angiogenesis and cell proliferation were significantly upregulated.

[0101] These results indicate that the CRISPR / dCas9-SunTag-mediated RBM25 activation system can effectively promote myocardial repair in ischemic heart failure, and its mechanisms include enhancing angiogenesis, promoting cardiomyocyte proliferation, and reducing myocardial fibrosis.

[0102] Example 12: Safety and Immunogenicity Evaluation

[0103] This example relates to the safety and immunogenicity evaluation of the CRISPR / dCas9-SunTag-mediated RBM25 activation system.

[0104] First, off-target effect analysis was performed. The myocardial and major organ tissues of the mice in the treatment group were analyzed using GUIDE-seq technology and high-throughput sequencing to detect potential off-target sites. The results showed that no significant off-target editing was detected based on the predicted potential off-target sites, and the off-target rate was less than 0.1%. RNA-seq analysis showed that the expression of non-target genes did not show significant abnormalities.

[0105] Secondly, immunogenicity was evaluated. The production of anti-dCas9 and anti-TET1 antibodies in the serum was detected by ELISA. Serum samples were collected before treatment, 2 weeks, 4 weeks, and 8 weeks after treatment. At the same time, the reactivity of splenic T cells to dCas9 and TET1 antigens was detected using ELISPOT. The results showed that only about 15% of the mice produced low-titer anti-dCas9 antibodies, and no significant T cell response was detected.

[0106] Then, systematic toxicity assessment was conducted. Histological changes in major organs (liver, spleen, lung, kidney, brain) were examined, and changes in blood biochemical indices (ALT, AST, TBIL, BUN, Cr, cTnI, CK-MB) and hematological indices were monitored. The results showed that no significant organ toxicity was found except for a transient mild increase in liver enzymes at the initial stage of treatment.

[0107] Finally, long-term safety observation was carried out. Some mice were observed up to 6 months after treatment, and cardiac function and histological changes were regularly evaluated. No tumor formation or other adverse reactions were found during long-term follow-up, demonstrating the good long-term safety of this treatment system.

[0108] Example 13: Translational research in a porcine model

[0109] This example involves translational research in a large animal (porcine) model to further verify the effectiveness and safety of the CRISPR / dCas9-SunTag-mediated RBM25 activation system.

[0110] First, a porcine myocardial infarction model was constructed. Miniature pigs aged 3 - 6 months with a body weight of 25 - 30 kg were used. Myocardial infarction was induced by overexpansion of the coronary artery with a balloon catheter. The successful establishment of the model was confirmed by echocardiography, serum cTnI index, and MRI.

[0111] Secondly, the large animal LNP / ultrasound administration protocol was optimized. The LNP dose range was 0.3 - 0.7 mg / kg, and it was administered by peripheral intravenous injection. The ultrasound parameters were optimized to 1.2 MHz, power 1.5 W / cm 2 , duty cycle 45%, for 5 minutes, synchronized with the cardiac cycle, and administered during diastole. The treatment was carried out 5 days after myocardial infarction.

[0112] Then, efficacy evaluation was conducted. Follow-up was carried out at 1, 3, and 6 months after treatment. Cardiac MRI was used to evaluate LVEF, scar area size, and ventricular remodeling. At the study endpoint (6 months), the animals were sacrificed for gross specimen preparation and myocardial section analysis to evaluate angiogenesis, cell proliferation, and fibrosis degree.

[0113] The results showed that the LVEF of the pigs in the treatment group increased by 15 percentage points compared with the control group, the scar area size decreased by 25%, and ventricular remodeling was significantly improved. Histological analysis confirmed an increase in microvascular density and a reduction in fibrosis. Safety monitoring found no serious adverse reactions, supporting the effectiveness and safety of this system in a large animal model.

[0114] Example 14: System for activating RBM25 gene expression in vitro

[0115] This example relates to an RBM25 gene expression activation system for in vitro research, including a transfection system composed of three expression vectors.

[0116] First, construct the following three expression vectors according to the methods of Examples 1-3: (1) pAAV-TRE3G-dCas9-SunTag-2A-mCherry vector containing the coding sequence of dCas9-SunTag-2A-mCherry controlled by the TRE3G promoter; (2) pAAV-EF1α-scFv-TET1-P2A-Hygro vector containing the coding sequence of scFv-TET1-P2A-Hygro controlled by the EF1α promoter; (3) pAAV-U6-gRNA-RBM25-1 vector containing the coding sequence of gRNA targeting the RBM25 promoter region controlled by the U6 promoter.

[0117] Second, mix these three vectors at a mass ratio of 1:1:0.5 and transfect HEK293T cells or primary cardiomyocytes using Lipofectamine 3000. Add Dox (100 - 500 ng / mL) 24 hours after transfection to induce the expression of dCas9-SunTag-2A-mCherry.

[0118] Then, observe the mCherry signal through a fluorescence microscope to monitor the transfection efficiency and the expression of dCas9-SunTag. Analyze the changes in RBM25 mRNA and protein levels by qRT-PCR and Western Blot. Analyze the changes in the methylation status of the RBM25 promoter region by methylation DNA immunoprecipitation (MeDIP).

[0119] The results show that this in vitro system can successfully activate RBM25 gene expression, with the RBM25 mRNA level increasing by 5 times and the protein level increasing by 3.5 times compared to the control group. MeDIP analysis confirms that the DNA methylation level in the RBM25 promoter region is significantly reduced, verifying the epigenetic regulation mechanism. This in vitro system provides a powerful tool for studying the mechanism of action of RBM25 in myocardial repair.

[0120] Example 15: Study on the mechanism of RBM25 regulating myocardial repair

[0121] This example relates to the study on the mechanism of action of RBM25 in myocardial repair, aiming to reveal the downstream effects and molecular mechanisms activated by RBM25.

[0122] First, RNA targets bound by RBM25 were identified by RNA-binding protein immunoprecipitation sequencing (RIP-seq). Immunoprecipitation with an anti-RBM25 antibody was performed using myocardial tissues from the RBM25 activation group and the control group, followed by high-throughput sequencing. Through bioinformatics analysis, RNA sequences and target genes preferentially bound by RBM25 were identified.

[0123] Second, changes in alternative splicing events before and after RBM25 activation were analyzed by RNA-seq. The regulation of RNA splicing by RBM25 was analyzed using rMATS and MAJIQ software. The screening criteria were: a change in splicing ratio > 20%, FDR < 0.05, and high expression in cardiomyocytes. Changes in key splicing events were verified by RT-PCR.

[0124] Then, the interaction between RBM25 and key pathways of myocardial repair was studied. The VEGF / PDGF signaling pathway, cell cycle regulation pathway, and pathways related to mitochondrial function were mainly analyzed. The activation status of these pathways was verified by Western Blot, fluorescence immunostaining, and functional experiments.

[0125] The results showed that RBM25 mainly regulated the alternative splicing of genes related to angiogenesis, cell proliferation, and metabolism. In particular, RBM25 activation promoted the expression of specific isoforms of VEGF mRNA, enhancing its stability and translation efficiency. At the same time, RBM25 regulated the alternative splicing of cell cycle proteins such as CyclinD1, promoting cardiomyocyte proliferation. In addition, RBM25 was also involved in the regulation of the expression of genes related to mitochondrial function, improving cardiomyocyte energy metabolism. These mechanisms together promoted angiogenesis, cell proliferation, and functional recovery of ischemic myocardium.

[0126] In summary, the present invention provides a technology for controllable activation of the RBM25 gene based on the CRISPR / dCas9-SunTag system and its application in the treatment of ischemic heart failure. This system precisely activates the expression of the RBM25 gene through epigenetic mechanisms, promoting myocardial repair and regeneration. The innovative LNP-ultrasound microbubble combined delivery strategy significantly improves the myocardial delivery efficiency of gene therapy, while the Dox-inducible expression system enables precise temporal control of therapeutic intervention. The present invention provides a new technical platform and treatment strategy for the gene therapy of ischemic heart failure, with broad clinical application prospects.

[0127] The above are only embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the scope of the claims of the present invention.

Claims

1. CRISPR / dCas9-SunTag-mediated RBM25 controllable activation system, characterized in that: include: (a) A protein comprising an inactivated Cas9 (dCas9) fused to a plurality of GCN4 peptide repeat sequences, wherein the number of the GCN4 peptide repeat sequences is 8-12, and the dCas9 contains a double mutation of D10A and H840A; (b) a protein comprising an anti-GCN4 single-chain antibody fragment (scFv) fused to the TET1 catalytic domain; (c) at least one gRNA targeting the promoter region of the RBM25 gene, wherein the targeting sequence of the gRNA is selected from the sequence shown in SEQ ID NO: 1-3; and (d) A lipid nanoparticle (LNP) system for delivering (a), (b) and (c), wherein the LNP system comprises an ionic lipid, an auxiliary lipid, cholesterol and a PEGylated lipid, wherein the mass ratio of the ionic lipid, the auxiliary lipid, cholesterol and the PEGylated lipid is 45-55:35-42:8-12:1-2.

2. The RBM25 controllable activation system according to claim 1, characterized in that: The ionic lipid is SM-102, the auxiliary lipid is DSPC, and the PEGylated lipid is DMG-PEG2000.

3. The RBM25 controllable activation system according to claim 1 or 2, characterized in that: The LNP system further comprises a CRPPR myocardial targeting peptide modification, wherein the CRPPR myocardial targeting peptide is coupled to the PEGylated lipid at a molar ratio of 0.1-0.3%.

4. The RBM25 controllable activation system according to claim 3, characterized in that: Wherein (a) and (b) are provided in the form of mRNA, wherein the mRNA comprises a 5' cap structure, an optimized 5' UTR, a coding sequence, an optimized 3' UTR and a poly A tail, and the uridine in the mRNA is completely replaced by 5-methoxyuridine.

5. The RBM25 controllable activation system according to claim 4, characterized in that: Also included are ultrasound microbubbles, which contain: (i) a lipid shell formed by 60-65% (w / w) DPPC, 4-6% (w / w) DSPE-PEG2000 and 30-35% (w / w) cholesterol; and (ii) The inner core is sulfur hexafluoride gas; The particle size of the ultrasonic microbubbles is 1.0-2.0 μm.

6. A method for preparing the CRISPR / dCas9-SunTag-mediated RBM25 controllable activation system according to any one of claims 1 to 5, comprising the following steps: (a) The coding sequences of dCas9-SunTag, scFv-TET1 and gRNA were cloned into expression vectors respectively; (b) preparing dCas9-SunTag mRNA and scFv-TET1 mRNA by in vitro transcription, comprising: (i) linearizing the expression vector using restriction endonucleases; (ii) performing a transcription reaction using an in vitro transcription kit, wherein the reaction system comprises an ARCA cap analog, ATP, CTP, GTP, and 5-methoxy UTP; (iii) DNaseI treatment to remove template DNA; and (iv) purifying and quantifying the resulting mRNA; (c) preparing lipid nanoparticles, comprising: (i) dissolving the ionic lipid, the helper lipid, cholesterol and the PEGylated lipid in ethanol; (ii) dissolving dCas9-SunTag mRNA, scFv-TET1 mRNA and gRNA in a pH 4.0 sodium acetate buffer at a mass ratio of 1:1:3; (iii) mixing using a microfluidic mixing device at a flow rate ratio of 1:3 (lipid:RNA); and (iv) dialyzing to remove the organic solvent and adjusting the pH to 7.4; and (d) when ultrasound microbubbles are included, further comprising the following steps: (i) preparing lipid films composed of DPPC, DSPE-PEG2000 and cholesterol; (ii) hydrating the lipid film and sonicating it; (iii) replacing air with sulphur hexafluoride gas; and (iv) Microbubbles were prepared by shaking and size screening.

7. A method for treating ischemic heart failure, comprising administering to a patient the CRISPR / dCas9-SunTag-mediated RBM25 controllable activation system according to any one of claims 1 to 5, wherein: (a) the composition is administered by intravenous injection; and (b) Apply ultrasound to the patient's heart area within 5 minutes after injection, with ultrasound parameters of: frequency 0.8-1.2 MHz, intensity 1.0-2.0 W / cm 2 , duty cycle 40-60%, duration 3-7 minutes.

8. The method according to claim 7, further comprising administering a tetracycline compound to the patient to induce the expression of dCas9-SunTag, wherein the tetracycline compound is doxycycline, administered at a dose of 2-10 mg / kg / day.

9. The method of claim 7 or 8, wherein the composition is administered to the patient within 3-7 days after myocardial infarction.

10. A system for activating RBM25 gene expression in vitro, comprising: (a) A first expression vector comprising a dCas9-SunTag-2A-mCherry coding sequence controlled by a TRE3G promoter; (b) a second expression vector comprising the scFv-TET1-P2A-Hygro coding sequence controlled by the EF1α promoter; and (c) a third expression vector comprising a gRNA coding sequence targeting the RBM25 promoter region controlled by a U6 promoter; Wherein, the expression of the dCas9-SunTag-2A-mCherry is controlled by the induction of tetracycline or its derivatives.