A controllable gene expression system based on homology-directed repair and its applications

Abstract

This invention discloses a controllable gene expression system based on homology-directed repair and its application, belonging to the field of gene therapy technology. The system is delivered via separate first and second vectors and includes: a targeting unit containing homologous arm sequences flanking a target gene locus exhibiting pathological state-dependent expression characteristics; an editing unit containing a CRISPR / Cas gene editing tool; and an effector unit containing a repair template composed of a foreign coding gene held by the homologous arm sequences. After the editing unit breaks at the target gene locus, the repair template integrates into the genome through homology-directed repair, allowing the expression of the foreign gene to be controlled by the regulatory elements of the endogenous target gene. This invention achieves intelligent dynamic regulation of the specific activation of exogenous therapeutic genes under pathological conditions and silencing them under normal conditions. Furthermore, through the division of labor between the two vectors and the synergy with tissue-specific promoters, it greatly improves the accuracy and safety of gene editing.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and more specifically, to a controllable gene expression system based on homology-directed repair and its applications. Background Technology

[0002] Gene therapy, a revolutionary technology in the biomedical field, offers a novel treatment approach for diseases that are difficult to cure with traditional methods, such as hereditary diseases and chronic degenerative diseases (e.g., cardiomyopathy, organ fibrosis, and neuroinflammation). Its core principle is to precisely intervene in diseases by delivering functional nucleic acid molecules (genes, RNA, etc.) to correct gene defects or regulate gene expression. In recent years, gene therapy has made breakthrough progress in clinical translation. For example, adeno-associated virus (AAV) vector gene therapy for spinal muscular atrophy and Duchenne muscular dystrophy has been approved for marketing, validating the feasibility and enormous potential of viral vector-mediated gene delivery systems in clinical applications.

[0003] AAV (autologous adenovirus) has become the most commonly used "golden vector" in gene therapy due to its advantages such as non-pathogenicity, low immunogenicity, diverse tissue targeting, and persistent gene expression. However, traditional AAV-mediated gene therapy still faces some core challenges that severely limit its efficacy and safety:

[0004] First, its expression regulation lacks specificity. Traditional gene therapy often uses broad-spectrum promoters (such as CMV) to drive the continuous expression of therapeutic genes, which cannot achieve dynamic regulation of "high expression in pathological states and low expression in normal states". This leads to the ectopic expression of therapeutic genes in normal tissues or continued overexpression after disease remission, causing side effects such as cytotoxicity and immune responses.

[0005] Secondly, its tissue targeting is insufficient. Even when tissue-specific promoters are used, their sequence length is usually 1-2kb, which not only occupies the limited packaging capacity of AAV (about 4.7kb), but also has problems such as weak transcriptional activity and high off-target risk, making it difficult to accurately target diseased tissues;

[0006] Finally, there is an imbalance between gene editing efficiency and safety. CRISPR / Cas system-mediated gene editing technology provides a precise tool for gene therapy, but traditional homology-directed repair (HDR) technology relies on continuously expressed Cas proteins and sgRNAs, which can easily lead to off-target mutations, chromosomal abnormalities, and other risks, and lacks temporal control over the repair process.

[0007] HDR, as a precise gene editing method mediated by the CRISPR / Cas system, can achieve targeted integration of exogenous genes. However, its clinical application is limited by problems such as low repair efficiency and a lack of regulatory mechanisms. In existing technologies, HDR-mediated gene expression is mostly in a "static mode," unable to dynamically adjust the expression level of therapeutic genes according to the occurrence and development of the disease, leading to undertreatment or overtreatment. At the same time, the components of traditional HDR systems (Cas protein, sgRNA, repair template) are usually delivered through a single vector. Due to the limited packaging capacity of AAV, it is difficult to load large therapeutic genes or complex regulatory elements, further limiting its application scope. Summary of the Invention

[0008] To address the aforementioned issues, this application provides a controllable gene expression system based on homology-directed repair and its application. This combination of elements, by screening target loci with pathology-dependent expression characteristics and combining editing tools with regulatory elements, achieves precise expression of exogenous genes at specific pathological stages, while maintaining low or no expression under normal physiological conditions, overcoming the limitations of "static regulation" in traditional gene therapy. Simultaneously, it balances gene editing efficiency and safety, providing a novel technical solution for targeted therapy of multiple tissue diseases.

[0009] The technical solution adopted in this application is as follows:

[0010] In a first aspect, this application provides a controllable gene expression system based on homology-directed repair, which is delivered via separate first and second vectors and comprises:

[0011] (i) A targeting unit comprising a first homologous arm sequence and a second homologous arm sequence that are homologous to sequences flanking a target locus in the genome of the target organism;

[0012] (ii) An editing unit comprising a CRISPR / Cas gene editing tool capable of specifically cutting the target locus;

[0013] (iii) An effector unit comprising a repair template, the repair template comprising at least one exogenous protein coding gene having therapeutic or indicative functions, wherein the 5' end of the exogenous coding gene is connected to the first homologous arm sequence and the 3' end is connected to the second homologous arm sequence;

[0014] The target locus has pathological state-dependent expression characteristics;

[0015] The repair template is configured to integrate into the genome via a homology-directed repair mechanism after the editing unit generates a double-strand break at the target locus, thereby controlling the expression of the exogenous gene to the expression regulatory elements of the target locus.

[0016] Furthermore, the CRISPR / Cas gene editing tool in the above-mentioned editing unit includes a Cas protein-coding sequence and an sgRNA sequence targeting the locus;

[0017] The Cas protein coding sequence is driven by a tissue-specific promoter and loaded into the first vector; the sgRNA sequence and the repair template are loaded into the second vector.

[0018] Furthermore, the aforementioned pathology-dependent expression characteristics refer to the following: the expression level of the target locus is lower than a first threshold under the normal physiological state of the corresponding tissue, and the expression level is higher than a second threshold under a specific pathological state, and the second threshold is significantly higher than the first threshold.

[0019] Furthermore, the target loci mentioned above are selected from at least one of the following:

[0020] (a) In cardiac tissue, selected from Myh7, NPPA, or NPPB loci;

[0021] (b) In liver tissue, selected from the CYP3A4 locus;

[0022] (c) In neural tissue, selected from the GFAP locus.

[0023] Furthermore, the lengths of the first homologous arm sequence and the second homologous arm sequence are each independently 200-1500 base pairs upstream and downstream of the target locus editing site; preferably, each is independently about 200 base pairs.

[0024] Furthermore, the protein encoded by the exogenous coding gene in the above-mentioned effector unit is selected from at least one of the following:

[0025] (a) Therapeutic proteins, including R-Tf-D-LP4, Beclin1, IL-10, AIP, BDNF, Bcl-2 or GST, or functional fragments, variants or fusion proteins of the same therapeutic function;

[0026] (b) Reporter proteins, including fluorescent or luminescent proteins.

[0027] Furthermore, the aforementioned tissue-specific promoters include any one of the myocardial-specific promoter cTnT, the liver-specific promoter ALB, and the universal strong promoter CMV.

[0028] Furthermore, both the first and second vectors are delivered via adeno-associated virus vectors;

[0029] The expression cassettes of the first vector, from 5' to 3', are: AAV ITR-tissue-specific promoter-Cas-PolyA-AAV ITR;

[0030] The AAV-ITR sequence, a unique genetic component shared by both AAV recombination and wild-type AAV, plays a crucial role in AAV genome replication, progeny genome generation and packaging, and sustained expression of exogenous genes. The tissue-specific promoter, as described above, can be the myocardial-specific promoter cTnT, the liver-specific promoter ALB, etc. The Cas protein coding sequence, as described above, can be spCas9 (4.2kb) or Cas12f1 (<1.5kb). The Poly A sequence includes, but is not limited to, polyadenylated sequences of various mRNAs. Preferably, the Poly A sequence described in this invention is SV40 PolyA (200bp), which reduces vector capacity usage while ensuring mRNA stability.

[0031] The expression cassette of the second vector, from 5' to 3', consists of: AAV ITR-U6 promoter - sgRNA - first homologous arm sequence at the 5' end of the target locus - foreign coding gene sequence - second homologous arm sequence at the 3' end of the target locus - AAV ITR;

[0032] The first and second vectors work synergistically to achieve Cas-mediated homologous targeted repair.

[0033] The U6 promoter is a commonly used RNA polymerase III promoter, mainly used to express small RNA molecules such as shRNA and sgRNA. The sgRNA sequence is selected based on high predicted editing efficiency and low predicted off-target risk, and a guanine base (G) is added to its 5' end to meet the transcription requirements of the U6 promoter. The homologous sequence, as described above, is the base sequence flanking the target gene site. Preferably, the homologous sequence in this invention is 200 bp, which can ensure gene editing efficiency while reducing vector capacity. The exogenous gene sequence, as described above, is the base sequence with a specific function required for the example.

[0034] Secondly, this application provides a recombinant nucleic acid molecule comprising an expression cassette selected from one of the following:

[0035] (1) An expression cassette containing a tissue-specific promoter and an operatively linked Cas protein-coding sequence; or

[0036] (2) An expression cassette containing an sgRNA expression element and a repair template sequence consisting of a first homologous arm sequence, a foreign coding gene, and a second homologous arm sequence connected in sequence;

[0037] Furthermore, the expression cassette is connected to adeno-associated virus inverted terminal repeat sequences on both sides.

[0038] Thirdly, this application provides a method for constructing the above-mentioned controllable gene expression system, characterized by comprising the following steps:

[0039] (a) Screening for target loci with pathological state-dependent expression characteristics;

[0040] (b) Construct recombinant nucleic acid molecules containing the Cas protein coding sequence in the editing unit, the sgRNA expression element, and the repair template, respectively;

[0041] (c) The recombinant nucleic acid molecules are packaged into AAV protein capsids to prepare the first vector and the second vector.

[0042] Furthermore, the aforementioned AAV protein capsid is selected from one or more of AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-PHP.eB, and / or MyoAAV. The AAV protein capsid described in this invention is MyoAAV. As a muscle-specific AAV serotype, MyoAAV's capsid protein can specifically recognize muscle-specific receptors (such as integrin α7β1) on the surface of cardiomyocyte membranes, achieving an infection efficiency of over 85% in cardiomyocytes, significantly higher than conventional AAV9 (70%), making it a preferred vector for targeted myocardial gene editing.

[0043] The aforementioned ITR sequence includes one or more of ITR1, ITR2, truncated ITR2, ITR3, ITR5, ITR4, ITR6, and ITR7. Preferably, the ITR sequence described in the invention is ITR2.

[0044] The AAV can be administered via intravenous injection, local injection (such as intramyocardial or intrahepatic injection), or intrathecal injection. The specific route of administration is selected based on the characteristics of the target tissue (e.g., tail vein injection is preferred for cardiac diseases, while intrathecal injection is preferred for neurological diseases).

[0045] Furthermore, the AAV vector was packaged in HEK293T cells using a three-plasmid method: the recombinant nucleic acid molecule containing the AAV genome was co-transfected into HEK293 cells along with two other plasmids required for AAV packaging, Rep-Cap and AdHelper, to generate AAV viral particles through intracellular assembly mechanisms.

[0046] The purification process of the AAV vector is as follows: First, HEK293T cells transfected with three plasmids were collected using the PEG8000 precipitation method; then, the cells were ruptured by repeated freeze-thaw cycles (usually 3-5 times) to release intracellular AAV virus particles; next, the virus was initially purified using iodixanol gradient centrifugation to remove most cellular impurities; finally, ultrafiltration centrifugation was performed using ultrafiltration tubes to further purify and concentrate the AAV virus, ultimately obtaining a high-purity, high-titer AAV vector (1×10⁻⁶). 13 (vg / mL).

[0047] In summary, this application has the following beneficial effects:

[0048] 1. This application achieves automatic synchronization between the expression level of the therapeutic gene and the severity of the disease by precisely integrating exogenous therapeutic genes into endogenous gene sites with "pathology-dependent high expression" characteristics (such as NPPB in heart failure): disease onset → endogenous gene activation → high expression of the therapeutic gene; disease remission → endogenous gene silencing → low expression of the therapeutic gene. This fundamentally solves the problem of "continuous overexpression" caused by traditional broad-spectrum promoters, avoids cytotoxic or immune side effects caused by ectopic and continuous expression of the therapeutic gene in normal tissues or after disease remission, and realizes "on-demand" gene therapy.

[0049] 2. This application employs a dual-vector strategy: the first vector uses a tissue-specific promoter (such as myocardial cTnT) to drive the Cas protein, strictly limiting the activity of the gene "scissors" to the target diseased tissue, thereby reducing the risk of off-target editing in other organs from the source. Simultaneously, the expression regulation of the therapeutic gene further relies on endogenous regulatory elements specifically activated in the target tissue under pathological conditions, achieving dual tissue targeting of editing and expression, greatly improving the precision and safety of the treatment.

[0050] 3. This application cleverly solves the industry bottleneck of insufficient packaging capacity (~4.7kb) of a single AAV vector by splitting the massive CRISPR-Cas9 system (Cas protein + sgRNA + homologous arm + therapeutic gene) into two AAV vectors. This design allows for the loading of larger or more complex therapeutic genes (such as ~1kb gene sequences) and is compatible with longer homologous arms to ensure editing efficiency, providing a universal platform for complex gene editing and gene replacement therapy using AAV delivery systems.

[0051] 4. This system adopts a modular design of "general-purpose editing tools + interchangeable target / treatment modules". By changing tissue-specific promoters, pathologically responsive target loci, and exogenous therapeutic genes, the same technology platform can be quickly adapted to different diseases (heart failure, liver fibrosis, neuroinflammation, etc.) in different tissues (heart, liver, nerves, etc.). This design endows the technical solution with strong versatility and scalability, not only targeting specific diseases, but also providing a new treatment strategy and drug development platform for a range of chronic diseases caused by "dysregulation of gene expression" or "requiring dynamic intervention".

[0052] In summary, this invention, through ingenious system design, simultaneously solves several key challenges that have long existed in the field of gene therapy, such as uncontrollable expression, imprecise targeting, unsafe editing, and limited vector capacity, bringing significant progress in terms of safety, efficacy, and versatility. Attached Figure Description

[0053] Figure 1 This is a diagram showing the experimental results of Example 1, in which gene editing targeting the Myh7 locus was performed on mice after transverse aortic arch coarctation (TAC surgery) to drive the efficient expression of the exogenous small molecule therapeutic protein MP1 in a cardiac pathological state to improve myocardial fibrosis.

[0054] Figure 2 The results of pathological staining of mouse heart tissue and the quantitative statistical results of the fibrosis area in Example 1 are shown.

[0055] Figure 3 This is the statistical result of the relative expression levels of heart failure and fibrosis-related genes detected by qPCR after RNA extraction from mouse heart tissue and reverse transcription into cDNA in Example 1.

[0056] Figure 4 This is a diagram showing the experimental results of gene editing targeting the Myh7 locus in Example 2 to achieve controllable expression of the small molecule therapeutic protein MP1. Detailed Implementation

[0057] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0058] The technical solution of this invention is as follows:

[0059] Unless otherwise stated, the practice of the present invention will employ conventional techniques in tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the scope of the art.

[0060] All technical solutions of this invention can be used for preventive or therapeutic purposes or for purposes other than preventive or therapeutic purposes.

[0061] The term "target locus" used in this invention refers to a gene obtained through screening that has the characteristics of "high expression in pathology, low expression in normal conditions, and tissue specificity," whose regulatory sequence (promoter + enhancer) can drive the specific expression of exogenous genes in disease states.

[0062] The term "homologous sequence" used in this invention refers to the DNA sequence upstream and downstream of the target locus editing site, typically 500-1500 bp in length, preferably 200 bp, used to mediate homologous recombination during the HDR process to ensure the targeted integration of exogenous genes.

[0063] The term "dual AAV system" used in this invention refers to two AAV vectors with clearly defined functions, which deliver Cas protein (vector 1) and sgRNA + homologous sequence + editing template (vector 2) respectively. Through synergistic action, they achieve precise HDR editing and avoid the capacity limitations of a single vector.

[0064] The term "ITR (inverted terminal repeat)" used in this invention refers to the inverted symmetric repeat sequences at both ends of the AAV genome, which are about 145 bp in length and play a key role in viral replication, packaging and sustained expression of exogenous genes. The ITR2 sequence is preferred in this invention.

[0065] The term "MyoAAV" used in this invention refers to a skeletal muscle / myocardial specific AAV serotype, whose capsid protein can specifically recognize muscle cell-specific receptors (such as integrin α7β1) on the surface of cardiomyocyte membranes, and the cardiomyocyte infection efficiency reaches more than 85%, which is significantly higher than that of conventional AAV9.

[0066] The term "repair template sequence" as used in this invention refers to an exogenous protein coding sequence containing a therapeutic function (MP1 small molecule protein) or an indicator function (mScarlet red fluorescent protein), which can be directionally integrated into the target gene locus via HDR and expressed in a controlled manner under the control of the target gene regulatory sequence.

[0067] The following table (Table 1) lists the nucleic acid sequence numbers and corresponding sequences used in the description of this invention (where the underlined and italicized indexes represent the "tags" used in the primers used for high-throughput sequencing to distinguish samples, also known as barcodes, which can be freely selected by high-throughput sequencing technicians as needed).

[0068] Table 1. Nucleic acid sequence numbers and their corresponding sequences used in this invention.

[0069]

[0070]

[0071] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0072] Example 1

[0073] Construction of a controllable gene expression system targeting the Myh7 locus and its application in the treatment of myocardial fibrosis:

[0074] This embodiment aims to verify the controllable gene expression combination based on the Myh7 locus (a pathologically highly expressed gene in myocardial fibrosis), and to achieve pathology-dependent expression of the therapeutic protein MP1 through a dual AAV system in TAC model mice, thereby improving myocardial fibrosis.

[0075] 1. Experimental Materials

[0076] Animals: C57BL / 6 mice (8 weeks old, male), purchased from Chengdu Dashuo Experimental Animal Co., Ltd., with a certificate of qualification for experimental animals obtained;

[0077] Cells: HEK293T cells, purchased from the ATCC cell bank;

[0078] Vector backbone: pAAV-ITR2 vector plasmid, Rep-Cap (MyoAAV serotype) plasmid, Ad Helper plasmid, purchased from Addgene;

[0079] Reagents: Restriction endonucleases (KpnI, NheI, RsrII, etc., NEB), seamless cloning kit (NEB), plasmid extraction kit (Tiangen), iodixanol (OptiPrep), PEG8000, Masson staining kit, Sirius red staining kit, WGA fluorescent dye, purchased from regular commercial channels;

[0080] Instruments: cell culture incubator, ultracentrifuge, confocal microscope, echocardiography, qPCR instrument, etc.

[0081] 2. Experimental Methods

[0082] (1) Target locus screening and validation

[0083] By analyzing the gene expression profiles of cardiac tissues from normal mice and transverse aortic arch constriction (TAC) induced myocardial hypertrophy mice using RNA-seq, the myocardial-specific gene Myh7, which is "lowly expressed in normal state and highly expressed in pathological state", was screened out.

[0084] A lentiviral vector consisting of the Myh7 promoter, enhancer, and luciferase reporter was constructed and transfected into cardiomyocytes (HL1). After Ang II-induced fibrosis, luciferase activity was detected. The results showed that the activity was significantly enhanced under pathological conditions, meeting the screening criteria.

[0085] (2) Construction of dual AAV vectors

[0086] Construction of AAV vector 1 (pAAV-cTnT-SpCas9):

[0087] Using pAAV-ITR2 as the backbone, the vector was digested with restriction endonucleases KpnI and RsrII, and the backbone fragment was recovered. The cTnT promoter, SpCas9 coding sequence, and SV40 PolyA sequence were amplified by PCR. The above fragments were inserted into the vector backbone in the order of "ITR2-cTnT-SpCas9-PolyA-ITR2" using seamless cloning technology. Stable3 competent cells were transformed, and single clones were selected and verified by Sanger sequencing to obtain the correct recombinant plasmid.

[0088] Construction of AAV vector 2 (pAAV-U6-sgRNA-Myh7-HDR-MP1):

[0089] Using pAAV-ITR2 as the backbone, after enzyme digestion and recovery, the U6 promoter, Myh7-sgRNA (with a G base added to the 5' end to adapt to the U6 promoter), Myh7-5' homologous sequence, MP1 coding sequence, and Myh7-3' homologous sequence were amplified by PCR. The sequence was then cloned into the vector in the order of "ITR2-U6-sgRNA-Myh7 gene 5' homologous sequence-P2A-MP1-Myh7 gene 3' homologous sequence-ITR2". After sequencing verification, the sequence was used for later use.

[0090] (3) MyoAAV virus packaging and purification

[0091] The virus was packaged using a three-plasmid method: HEK293T cells were cultured to 80-90% confluence. 7 μg of recombinant plasmid (AAV vector 1 or vector 2), 7 μg of Rep-Cap (MyoAAV) plasmid, and 20 μg of Ad Helper plasmid were added to each 15 cm culture dish. This mixture was then mixed with PEI transfection reagent and used to transfect the cells. After culturing at 37°C for 8 hours, the medium was replaced with serum-free DMEM, and the cells were cultured for another 64 hours. Cells and supernatant were collected. The cell pellet was resuspended in AAV lysis buffer (20 mM Tris pH 8.0, 1 mM MgCl2, 150 mM NaCl), and 40% PEG8000 solution (containing 2.5 M NaCl) was added. The mixture was incubated at 4°C for 2 hours, and the pellet was collected by centrifugation. The resuspended pellet was mixed with cell lysis buffer and subjected to three freeze-thaw cycles (-80°C / 37°C). Impurities were digested with a totipotent nuclease. The cells were then subjected to gradient ultracentrifugation with iodixanol (35000 rpm, 2...). The virus was purified (hours), concentrated in an ultrafiltration tube, and the titer was determined by qPCR to finally obtain MyoAAV vector 1 and vector 2 with a titer ≥1×101³vg / mL.

[0092] (4) Animal experimental design

[0093] C57BL / 6 mice were randomly divided into 4 groups (n=6 / group):

[0094] Sham group: only open-chest surgery without TAC, saline was injected via tail vein;

[0095] TAC group: TAC surgery was performed to induce myocardial hypertrophy, followed by injection of normal saline via the tail vein;

[0096] TAC+AAV empty vector group: TAC surgery + tail vein injection of MyoAAV empty vector (1×10¹¹ vg / g);

[0097] TAC+AAV treatment group: TAC surgery + tail vein injection of MyoAAV carrier 1 + carrier 2 (5×10¹⁰ vg / g each, volume ratio 1:1).

[0098] Viral injections were administered on the 3rd day after surgery, and relevant indicators were tested 42 days (6 weeks) after surgery.

[0099] (5) Detection methods

[0100] Cardiac function testing: Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (FS) were measured by echocardiography 42 days post-surgery; Pathological morphology testing: After mouse sacrifice, heart tissue was weighed, and the heart weight / body weight ratio (HW / BW) was calculated; paraffin sections were prepared, stained with Masson and Sirius red, and the fibrosis area was analyzed using ImageJ software; Cardiac cell area detection: Frozen sections were stained with WGA fluorescent dye, observed under a confocal microscope, and the cross-sectional area of ​​myocardial fibers was counted; Gene expression detection: RNA was extracted from heart tissue, reverse transcribed into cDNA, and the relative expression levels of heart failure-related genes (ANP, BNP) and fibrosis-related genes (TGF-β, Col-I) were detected by qPCR; Editing efficiency detection: Genomic DNA was extracted from heart tissue, Myh7 editing sites were amplified by PCR, and amplicon sequencing was used to analyze HDR efficiency and splicing site base mutation efficiency.

[0101] 3. Experimental Results:

[0102] Figure 1-3 Figure showing the experimental results of gene editing targeting the Myh7 locus in mice after transverse aortic arch coarctation (TAC) surgery to drive the efficient expression of the exogenous small molecule therapeutic protein MP1 in cardiac pathology to improve myocardial fibrosis:

[0103] Figure 1 Figure 1A shows the gene editing effect of the viral vector MyoAAV-U6-sgRNA-Myh7-HDR-MP1, constructed targeting Myh7, injected into TAC mice via the tail vein; Figure 1B shows a comparison of the gross morphology of the mouse heart, showing that the heart volume in the treatment group was significantly reduced; Figure 1C shows the heart-to-body weight ratio, indicating that the treatment group significantly reduced the heart-to-body weight ratio (P<0.01); Figures 1D and 1E show the results of echocardiography, suggesting that the treatment group significantly improved the decline in cardiac function (P<0.01); Figure 2A shows the results of pathological staining (Masson staining and Sirius red staining) of mouse heart tissue, visually demonstrating the degree of myocardial fibrosis; Figure 2B shows the quantitative statistics of the fibrosis area after pathological staining, showing that the treatment group significantly alleviated myocardial fibrosis (P<0.01); Figure 3 shows the qPCR results after RNA extraction from mouse heart tissue, reverse transcription into cDNA. Statistical results of the relative expression levels of heart failure and fibrosis-related genes (ANP, BNP, TGF-β, Col-I, Col-III, CTGF, POSTN, β-MHC, α-SMA) showed that the treatment group could significantly downregulate the expression levels of myocardial fibrosis-related genes (P<0.01).

[0104] This demonstrates that the controllable gene expression combination based on the Myh7 locus constructed in this embodiment achieves specific high expression of the therapeutic protein MP1 under myocardial pathological conditions through the dual MyoAAV system, effectively improving myocardial fibrosis and cardiac function in TAC mice, with low off-target risk, thus verifying the effectiveness and safety of the system in the treatment of heart diseases.

[0105] Example 2

[0106] Validation of Myh7 locus-mediated controlled expression of exogenous genes

[0107] This embodiment aims to verify the pathological dependence of exogenous gene expression, that is, the expression level of therapeutic genes is positively correlated with the severity of the disease, and low expression is observed under normal conditions.

[0108] 1. Experimental Materials

[0109] Animals: C57BL / 6 mice (6-8 weeks old, male);

[0110] Vector: MyoAAV vector 2 (pAAV-U6-sgRNA-Myh7-HDR-P2A-mScarlet, where mScarlet is a red fluorescent protein) was constructed, and the other materials were the same as in Example 1.

[0111] 2. Experimental Methods

[0112] (1) Vector construction and virus packaging

[0113] Following the method in Example 1, the editing template sequence was replaced with "P2A-mScarlet" (P2A is a self-cleaving peptide) to construct MyoAAV vector 2, with a titer ≥1×10¹³vg / mL after packaging.

[0114] (2) Animal experiment design

[0115] Grouping: Sham group, TAC group, TAC+Ang II re-intervention group (Ang II infused via osmotic pump 28 days post-operation to enhance pathological status), n=3 in each group;

[0116] Treatment: All groups were injected via tail vein with MyoAAV vector 1 + vector 2 (1×10¹³ vg / g each). Samples were taken at 0, 14, 28, 42 and 70 days postoperatively, and MP1 expression was detected by qPCR.

[0117] (3) Detection method

[0118] Fluorescence detection: Frozen sections were stained with DAPI and WGA, and the fluorescence intensity of mScarlet was observed by confocal microscopy to quantitatively analyze the expression level of exogenous genes; qPCR detection: RNA was extracted from heart tissue at different time points, and the relative expression level of MP1 was detected by qPCR; Pathological verification: After Ang II infusion, the correlation between the degree of myocardial fibrosis and the expression level of MP1 was detected.

[0119] 3. Experimental Results

[0120] Figure 4 shows the experimental results of gene editing targeting the Myh7 locus to achieve controllable expression of the small molecule therapeutic protein MP1. Figure 4A: Gene editing effect after injection of the MyoAAV-U6-sgRNA-Myh7-HDR-mScarlet designed to target Myh7 into TAC mice, showing a schematic diagram of controllable expression of the target gene; Figure 4B: Transcriptional maps of myocardial tissue in TAC and Sham mice, showing that Myh7 is significantly highly expressed during the occurrence of myocardial fibrosis; Figure 4 C: Fluorescence imaging results, showing the distribution of mScarlet-positive cells in different dose TAC groups and Sham groups; Figure 4D: Quantitative statistics of the ratio of mScarlet-positive cells in different dose TAC groups and Sham groups; Figure 4E: Experimental procedure and timetable of MP1 expression level autonomously regulated by the pathological state of mouse heart; Figure 4F: Schematic diagram of the time dynamic change of relative MP1 expression level in mouse heart tissue detected by qPCR.

[0121] Figure 4 The results showed that, in terms of fluorescence intensity, the mScarlet fluorescence intensity of the TAC+Ang II group was significantly higher than that of the TAC group (P<0.01), while the Sham group showed almost no fluorescence signal. In terms of dynamic expression characteristics, qPCR results showed that MP1 expression gradually increased with the extension of postoperative time, with the TAC+Ang II group reaching the peak expression at 70 days postoperatively. Moreover, MP1 expression was significantly positively correlated with the degree of myocardial fibrosis (r=0.87, P<0.001).

[0122] The above results confirm that the expression of the exogenous gene MP1 depends on the pathological activation of the Myh7 locus: the higher the degree of disease, the higher the expression level of MP1, while it is low under normal physiological conditions. This successfully achieves dynamic regulation of "expression on demand", further verifying the specificity and effectiveness of the controllable gene expression system of this invention.

[0123] Example 3

[0124] This embodiment provides a method for constructing a controllable gene expression system targeting the ANP locus.

[0125] This embodiment aims to verify the controllable gene expression combination based on the CYP3A4 locus (highly expressed during myocardial infarction), and to achieve pathological-dependent expression of the therapeutic protein YAP5SA in a myocardial infarction model mouse through the delivery of the therapeutic protein YAP5SA via a dual AAV system, thereby promoting the regeneration of cardiomyocytes in the infarcted area.

[0126] The experimental procedure was basically the same as in Example 1, except for the construction of the dual AAV vector, specifically:

[0127] (1) Construction of AAV vector 1 (pAAV-cTnT-SpCas9):

[0128] Using pAAV-ITR2 as the backbone, the vector was digested with restriction endonucleases KpnI and RsrII, and the backbone fragment was recovered. The cTnT promoter, SpCas9 coding sequence, and SV40 PolyA sequence were amplified by PCR. The above fragments were inserted into the vector backbone in the order of "ITR2-cTnT-SpCas9-PolyA-ITR2" using seamless cloning technology. Stable3 competent cells were transformed, and single clones were selected and verified by Sanger sequencing to obtain the correct recombinant plasmid.

[0129] (2) Construction of AAV vector 2 (pAAV-U6-sgRNA-ANP-HDR-YAP5SA):

[0130] Using pAAV-ITR2 as the backbone, after enzyme digestion and recovery, the U6 promoter, ANP-sgRNA (with a G base added at the 5' end to adapt to the U6 promoter), Myh7-5' homologous sequence, MP1 coding sequence, and Myh7-3' homologous sequence were amplified by PCR. The sequences were then cloned into the vector in the order of "ITR2-U6-sgRNA-ANP gene 5' homologous sequence-P2A-YAP5SA-ANP gene 3' homologous sequence-ITR2". After sequencing verification, the sequences were used for later use.

[0131] Example 4

[0132] This embodiment provides a method for constructing a controllable gene expression system targeting the COL1A1 gene locus.

[0133] This embodiment aims to verify the controllable gene expression combination based on the COL1A1 locus (highly expressed in liver fibrosis), and to achieve pathological-dependent expression of the therapeutic protein KP1 in a liver fibrosis model mouse through a dual AAV system, thereby improving liver fibrosis.

[0134] The experimental procedure was basically the same as in Example 1, except for the construction of the dual AAV vector, specifically:

[0135] (1) Construction of AAV vector 1 (pAAV-TBG-SpCas9):

[0136] Using pAAV-ITR2 as the backbone, the vector was digested with restriction endonucleases KpnI and RsrII, and the backbone fragment was recovered. The TBG promoter, SpCas9 coding sequence, and SV40 PolyA sequence were amplified by PCR. The above fragments were inserted into the vector backbone in the order of "ITR2-TBG-SpCas9-PolyA-ITR2" using seamless cloning technology. Stable3 competent cells were transformed, and single clones were selected and verified by Sanger sequencing to obtain the correct recombinant plasmid.

[0137] (2) Construction of AAV vector 2 (pAAV-U6-sgRNA-COL1A1-HDR-KP1):

[0138] Using pAAV-ITR2 as the backbone, after enzyme digestion and recovery, the U6 promoter, COL1A1-sgRNA (with a G base added at the 5' end to adapt to the U6 promoter), Myh7-5' homologous sequence, MP1 coding sequence, and Myh7-3' homologous sequence were amplified by PCR. The sequences were then cloned into the vector in the order of "ITR2-U6-sgRNA-COL1A1 gene 5' homologous sequence-P2A-KP1-COL1A1 gene 3' homologous sequence-ITR2". After sequencing verification, the sequences were used for later use.

[0139] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A controllable gene expression system based on homology-directed repair, characterized in that, The system is delivered via separate first and second carriers and includes: (i) A targeting unit comprising a first homologous arm sequence and a second homologous arm sequence that are homologous to sequences flanking a target locus in the genome of the target organism; (ii) An editing unit comprising a CRISPR / Cas gene editing tool capable of specifically cutting the target locus; (iii) An effector unit comprising a repair template, the repair template comprising at least one exogenous protein coding gene having therapeutic or indicative functions, wherein the 5' end of the exogenous coding gene is connected to the first homologous arm sequence and the 3' end is connected to the second homologous arm sequence; The target locus has pathological state-dependent expression characteristics; The repair template is configured to integrate into the genome via a homology-directed repair mechanism after the editing unit generates a double-strand break at the target locus, thereby controlling the expression of the exogenous coding gene to the expression regulatory elements of the target locus.

2. The controllable gene expression system based on homology-directed repair according to claim 1, characterized in that, The CRISPR / Cas gene editing tool in the editing unit includes a Cas protein-coding sequence and an sgRNA sequence targeting the locus. The Cas protein coding sequence is driven by a tissue-specific promoter and loaded into the first vector; the sgRNA sequence and the repair template are loaded into the second vector.

3. The controllable gene expression system based on homology-directed repair according to claim 1, characterized in that, The pathology-dependent expression characteristic refers to the following: the expression level of the target locus is lower than a first threshold under normal physiological conditions of the corresponding tissue, and the expression level is higher than a second threshold under specific pathological conditions, and the second threshold is significantly higher than the first threshold.

4. The controllable gene expression system based on homology-directed repair according to claim 3, characterized in that, The target locus is selected from at least one of the following: (a) In cardiac tissue, selected from Myh7, NPPA, or NPPB loci; (b) In liver tissue, selected from the CYP3A4 locus; (c) In neural tissue, selected from the GFAP locus.

5. The controllable gene expression system based on homology-directed repair according to claim 1, characterized in that, The lengths of the first homologous arm sequence and the second homologous arm sequence are each independently 200-1500 base pairs upstream and downstream of the target locus editing site.

6. The controllable gene expression system based on homology-directed repair according to claim 1, characterized in that, The protein encoded by the exogenous coding gene in the effector unit is selected from at least one of the following: (a) Therapeutic proteins, including R-Tf-D-LP4, Beclin1, IL-10, AIP, BDNF, Bcl-2 or GST, or functional fragments, variants or fusion proteins of the same therapeutic function; (b) Reporter proteins, including fluorescent or luminescent proteins.

7. The controllable gene expression system based on homology-directed repair according to claim 2, characterized in that, The tissue-specific promoters include any one of the myocardial-specific promoter cTnT, the liver-specific promoter ALB, and the universal strong promoter CMV.

8. The controllable gene expression system based on homology-directed repair according to any one of claims 1-7, characterized in that, Both the first and second vectors are delivered via adeno-associated virus vectors; The expression cassettes of the first vector, from 5' to 3', are: AAV ITR-tissue-specific promoter-Cas-PolyA-AAVITR; The expression cassette of the second vector, from 5' to 3', consists of: AAV ITR-U6 promoter - sgRNA - first homologous arm sequence at the 5' end of the target locus - foreign coding gene sequence - second homologous arm sequence at the 3' end of the target locus - AAV ITR; The first and second vectors work synergistically to achieve Cas-mediated homologous targeted repair.

9. A method for constructing a controllable gene expression system as described in any one of claims 1-8, characterized in that, Including the following steps: (a) Screening for target loci with pathological state-dependent expression characteristics; (b) Construct recombinant nucleic acid molecules containing the Cas protein coding sequence of the editing unit, the sgRNA expression element, and the repair template, respectively; (c) The recombinant nucleic acid molecules are packaged into AAV protein capsids to prepare the first vector and the second vector.

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