Regulation element combination for driving nucleic acid expression based on ITR-enhancer and application of regulation element combination

Abstract

The invention relates to the field of biological medicine, and particularly discloses a regulatory element combination for driving nucleic acid expression based on ITR-enhancer and application of the regulatory element combination. The regulatory element combination for driving nucleic acid expression based on an ITR-enhancer comprises at least one inverted terminal repeat (ITR) and at least one enhancer, the regulatory element combination is delivered through an AAV vector. The invention also provides a recombinant nucleic acid molecule for improving specific delivery of the target gene. The recombinant nucleic acid molecule comprises the regulatory element combination, an exogenous target gene and polyadenylic acid. The AAV vector provided by the invention can be used for driving the expression of a target gene without the action of a traditional promoter, and has the advantages of enhancer tissue specificity, short sequence (50-100bp), pathological microenvironment response and the like, thereby breaking through the capacity, targeting and dynamic limitation of the traditional AAV vector, improving the DNA (Deoxyribose Nucleic Acid) carrying capacity of the AAV vector, and simultaneously improving the expression of the target gene. The tissue and spatio-temporal expression specificity of a target gene is realized.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and more specifically, to a regulatory element combination based on ITR-enhancer driven nucleic acid expression and its application. Background Art

[0002] Gene therapy offers a highly promising solution for genetic diseases (such as those caused by single gene mutations). Its core concept involves treating genetic diseases through three approaches: 1) gene augmentation, which delivers a functioning gene to the patient's cells to compensate for the defective original gene; 2) gene silencing, which inhibits the expression of the abnormal gene through techniques such as RNA interference, blocking its harmful effects; and 3) gene editing, which precisely corrects the disease-causing mutation (such as base substitution or fragment repair) to restore the gene sequence at its source. For example, Zolgensma, a gene therapy approved by the US FDA in 2018 for the treatment of spinal muscular atrophy (SMA), uses an AAV vector to deliver the SMN1 gene to spinal cord neurons. This gene therapy replenishes the SMN1 protein expression that is blocked by the mutation, thereby increasing the expression of a fully functional SMN protein and ultimately curing the disease. The application of gene therapy has the potential to achieve a radical cure for genetic diseases, even a one-time cure.

[0003] Gene therapy intervenes in diseases by delivering biomacromolecules such as DNA, RNA, or proteins. DNA delivery is the most widely used clinical strategy due to its long-lasting efficacy, strong stability, and ability to encode complex functional genes. Its core mechanism is to deliver functional gene fragments into the patient's cells in the form of exogenous DNA, thereby achieving gene repair or compensation. Among various gene therapy vectors, adeno-associated virus (AAV) is widely used due to its unique advantages.

[0004] AAV is a defective single-stranded DNA virus that occurs naturally in humans. It possesses the following characteristics: 1) Non-pathogenicity: AAV does not cause human disease, making it a naturally occurring and highly biosafe viral vector. 2) Low immunogenicity: AAV exhibits low immunogenicity, preventing therapeutic failure or severe side effects due to an overreaction of the host immune system, even with multiple deliveries. 3) Diverse tissue targeting: AAV vectors have variable tissue targeting, which can be manipulated through modification of their capsid proteins. Numerous AAV serotypes have been identified and developed (e.g., AAV1, AAV2, and AAV9), each with highly specific infectivity for different tissues (e.g., heart, liver, central nervous system, muscle, etc.). 4) Long-lasting gene expression: AAV-delivered genes can be expressed long-term in target cells. Although the AAV genome typically exists as a circular epichromosome within host cells (rather than integrating into the host genome), it is stable enough to support gene expression for months or even years. These unique biological properties have made it a dominant player in the field of DNA delivery and are considered the "gold standard vector" for gene therapy.

[0005] The wild-type AAV genome is approximately 4.7 kb in size and contains three promoters (P5, P19, and P40), flanked by 145-nt inverted terminal repeats (ITRs) and the Rep and Cap genes in between. Based on the AAV genome structure, engineered recombinant AAV vectors replace the endogenous Rep and Cap genes with an expression cassette. Traditionally, the expression cassette consists of a promoter-driven target gene and a poly(A) tail. However, the design of traditional AAV vectors (promoter + target gene) still faces three key challenges in clinical application: 1) Gene loading capacity: AAV packaging capacity is only approximately 4.7 kb, and classic strong promoters (such as the CMV promoter, which is 600-800 bp in length) occupy a significant amount of space, limiting the loading of large therapeutic genes; 2) Lack of tissue specificity: Non-targeted expression driven by broad-spectrum promoters may induce toxicity. While tissue-specific promoters can improve targeting, their weak transcriptional activity and large size (1-2 kb) further exacerbate the "load-specificity" imbalance. 3) Cytotoxicity: Long-term continuous expression of genes in AAV vectors may cause cytotoxicity, affecting therapeutic efficacy and safety.

[0006] Therefore, breaking through the gene load limitation and insufficient tissue specificity of AAV vectors, solving the toxicity problem caused by sustained expression, and designing a new gene delivery system with efficient expression, precise targeting and dynamic regulation capabilities are key issues that need to be urgently addressed in the current AAV gene therapy field. Summary of the Invention

[0007] In order to solve the above problems, the present application provides a regulatory element combination based on ITR-enhancer driven nucleic acid expression and its application.

[0008] After extensive experimentation and research, the inventors have creatively developed an adeno-associated virus (AAV) expression vector based on an ITR-enhancer combination to regulate nucleic acid expression. Compared to traditional AAV expression vectors, this system can remove the exogenous promoter, releasing a 600-2000 bp vector capacity, significantly increasing the ability to carry large therapeutic genes. Furthermore, it leverages the tissue-specificity, short sequence length (50-100 bp), and pathological microenvironmental responsiveness of the enhancer to achieve tissue-specific and disease-responsive gene expression. Demonstrated versatility in driving tissue-specific expression of large genes (Cas9) has been demonstrated, demonstrating its broad application prospects.

[0009] This application adopts the following technical solutions: In a first aspect, the present application provides a regulatory element combination based on ITR-enhancer driven nucleic acid expression, comprising: At least one inverted terminal repeat (ITR); at least one enhancer; The combination of regulatory elements is delivered via an AAV vector.

[0010] In some embodiments of the present invention, the ITR-enhancer-driven nucleic acid expression regulatory elements are inverted symmetrical repeat sequences at both ends of the AAV genome, which can form a highly stable secondary structure and play an essential role in the replication and packaging of the virus. In the present invention, the ITR is also the core element that drives the expression of the exogenous target gene.

[0011] Furthermore, the ITRs include one or more of ITR1, ITR2, ITR3, ITR5, ITR4, ITR6, and ITR7. Preferably, the ITR sequence described herein is an ITR2 sequence. The ITR2 sequence includes a common wild-type ITR2 sequence (145 bp) and a truncated ITR2 sequence (where a portion of the palindrome is deleted, reducing the sequence length from 145 bp to 130 bp). More preferably, the ITR2 sequence used in the present invention is a truncated ITR2 sequence.

[0012] In some embodiments of the present invention, based on ITR-enhancer-driven nucleic acid expression regulatory elements, the enhancer sequence is a non-coding DNA sequence that can enhance the transcription efficiency of the target gene, including one or more broad-spectrum enhancers, specific enhancers, and pathological responsive enhancers. Among them, specific enhancers include enhancers that have specific enhancing effects on the heart, liver, skeletal muscle, lungs, spleen, kidneys, and / or brain. In the present invention, the enhancer and ITR combination drive efficient and specific expression of the target gene.

[0013] Preferably, the enhancer includes at least one of a broad-spectrum enhancer, a tissue-specific enhancer, and an inducible enhancer.

[0014] Preferably, the broad-spectrum enhancer includes the CMV enhancer; the tissue-specific enhancer includes at least one of the My17 atrial-specific enhancer, the My13 ventricular-specific enhancer, the liver-specific enhancer, the lung-specific enhancer, the skeletal muscle-specific enhancer and the neuron-specific enhancer; the inducible enhancer includes at least one of the corresponding tissue damage enhancer, the corresponding pathological microenvironment enhancer and the corresponding environmental stress enhancer.

[0015] More preferably, the enhancer is the My17 enhancer or the My13 enhancer.

[0016] In some embodiments of the present invention, in the ITR-enhancer-driven nucleic acid expression regulatory element, the enhancer sequence length is 50-500 bp, preferably, the enhancer length of the present invention is 100 bp.

[0017] In some embodiments of the present invention, in the ITR-enhancer-driven nucleic acid expression regulatory element, the enhancer is located at the 3' end of the 5' ITR sequence of the AAV genome, the 5' end of the start codon of the target gene, or the 3' end of PolyA and the 5' end of the 3' ITR sequence. Preferably, the enhancer of the present invention is located at the 3' end of PolyA of the AAV genome and the 5' end of the 3' ITR sequence.

[0018] Furthermore, the above also includes: (iii) the target sequence of miR122.

[0019] The miR122 target sequence (miR122TS) is one of the earliest discovered tissue-specific microRNAs. It is highly liver-specific and virtually undetectable in other tissues. The miR122 sequence is highly conserved across species. By inserting the miR122 target sequence into the aforementioned regulatory elements, it is possible to ensure that the therapeutic gene is primarily expressed in non-hepatic cells (e.g., other targeted tissues) or only under specific conditions, thereby improving the safety and specificity of the treatment.

[0020] In a second aspect, the present application provides a recombinant nucleic acid molecule for improving the specific delivery of a target gene, wherein the recombinant nucleic acid molecule comprises: the above-mentioned regulatory element combination based on ITR-enhancer driven nucleic acid expression, an exogenous target gene, and polyadenylation.

[0021] Furthermore, the exogenous target gene includes at least one of a therapeutic gene for gene therapy, a Cas gene in a gene editing system, and a Cre gene in a Cre / Loxp system. The exogenous target gene may also be any target gene delivered via AAV for biological or medical research. Preferably, the exogenous target genes of the present invention are Cre and Cas9 genes.

[0022] Furthermore, the above-mentioned recombinant nucleic acid molecule is composed of at least four parts: ITR, enhancer, exogenous target gene and polyadenylic acid (PolyA, pA), wherein the pA sequence includes but is not limited to the polyadenylic acid sequence of various mRNAs. Preferably, the pA sequence described in the present invention is SV40 PolyA and a short sequence PolyA.

[0023] Furthermore, the recombinant nucleic acid molecule comprises, from the 5' end to the 3' end, in sequence: inverted terminal repeat sequence ITR-5'UTR-exogenous target gene-3'UTR-3' terminal poly A tail-enhancer-inverted terminal repeat sequence ITR.

[0024] In a third aspect, the present application provides a recombinant adeno-associated virus for improving target gene specific delivery, comprising: AAV protein capsid; The recombinant nucleic acid molecule is the above-mentioned recombinant nucleic acid molecule that improves the specific delivery of the target gene.

[0025] Furthermore, the 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, preferably, the AAV protein capsid of the present invention is AAV9.

[0026] Preferably, the above-mentioned ITR sequence includes one or more of ITR1, ITR2, truncated ITR2, ITR3, ITR5, ITR4, ITR6, and ITR7.

[0027] In a fourth aspect, the present application provides a pharmaceutical composition comprising the aforementioned combination of regulatory elements, or the aforementioned recombinant nucleic acid molecule, or the aforementioned recombinant adeno-associated virus, and a pharmaceutically acceptable excipient. In some preferred embodiments, the pharmaceutical composition can be formulated for administration by intravenous injection, oral administration, inhalation, intranasal administration, intratracheal administration, intraarterial administration, intraocular administration, intramuscular administration, intraperitoneal administration, and / or other parenteral routes.

[0028] In a fifth aspect, the present application provides a method for constructing an adeno-associated virus vector for regulating nucleic acid expression based on an ITR-enhancer combination, comprising: (i) preparing a DNA molecule comprising at least one ITR and an enhancer; and (ii) packaging the DNA molecule using AAV virus.

[0029] Furthermore, the method for constructing an adeno-associated virus vector comprises the following steps: Preparation of plasmid DNA containing AAV genome: The AAV plasmid DNA contains all the DNA sequences described in the present invention, and the enhancer sequence, exogenous target gene sequence and PolyA sequence are located between the ITR sequences.

[0030] AAV vector packaging in HEK293 cells using the three-plasmid method: The three-plasmid method involves co-transfecting HEK293 cells with plasmid DNA containing the AAV genome and two auxiliary packaging plasmids (Rep-Cap plasmid and Ad Helper plasmid) required for AAV packaging to produce AAV particles; Purification of AAV vectors: The method for purifying AAV vectors includes using PEG8000 to collect cell pellets of HEK293 cells transfected with three plasmids, repeatedly freezing and thawing to release AAV from the cell pellets, initially purifying AAV using iodixanol gradient centrifugation, and then further purifying and concentrating AAV by ultrafiltration centrifugation using ultrafiltration tubes.

[0031] In a sixth aspect, the present application provides an application of an adeno-associated virus vector for regulating nucleic acid expression based on an ITR-enhancer combination, wherein the application includes one or more of the following: 1) driving the expression of an exogenous target gene; 2) driving the expression of an exogenous target gene in a specific tissue; 3) driving the expression of an exogenous target gene in a specific period (such as expression during a disease stage).

[0032] In some embodiments of the present invention, the specific tissues include, but are not limited to, one or more of heart, liver, skeletal muscle, lung, spleen, kidney and / or brain tissues.

[0033] In some embodiments of the present invention, the specific time includes but is not limited to the onset of the disease (such as the specific time expression when heart failure occurs).

[0034] In summary, this application has the following beneficial effects: (1) The present invention provides an element combination based on ITR-enhancer for regulating nucleic acid expression, and constructs a new gene expression regulation system without exogenous promoter. By deleting the exogenous promoter, the 600-2000 bp vector capacity is released, which greatly improves the loading capacity of large therapeutic genes. It is particularly suitable for application scenarios of large gene expression, such as using ITR-enhancer to drive the expression of the classic CRISPR / Cas9 system in a single AAV system for gene knockout, realizing applications that traditionally require dual AAV systems.

[0035] (2) The present invention provides an element combination for regulating nucleic acid expression based on ITR-enhancer, which uses ITR-enhancer to drive the expression of exogenous target genes. Compared with the shortcomings of traditional tissue-specific promoters such as long sequences (1-2 kb) and low tissue specificity, the enhancer has high tissue specificity and short sequences, which can achieve specific expression of exogenous target genes.

[0036] (3) The present invention provides an element combination based on ITR-enhancer for regulating nucleic acid expression, which uses ITR-enhancer to drive the expression of exogenous target genes. Compared with the safety issues such as cytotoxicity that may be caused by the continuous expression of traditional AAV, the pathological responsiveness of the enhancer can achieve dynamic expression of exogenous target genes, thereby improving the safety of AAV vectors for gene therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The experimental results in Example 1 show that AAV9-Cre-pA-CMVEnh can drive the efficient expression of the target gene Cre: Figure A shows the results of AAV9-Cre-pA and AAV9-Cre-pA-CMVEnh injected into Rosa fsCas9-tdTomato Flowchart for the design of reporter gene mouse experiments; Figure B shows the expression of TOM (red) reporter gene fluorescence signals in frozen sections of the heart, liver, lung, spleen, kidney, brain, and skeletal muscle of mice after AAV9 injection of two AAV9s, AAV9-Cre-pA and AAV9-Cre-pA-CMVEnh, on the 7th day; Figure C shows the expression of TOM fluorescence signals in Rosa26 fsCas9-tdTomatoQuantitative analysis of frozen sections of various reporter mouse tissues (atria, ventricles, liver, spleen, lung, kidney, brain, and skeletal muscle) (n = 3). Figure D shows two cell-based experiments confirming that ITR transcriptional activity in AAV9-Cre-pA-CMVEnh is dependent on RNA polymerase II (Pol II). In Figures A, B, C, and D, red dots represent AAV9-Cre-pA (control group), and green dots represent AAV9-Cre-pA-CMVEnh (experimental group). Points in the scatterplots represent n values. TOM: tdTomato. Student's t-test, *P < 0.05, **P < 0.01.

[0038] Figure 2 The experimental results in Example 2 show that AAV9-Cre-pA-miR122TS-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh can drive the target gene Cre to be specifically expressed in the atrium and ventricle respectively: Figure A shows the results of AAV9-Cre-pA-miR122TS-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh injected into Rosa fsCas9-tdTomato Flowchart of experimental design of reporter gene mice; Figure B shows subcutaneous injection of AAV9-Cre-pA-miR122TS-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh into Rosa fsCas9-tdTomato Reporter gene neonatal mice and tail vein injection Rosa fsCas9-tdTomato Reporter gene adult mice, showing the results of TOM (red) reporter gene fluorescence signal expression in the mouse atrium and ventricle; Figure C shows AAV9-Cre-pA-miR122TS-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh injected subcutaneously in Rosa fsCas9 -tdTomato Reporter gene neonatal mice and tail vein injection Rosa fsCas9-tdTomato The results of TOM (red) reporter gene fluorescence signal expression were shown in frozen sections of liver, spleen, lung, kidney, brain, and skeletal muscle of adult mice; Figure D shows the expression of TOM (red) reporter gene fluorescence signal in AAV9-Cre-pA-miR122TS-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh. Rosa26 fsCas9-tdTomato(n=3) Quantitative analysis of frozen sections of various tissues (atrium, ventricle, liver, spleen, lung, kidney, brain, and skeletal muscle) of newborn mice expressing the reporter gene; Figure E shows the expression of AAV9-Cre-pA-miR122TS-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh in the Rosa26 fsCas9-tdTomato (n=3) Quantitative analysis results of frozen sections of various tissues (atrium, ventricle, liver, spleen, lung, kidney, brain, and skeletal muscle) of reporter gene adult mice; red dots in C, D, and E represent AAV9-Cre-pA-miR122TS-Myl7Enh (Myl7Enh group), and green dots represent AAV9-Cre-pA-miR122TS-Myl3Enh (Myl3Enh group); the number of points in the scatter plots represents the n value; TOM: tdTomato.

[0039] Figure 3 In Example 3, a mouse atrial fibrillation model was prepared by using the ITR-Myl3Enh combination to achieve single AAV delivery of classic CRISPR / Cas9 specific expression: Figure A is a flowchart of the experimental design of C57 wild-type mice injected with AAV9-Cas9-miR122TS-pA-Myl7Enh-U6-Scn5a.gRNA; Figure B is the electrocardiogram results of mice injected with AAV9-Cas9-miR122TS-pA-Myl7Enh-U6-Scn5a.gRNA; Figure C: Amplicon sequencing detection results of gene knockout in the atrium, ventricle, liver, lung, spleen, kidney, brain and skeletal muscle of mice injected with AAV9-Cas9-miR122TS-pA-Myl7Enh-U6-Scn5a.gRNA; the points in the scatter bar graph represent the n value; Figure D is an immunofluorescence image of atrial-specific knockout of Scn5a; Figure E is a WB image of atrial-specific knockout of Scn5a. DETAILED DESCRIPTION

[0040] 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 used to illustrate the present invention and should not be construed as limiting the scope of the present invention. Specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0041] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art.

[0042] All technical solutions of the present invention can be used for preventive or therapeutic purposes or for non-preventive or non-therapeutic purposes.

[0043] The term "ITR" as used herein refers to inverted symmetric repeats, a fundamental component of the AAV genome. These sequences form a highly stable secondary structure and play an essential role in viral replication and packaging. While the overall structure of ITRs is highly conserved across different serotypes, some subtypes or variants exist, primarily categorized as naturally occurring serotype-associated ITRs and artificially modified ITRs. While there are minor differences in ITR sequences between different AAV serotypes, the core structure (e.g., palindromic sequences and Rep binding sites) remains conserved.

[0044] Among them, ITR1, ITR2, ITR3, ITR4, ITR5, and ITR6 are natural serotype-related ITRs, and truncated ITR2 and ITR7 are artificially modified ITRs. Specifically: ITR1 and ITR2 refer to ITRs derived from AAV serotype 2 (AAV2). A typical AAV2 genome has one ITR at each end. The sequences of these two ITRs are not completely identical, so they are named ITR1 and ITR2, respectively.

[0045] ITR3 refers to the ITR derived from AAV serotype 3 (AAV3); ITR4 refers to the ITR derived from AAV serotype 4 (AAV4); ITR5 refers to the ITR derived from AAV serotype 5 (AAV5); ITR6 refers to the ITR derived from AAV serotype 6 (AAV6).

[0046] Truncated ITR2 refers to an artificial variant obtained by deleting part of the sequence based on the natural AAV2 ITR2; ITR7 refers to synthetic or significantly modified ITR.

[0047] The term "Enh" as used in the present invention refers to an enhancer, which is a non-coding DNA sequence that can enhance the transcription efficiency of a target gene. The term "CMVEnh" refers to the enhancer sequence of the universal promoter CMV, which is a broad-spectrum enhancer with no tissue or spatiotemporal specificity. The term "Myl7Enh" refers to an atrial-specific enhancer, a non-coding sequence that enhances the atrial-specific expression of an exogenous target gene. The term "Myl3Enh" refers to a ventricular-specific enhancer, a non-coding sequence that enhances the ventricular-specific expression of an exogenous target gene.

[0048] The term "exogenous target gene" used in the present invention refers to a target gene whose expression is driven by an ITR-enhancer. The gene can be a therapeutic gene for gene therapy, a Cas gene in a gene editing system, a Cre gene in a Cre / Loxp system, or various target genes delivered by AAV for biological or medical research.

[0049] The term "pA" used in the present invention refers to a polyadenylation sequence, which is a termination signal sequence for mRNA transcription.

[0050] The term "AAV9" as used in the present invention refers to recombinant AAV9 (rAAV9) engineered from wild-type AAV9 (human adeno-associated virus type 9) capable of delivering a target gene, and any complete or partial variants thereof that have been obtained through various engineering, recombination, transformation, modification, and alteration while retaining affinity for the heart. In the AAV vectors of the present invention, including AAV9, the DNA sequence of the AAV vector comprises the following components from the 5' end to the 3' end: inverted terminal repeat (ITR) - 5' untranslated region (5'UTR) - target gene - 3' untranslated region (3'UTR) - 3' terminal poly A tail (PolyA) - enhancer - inverted terminal repeat (ITR).

[0051] The following table lists the nucleic acid sequence numbers and corresponding sequences used in the description of the present invention (wherein the underlined and italicized index represents the "tag" used in the primers used in high-throughput sequencing to distinguish samples, also known as barcodes, which can be freely selected by high-throughput sequencing technicians as needed).

[0052] Table 1 Nucleic acid sequence numbers and corresponding sequences used in the present invention

[0053]

[0054] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0055] Example 1

[0056] This embodiment provides an ITR-enhancer nucleic acid regulatory element combination, which can achieve efficient expression of AAV9 gene delivery in vivo in the Cre-Loxp system.

[0057] In this example, for the Cre-Loxp system, the inventors first constructed the AAV9-Cre-pA adeno-associated virus to evaluate the expression of the AAV9-Cre-pA driver gene in mice; and further constructed the AAV9-Cre-pA-CMVEnh to evaluate whether the ITR-enhancer nucleic acid regulatory element combination enhances the efficient expression of AAV9 gene delivery in vivo.

[0058] The specific process is as follows: 1. Construction of AAV9-Cre-pA and AAV9-Cre-pA-CMVEnh Plasmids The construct was based on the AAV-U6sgRNA-U6sgRNA-Tnnt2-Cre plasmid vector, which can be found in the public literature (Guo Y et al., Analysis of Cardiac Myocyte Maturation Using CASAAV, aPlatform for Rapid Dissection of Cardiac Myocyte Gene Function In Vivo. CircRes., 120(12): 1874-1888, 2017).

[0059] Preparation of AAV9-Cre-pA plasmid: Restriction endonucleases KpnI, NheI, and RsrII were selected as restriction sites in the AAV-U6sgRNA-U6sgRNA-Tnnt2-Cre plasmid vector. The following components were added and mixed: 1 μg of AAV-U6sgRNA-U6sgRNA-Tnnt2-Cre plasmid; 1 μL of KpnI; 1 μL of NheI; 1 μL of RsrII; 3 μL of 10× CutSmart buffer; and nuclease-free water, adding the total volume to 30 μL. The resulting mixture was incubated in a 37°C water bath for 60 min. The digested products were subjected to agarose gel electrophoresis and recovered.

[0060] The Cre sequence was amplified by PCR, and the upstream primer F sequence of the primer pair used was shown in SEQ ID NO: 1 in Table 1, and the downstream primer R sequence was shown in SEQ ID NO: 2 in Table 1; the pA sequence was amplified by PCR, and the primer pair sequences used were shown in SEQ ID NO: 3 (F) and SEQ ID NO: 4 (R) in Table 1.

[0061] The PCR product was recovered from the gel and cloned into the enzyme-digested plasmid using seamless cloning. The enzymes and seamless cloning kits used in the preparation process are commercially available (NEB, R3142S, R3131S, R0501S; Nanjing Novozymes Biotechnology Co., Ltd., C117-01). The ligation product was transformed into Stable3 competent cells and plated onto LB medium plates containing ampicillin. Single colonies were selected for Sanger sequencing to obtain the successfully cloned AAV9-Cre-pA plasmid for future use.

[0062] (2) Preparation of AAV9-Cre-pA-CMVEnh plasmid The AAV-U6sgRNA-U6sgRNA-Tnnt2-Cre plasmid enzyme digestion and gel recovery steps are the same as above.

[0063] The Cre sequence was amplified by PCR using the primer pairs shown in SEQ ID NO: 1 (F) and SEQ ID NO: 2 (R) in Table 1 ; The pA sequence was amplified by PCR using the primer pairs shown in Table 1 as SEQ ID NO: 3(F) and SEQ ID NO: 5(R); The CMVEnh sequence was amplified by PCR, and the primer pair sequences used were shown in Table 1 as SEQ ID NO: 6 (F) and SEQ ID NO: 7 (R).

[0064] The PCR product was recovered from the gel and cloned into the enzyme-digested plasmid using seamless cloning. The ligation product was transformed into Stable3 competent cells and plated onto LB medium plates containing ampicillin. A single colony was selected for Sanger sequencing to obtain the successfully cloned AAV9-Cre-pA-CMVEnh plasmid for future use.

[0065] 2. Packaging AAV9 Viruses AAV9-Cre-pA and AAV9-Cre-pA-CMVEnh In this step, the constructed AAV9-Cre-pA plasmid and AAV9-Cre-pA-CMVEnh plasmid were separately used for AAV9 viral packaging. HEK293T cells were used as host cells for AAV9 production. For high-quality extraction of the plasmids required for AAV9 packaging, an endotoxin-free plasmid extraction kit (Tiangen Biotech (Beijing) Co., Ltd., DP117) was used.

[0066] The specific steps of AAV packaging are: ① Remove the frozen HEK293T cells from liquid nitrogen and thaw them. Place them in a 15cm cell culture dish and culture them. When the cells grow to ~90% confluency, perform cell passage. Each AAV packaging requires 10 15cm cell culture dishes of HEK293T cells. When the cell density reaches ~90% confluency, add 1.8mL of transfection reagent PEI (1mg / mL), 7μg AAV plasmid, 7μg Rep2 / Cap9 plasmid and 20μg pHelper plasmid to each dish of cells. After culturing at 37℃ for 8-12 hours, replace the culture medium with serum-free high-glucose DMEM containing 1% double-antibody. Continue culturing at 37℃ for 66-72 hours, then collect the cells. ② Centrifuge the collected cell pellet at 3000 rpm for 5 minutes, discard the supernatant, resuspend the cell pellet in AAV lysis buffer (20 mM Tris pH 8.0, 1 mM MgCl2, 150 mM NaCl), and store at -80°C. ③ Add 1 / 4 volume of 40% PEG8000 solution containing 2.5M NaCl to the cell culture medium collected in step ②, let it stand at 4℃ for 2h, centrifuge at 4000rpm for 30min to precipitate, discard the supernatant, resuspend the precipitate in AAV lysis buffer, and mix it with the cell suspension in step ②. After adding 50μg / ml universal nuclease, the cell suspension was repeatedly frozen and thawed at 37℃ and -80℃ for 3 times to fully lyse the cells.

[0067] ④ Centrifuge at 4000 rpm for 30 min at 4°C, collect the supernatant, and layer it on an iodixanol gradient (OptiPrep) for AAV purification by density gradient ultracentrifugation; ⑤ In a centrifugal filter tube with a molecular cutoff of 100 kDa, AAV was washed and concentrated with a PBS solution containing 0.001% Pluronic F-68. The AAV titer was determined by real-time fluorescence quantitative PCR (RT-qPCR). The obtained AAV titer was above 1E+13 vg / mL.

[0068] 3. Packaging AAV-DJ Virus AAV-DJ-Cre-pA-CMVEnh The same method for preparing AAV9 virus as described in step (2) was used to replace the Rep2 / Cap9 plasmid with the Rep2 / Cap-DJ plasmid, thereby completing the AAV-DJ virus packaging of the plasmid (AAV-DJ is an artificially modified chimeric serotype with a broad tropism and high transduction efficiency) to obtain AAV-DJ-Cre-pA-CMVEnh.

[0069] ITR drives low expression of exogenous target genes Select the fluorescent reporter gene mouse with CAG promoter-Loxp-stop-Loxp-Cas9-2A-tdTomato knocked into the Rosa26 safety site (Jicui Yaokang, Strain No. T002249, Rosa26 fsCas9-tsTomato Indicated), this mouse has a fluorescent reporter gene that can be activated by Cre.

[0070] AAV9-Cre-pA was added at a rate of 5 × 10 10 Newborn Rosa26fsCas9-tdTomato mice (n=3) were subcutaneously injected with 100 μg / g (vg / g stands for vector genomes per gram body weight). Mice were sacrificed 7 days later, and the heart, liver, spleen, lung, kidney, brain, and skeletal muscle were obtained, fixed, dehydrated, embedded, and frozen sectioned (8 μm thickness). Tissue sections were stained with 4',6-diamidino-2-phenylindole (DAPI) and imaged using a confocal microscope. Cells expressing the tdTomato (TOM) reporter gene fluorescent protein were counted and statistically analyzed using Image J and Graphpad Prism software.

[0071] The results are as follows Figure 1 As shown in Figure C, the TOM-positive cell rate in the heart is approximately 5%, while in liver tissue sections, it reaches approximately 40%. In other organs, such as the spleen, lung, kidney, skeletal muscle, and brain, the positive cell rate is almost 0%. This indicates that the ITR can drive expression of the target gene Cre, indicating that the ITR has weak promoter transcriptional activity.

[0072] 4. ITR-enhancers significantly increase the expression of exogenous target genes Prepare in the same way as above Rosa26 fsCas9-tdTomato Fluorescent reporter gene mice.

[0073] AAV9-Cre-pA and AAV9-Cre-pA-CMVEnh were subcutaneously injected into two groups of Rosa26fsCas9-tdTomato newborn mice (n=3) at a dose of 5×1010 vg / g ( Figure 1(As shown in A), mice were sacrificed 7 days later. Hearts, livers, spleens, lungs, kidneys, brains, and skeletal muscles were obtained from both groups and fixed, dehydrated, embedded, and frozen sectioned. Tissue sections were stained with 4',6-diamidino-2-phenylindole (DAPI) and imaged using a confocal microscope. Cells positive for the fluorescent protein expressing the tdTomato (TOM) reporter gene were counted and statistically analyzed using Image J and Graphpad Prism software.

[0074] like Figure 1 B and Figure 1 As shown in C, compared with mice injected with AAV9-Cre-pA, the TOM-positive cell rate in heart and liver tissue sections of mice injected with AAV9-Cre-pA-CMVEnh reached almost 100%, the TOM-positive cell rate in the lung was ~25%, and the TOM-positive cell rate in the spleen was ~10%.

[0075] like Figure 1 As shown in D, AAV-DJ-Cre-pA-CMVEnh infected Neuro2a and 393T cells, respectively, and small molecule inhibitors of RNA polymerase I (Pol I), RNA polymerase II (Pol II), and RNA polymerase III (Pol III) were added, respectively. The results showed that only the Pol II inhibitor could significantly inhibit the transcriptional activity of the ITR-enhancer.

[0076] Conclusion: ITR-enhancer can significantly enhance the expression of target gene Cre, and ITR transcriptional activity is dependent on Pol II.

[0077] Example 2

[0078] This example provides ITR-enhancer nucleic acid regulatory elements to drive efficient expression of exogenous target genes in the heart The inventors selected atrial and ventricular-specific enhancers, and used the Cre-Loxp system to select the atrial-specific enhancer Myl7 Enhancer (Myl7Enh) and the ventricular-specific enhancer Myl3 Enhancer (Myl3Enh). At the same time, they selected miR122TS to inhibit the weak transcriptional activity of ITR in the liver. In this way, the AAV9-Cre-miR122TS-pA-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh adeno-associated viruses were constructed to evaluate whether the ITR-enhancer nucleic acid regulatory element combination drives tissue-specific high expression of AAV9 gene delivery in vivo.

[0079] The specific process is as follows: Construct AAV9-Cre-miR122TS-pA-Myl7Enh and AAV9-Cre-miR122TS- pA-Myl3Enh plasmid The AAV9-Cre-pA-CMVEnh plasmid constructed in Example 1 was selected, and the restriction endonuclease BamHI (NEB, R0136S) was used as the restriction endonuclease site. The following components were added and mixed: 1 μg of AAV9-Cre-pA-CMVEnh plasmid; 1 μL of BamHI; 3 μL of 10× CutSmart buffer; and nuclease-free water, adding up to 30 μL. The resulting mixture was incubated in a 37°C water bath for 60 min. The digestion products were subjected to agarose gel electrophoresis and recovered.

[0080] A chemically synthesized DNA fragment containing the 3× miR122TS gene sequence (SEQ ID NO: 8 in Table 1, commissioned by Hangzhou Youkang Biotechnology Co., Ltd.) was cloned into the AAV9-Cre-pA-CMVEnh vector to construct the AAV9-Cre-miR122TS-pA-CMVEnh plasmid. The AAV9-Cre-miR122TS-pA-CMVEnh plasmid was further digested with restriction enzymes AscI (NEB, R0558S) and RsrII. The following components were added and mixed: 1 μg of AAV9-Cre-miR122TS-pA-CMVEnh plasmid; 1 μL of AscI; 1 μL of RsrII; 3 μL of 10× CutSmart buffer; and nuclease-free water (to a final volume of 30 μL). The resulting mixture was reacted in a 37°C water bath for 60 min. The digestion products were subjected to agarose gel electrophoresis and recovered.

[0081] The chemically synthesized Myl7Enh DNA fragment (as shown in SEQ ID NO: 9 in Table 1) and the Myl3Enh DNA fragment (as shown in SEQ ID NO: 10 in Table 1) were cloned into the AAV9-Cre-pA-CMVEnh vector, respectively, to successfully construct the AV9-Cre-miR122TS-pA-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh plasmids.

[0082] (2) AAV9 virus packaging The same method for preparing AAV9 virus as in Example 1 was followed, except that the AAV plasmid was replaced with the plasmid in step (1) of this example, thereby completing the AAV9 virus packaging of the two plasmids and obtaining AAV9-Cre-miR122TS-pA-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh.

[0083] (3) Evaluation of the expression of the target gene Cre driven by ITR-Myl7Enh and ITR-Myl3Enh AAV9-Cre-miR122TS-pA-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh were injected into the 400 μg / mL PBS at 5 × 10 μg / mL. 10 vg / g were subcutaneously injected into Rosa26fsCas9-tdTomato (n=3) newborn mice (e.g. Figure 2 (As shown in A), mice were sacrificed 7 days later, and the heart, liver, spleen, lung, kidney, brain, and skeletal muscle were obtained, fixed, dehydrated, embedded, and frozen sectioned (8 μm thickness). Tissue sections were stained with 4',6-diamidino-2-phenylindole (DAPI) and imaged under a confocal microscope. Cells positive for the fluorescent protein of the tdTomato (TOM) reporter gene were counted and statistically analyzed using Image J and Graphpad Prism software.

[0084] The results are as follows Figure 2 As shown in Figures B, 2C, and 2D, in mice injected with AAV9-Cre-miR122TS-pA-Myl7Enh, the TOM-positive cell rate in the atria reached almost 95%, while in ventricular tissue sections, the positive cell rate was only about 5%, and in other organs such as the spleen, lung, kidney, skeletal muscle, and brain, the positive cell rate was almost 0%. In mice injected with AAV9-Cre-miR122TS-pA-Myl3Enh, the TOM-positive cell rate in the atria was only about 5%, while in ventricular tissue sections, the positive cell rate reached about 90%, and in other organs such as the spleen, lung, kidney, skeletal muscle, and brain, the positive cell rate was almost 0%.

[0085] In addition, to evaluate whether the expression of exogenous target genes driven by the nucleic acid regulatory elements of the TR-enhancer is affected by the age of the subjects and the mode of administration, the inventors injected the viruses AAV9-Cre-miR122TS-pA-Myl3Enh and AAV9-Cre-miR122TS-pA-Myl7Enh into the tail vein of adult Rosa26fsCas9-tdTomato mice (4 weeks old, adult group, n=3). The mice were killed one week later, and the heart, liver, spleen, lung, kidney, brain, and skeletal muscle tissues were frozen and stained with DAPI.

[0086] The results are as follows Figure 2As shown in Figures 2B, 2C, and 2E, in adult mice injected with AAV9-Cre-miR122TS-pA-Myl7Enh, the TOM-positive cell rate reached nearly 90% in the atria, but only about 5% in ventricular tissue sections, and almost 0% in other organs such as the spleen, lung, kidney, skeletal muscle, and brain. In adult mice injected with AAV9-Cre-miR122TS-pA-Myl3Enh, the TOM-positive cell rate was only about 5% in the atria, but reached about 80% in ventricular tissue sections, and almost 0% in other organs such as the spleen, lung, kidney, skeletal muscle, and brain.

[0087] Conclusion: ITR-enhancer nucleic acid regulatory elements can drive the expression of exogenous target genes in specific tissues by selecting different tissue-specific enhancers, and this regulatory gene expression is not affected by the route of administration and animal age.

[0088] Example 3

[0089] This example provides ITR-enhancer nucleic acid regulatory elements to implement a single AAV delivery CRISPR / spCas9 system In traditional AAV delivery systems, the nucleic acid regulatory element is an exogenous promoter. Since promoter sequences are generally longer than 500 base pairs (bp), they occupy more than 10% of the AAV genome (~4.7 kb). This limits the gene load of AAV and prevents the delivery of large exogenous target genes. Traditional AAV delivery of CRISPR / spCas9 requires dual AAVs: one AAV expresses spCas9 alone, while the other delivers the gRNA. The use of dual AAVs can pose safety concerns.

[0090] In this application, the ITR-enhancer nucleic acid regulatory element can maximize the DNA loading capacity of AAV by deleting the exogenous promoter and selecting a tissue-specific, short-sequence enhancer. Therefore, in this embodiment, the inventors selected a short-sequence Myl7Enh (abbreviated as Myl7ENh mini) and combined it with the ITR to form the ITR-Myl7Enh mini, which was delivered via a single AAV CRISPR / spCas9 system to achieve gene editing.

[0091] To this end, the inventors designed a spCas9 single-stranded guide RNA (sgRNA) targeting exon 1 of Scn5a, driven by the U6 promoter. Scn5a encodes the α subunit of the cardiac sodium channel Nav1.5, mediating rapid sodium influx (INa) and triggering cardiomyocyte depolarization. Mutations in this gene can lead to a range of inherited arrhythmias, including long QT syndrome type 3 (LQT3), Brugada syndrome, and progressive heart block (PCCD).

[0092] The specific implementation steps are as follows: Construct AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-Scn5a.sgRNA plasmid.

[0093] The AAV9-Cre-miR122TS-pA-Myl7Enh plasmid constructed in the example was selected. Restriction enzymes KpnI and HindIII (NEB, R0104S) were used as digestion sites. The following components were added and mixed: 1 μg of AAV9-Cre-miR122TS-pA-Myl7Enh plasmid; 1 μL of KpnI; 1 μL of HindIII; 3 μL of 10× CutSmart buffer; and nuclease-free water, adding the final volume to 30 μL. The resulting mixture was incubated in a 37°C water bath for 60 minutes. The digestion products were subjected to agarose gel electrophoresis and recovered.

[0094] The spCas9 sequence was amplified by PCR using the primer pairs shown in Table 1 (SEQ ID NO: 11(F) and SEQ ID NO: 12(R). The PCR product was recovered from gel and cloned into a restriction-digested plasmid vector using a seamless cloning kit (Nanjing Novozymes Biotechnology Co., Ltd., C117-01). The ligation product was transformed into Stable3 competent cells and plated on LB medium plates containing ampicillin. Single colonies were selected for Sanger sequencing to obtain the successfully cloned AAV9-spCas9-miR122TS-pA-Myl7Enh plasmid for future use.

[0095] Using the AAV9-spCas9-miR122TS-pA-Myl7Enh plasmid described above, with NheI and RsrII selected as restriction sites, the following components were added and mixed: 1 μg of AAV9-spCas9-miR122TS-pA-Myl7Enh plasmid; 1 μL of NheI; 1 μL of RsrII; 3 μL of 10× CutSmart buffer; and nuclease-free water, adding up to 30 μL. The resulting mixture was incubated in a 37°C water bath for 60 min. The digested products were subjected to agarose gel electrophoresis and recovered. The chemically synthesized pA-Myl7Enhmini-U6-sgRNA DNA fragment (as shown in SEQ ID NO: 13 in Table 1, commissioned to Hangzhou Youkang Biotechnology Co., Ltd. for synthesis) was cloned into the AAV9-spCas9-miR122TS-pA-Myl7Enh vector to successfully construct the AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-sgRNA plasmid.

[0096] Using the aforementioned AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-sgRNA plasmid, PaqCI was selected as the restriction site. The following components were added and mixed: 1 μg of AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-sgRNA plasmid; 1 μL of PaqCI; 3 μL of 10× CutSmart buffer; and nuclease-free water, adding the final volume to 30 μL. The resulting mixture was incubated in a 37°C water bath for 60 min. The digested products were subjected to agarose gel electrophoresis and recovered.

[0097] sgRNA was designed for the mouse Scn5a gene using the CRISPick software, and the optimal sgRNA located on exon 1 was screened out, as shown in SEQ ID NO: 14 in Table 1. The primers for Scn5a.sgRNA were synthesized from the successful AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-sgRNA constructed above, preferably using the PaqCI restriction site. The primer sequences are shown in SEQ ID NO: 15 (F) and SEQ ID NO: 16 (R) in Table 1, respectively. The AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-Scn5a.sgRNA plasmid was successfully cloned by T4 ligation.

[0098] (2) AAV9 virus packaging The same method for preparing AAV9 virus as in Example 1 was followed, except that the AAV plasmid was replaced with the plasmid in step (1) of this example, thereby completing the AAV9 virus packaging of the plasmid and obtaining AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-Scn5a.sgRNA.

[0099] (3) ITR-Myl7Enhmini drives atrial-specific expression of spCas9 and knocks out Scn5a to successfully establish a mouse atrial fibrillation model AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-Scn5a.sgRNA was added at a rate of 1×10 11 vg / g were injected subcutaneously into C57 newborn mice (n=3) (as Figure 3 As shown in A), 30 days later, the mice were anesthetized with tribromoethanol and the surface electrocardiogram of the mice was measured by Power Lab electrocardiograph. Figure 3 B, ECG results showed that mice injected with AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-Scn5a.sgRNA developed obvious atrial fibrillation.

[0100] After ECG testing, mice were sacrificed, and tissues from the atria, ventricles, liver, spleen, lungs, kidneys, brain, and skeletal muscle were collected for amplicon sequencing (Amplicon-Seq). The specific protocol is as follows: After removing tissues from the atria, ventricles, liver, spleen, lungs, kidneys, brain, and skeletal muscle of mice (n = 3), gDNA was extracted from heart and liver tissues using a tissue genomic DNA (gDNA) extraction kit (Shanghai Sangon Biotech Co., Ltd.).

[0101] The specific operation process was as follows: Primers were designed for high-throughput sequencing library construction of the target locus. The primer sequences are shown in Table 1 as SEQ ID NO: 17(F) and SEQ ID NO: 18(R), respectively. PCR amplification of the target fragment was performed using gDNA as a template. The following components were added: 2 μg gDNA; 1 μL upstream primer (F); 1 μL downstream primer (R); 10 μL 2× KeyPo Master Mix; and nuclease-free water to 20 μL. The added sample was placed in a thermal cycler and the PCR reaction program was as follows: 98°C for 30 s; 98°C for 10 s, 70°C for 30 s, 72°C for 60 s, touchdown PCR for 15 cycles; 98°C for 10 s, 70°C for 30 s, 72°C for 60 s, touchdown PCR for 15 cycles; 72°C for 5 min; hold at 12°C. The PCR product was purified. The second round of PCR amplification was performed using primers P5 and P7, provided by Mingma (Shanghai) Biotechnology Co., Ltd.; the primer sequences are shown in Table 1 as SEQ ID NO: 19 (F) and SEQ ID NO: 20 (R), respectively. PCR amplification of the target fragment was performed using the first-round PCR product as a template. The following components were added: 2 μg of PCR product; 1 μL of upstream primer (F); 1 μL of downstream primer (R); 10 μL of 2× KeyPo Master Mix; and nuclease-free water to a total of 20 μL. The added sample was placed in a thermal cycler and the PCR reaction program was as follows: 98°C for 30 s; 20 cycles of 98°C for 10 s, 55°C for 30 s, and 72°C for 60 s; 72°C for 5 min; and a hold at 12°C. After agarose gel electrophoresis of the PCR product, the target fragment was excised and purified by gel recovery. The purified fragment was subjected to second-generation sequencing (Beijing Novogene Technology Co., Ltd.). The sequencing results were analyzed using CRISPResso2, and statistical analysis and plotting were performed using Graphpad Prism software.

[0102] like Figure 3 As shown in C, the results showed that the mice mainly produced gene editing (insertion / deletion mutations, represented by indels) in the atrium, with the indels generation rate in atrial tissue being ~23%, and the indels generation rates in other tissues being less than ~5%.

[0103] like Figure 3 As shown in D and 3E, immunofluorescence and WB results indicated that Scn5a was only knocked out in the atrium, while the expression level of Scn5a in the ventricle did not change.

[0104] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A regulatory element combination based on ITR-enhancer driven nucleic acid expression, characterized in that: It includes: (i) at least one inverted terminal repeat (ITR), and (ii) at least one enhancer; The regulatory element combination is delivered via an AAV vector without an exogenous promoter.

2. The regulatory element combination based on ITR-enhancer driven nucleic acid expression according to claim 1, characterized in that: The enhancers include one or more of broad-spectrum enhancers, specific enhancers, and pathology-responsive enhancers.

3. The regulatory element combination based on ITR-enhancer driven nucleic acid expression according to claim 2, characterized in that: The enhancer includes at least one of a broad-spectrum enhancer, a tissue-specific enhancer, and an inducible enhancer.

4. The regulatory element combination based on ITR-enhancer driven nucleic acid expression according to claim 3, characterized in that: The enhancers are the atrial-specific enhancer My17 enhancer and the ventricular-specific enhancer My13 enhancer.

5. The regulatory element combination based on ITR-enhancer driven nucleic acid expression according to claim 3, characterized in that: Also includes: (iii) Target sequence of miR122.

6. A recombinant nucleic acid molecule for improving target gene specific delivery, characterized in that: The recombinant nucleic acid molecule comprises: the regulatory element combination based on ITR-enhancer driven nucleic acid expression according to any one of claims 1 to 5, an exogenous target gene and polyadenylation.

7. The recombinant nucleic acid molecule for improving target gene specific delivery according to claim 6, characterized in that: The recombinant nucleic acid molecule comprises, from 5' end to 3' end, in sequence: inverted terminal repeat sequence ITR-5'UTR-exogenous target gene-3'UTR-3' terminal poly A tail-enhancer-inverted terminal repeat sequence ITR.

8. The recombinant nucleic acid molecule for improving target gene specific delivery according to claim 7, characterized in that: The exogenous target gene is the Cre gene of the Cre / Loxp system.

9. The recombinant nucleic acid molecule for improving target gene specific delivery according to claim 8, characterized in that: The recombinant nucleic acid molecule is any one of the following: (i) Cre-miR122TS-pA-Myl7Enh; (ii) Cre-miR122TS-pA-Myl3Enh; (iii) Cre-miR122TS-pA-Myl7Enhmini; (iv) Cre-miR122TS-pA-Myl3Enhmini.

10. A recombinant adeno-associated virus for improving target gene specific delivery, characterized in that: It includes: (i) AAV protein capsid; (ii) A recombinant nucleic acid molecule, which is a recombinant nucleic acid molecule for improving target gene specific delivery according to any one of claims 6 to 9.

11. The recombinant adeno-associated virus for improving target gene specific delivery according to claim 10, characterized in that: The exogenous target gene includes at least one of a therapeutic gene for gene therapy, a Cas gene in a gene editing system, and a Cre gene in a Cre / Loxp system.

12. The recombinant adeno-associated virus for improving target gene specific delivery according to claim 10, characterized in that: The recombinant adeno-associated virus is any one of the following: (i) AAV9-Cre-miR122TS-pA-Myl7Enh; (ii) AAV9-Cre-miR122TS-pA-Myl3Enh; (iii) AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-Scn5a.sgRNA.

13. The recombinant adeno-associated virus for improving target gene specific delivery according to claim 10, characterized in that: The AAV is one or more selected from AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-PHP.eB and / or MyoAAV; The ITR sequence includes one or more of ITR1, ITR2, ITR3, ITR5, ITR4, ITR6, and ITR7.

14. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the regulatory element combination according to any one of claims 1 to 5, or the recombinant nucleic acid molecule according to any one of claims 6 to 9, or the recombinant adeno-associated virus according to any one of claims 10 to 13, and a pharmaceutically acceptable excipient.

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