A regulatory element combination based on itr-enhancer driven nucleic acid expression and application thereof

Abstract

The application relates to the field of biological medicine, and particularly discloses a regulatory element combination based on ITR-enhancer-driven nucleic acid expression and application thereof. The regulatory element combination based on ITR-enhancer-driven nucleic acid expression comprises at least one inverted terminal repeat (ITR) and at least one enhancer; and the regulatory element combination is delivered through an AAV vector. The application also provides a recombinant nucleic acid molecule for improving target gene-specific delivery, which comprises the above regulatory element combination, an exogenous target gene and a poly A. The AAV vector provided by the application can drive the expression of a target gene without the action of a traditional promoter, and has the advantages of enhancer tissue specificity, a short sequence (50-100 bp) and pathological microenvironment response, thereby breaking through the limitations of traditional AAV vector capacity, targeting and dynamics, improving the DNA load of the AAV vector, and realizing the tissue and space-time expression specificity of the target gene.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, more specifically, it relates to a combination of regulatory elements based on ITR-enhancer driven nucleic acid expression and its application. BACKGROUND

[0002] Gene therapy provides a promising solution for genetic diseases (such as diseases caused by single gene mutations), the core idea of which is mainly achieved by three ways to treat genetic diseases: 1) gene supplement, delivering functional normal genes to patient cells to make up for the defects of original genes; 2) gene silencing, inhibiting the expression of abnormal genes through RNA interference technology, etc. to block their harmful effects; 3) gene editing, accurately correcting pathogenic mutations (such as base substitution or fragment repair), and restoring gene sequences from the root. For example, Zolgensma gene therapy approved by the US FDA in 2018 to treat spinal muscular atrophy (SMA), which uses AAV vectors to deliver SMN1 genes to spinal cord neurons to supplement the inability of SMN to express normally due to gene mutations, thereby increasing the expression of SMN protein with complete function, and thus curing the disease in vivo gene therapy. The application of gene therapy is expected to achieve radical treatment of genetic diseases, and even achieve the effect of "one-time cure".

[0003] Gene therapy achieves intervention on diseases by delivering biological macromolecules such as DNA, RNA or protein, among which DNA delivery is the most widely used strategy in clinical application due to its long-acting, strong stability and the ability to encode complex functional genes. The core mechanism is to deliver functional gene fragments in the form of exogenous DNA to the patient's cells, 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 naturally exists in the human body. It has the following characteristics: 1) non-pathogenic, AAV does not cause human diseases, and is a naturally occurring and highly biologically safe viral vector; 2) low immunogenicity, AAV has low immunogenicity, and even in the case of multiple deliveries, it can avoid treatment failure or serious side effects caused by excessive reaction of the host immune system; 3) diverse tissue targeting, the tissue targeting of AAV vectors can be regulated by modification of its capsid protein. At present, a variety of AAV serotypes (such as AAV1, AAV2, AAV9) have been identified and developed, each of which has a high degree of specific infectivity for different tissues (such as heart, liver, central nervous system, muscle tissue, etc.); 4) persistent gene expression characteristics, AAV-delivered genes can be expressed in target cells for a long time. Although the genome of AAV usually exists in the host cell in the form of a circular chromatin (rather than integrated into the host genome), its stability is sufficient to support gene expression for several months or even years. With the above unique biological characteristics, it occupies a dominant position in the field of DNA delivery and is known as the "gold standard vector" of gene therapy.

[0005] The wild-type AAV genome has a package size of about 4.7 kb, contains three promoters (P5, P19, P40), and is composed of two terminal inverted repeat sequences (inverted terminal repeat, ITR) at both ends and the middle Rep, Cap genes. Based on the genome structure of AAV, the endogenous Rep and Cap genes of the engineered recombinant AAV vector are replaced by an expression cassette, and the traditional expression cassette is composed of a promoter driving a target gene and a polyA tail. However, the design of the traditional AAV vector (promoter + target gene) still faces three major challenges in clinical applications: 1) gene load limitation: the AAV packaging capacity is only about 4.7 kb, while the classic strong promoter (such as the CMV promoter, with a length of 600-800 bp) occupies a large capacity, limiting the loading of large therapeutic genes; 2) insufficient tissue specificity, non-targeted expression driven by broad-spectrum promoters can cause toxic reactions, while tissue-specific promoters can improve targeting, but their weak transcriptional activity and larger size (1-2 kb) further exacerbate the "load-specificity" imbalance. 3) cytotoxicity, long-term sustained expression of genes in AAV vectors can cause cytotoxicity, affecting treatment efficacy and safety.

[0006] Therefore, breaking through the gene load limitation and tissue specificity deficiency of AAV vectors, and solving the toxicity problem caused by sustained expression, designing a new type of gene delivery system with high expression, precise targeting and dynamic regulation ability, is a key problem to be solved in the current AAV gene therapy field. SUMMARY

[0007] To solve the above problems, the application provides a combination of regulatory elements for driving nucleic acid expression based on ITR-enhancer and application thereof.

[0008] The inventors have creatively invented an adeno-associated virus (AAV) expression vector based on ITR-enhancer combination for regulating nucleic acid expression through experiments and research. Compared with traditional AAV expression vectors, the system can delete exogenous promoters to release 600-2000 bp vector capacity, significantly improving the loading capacity of large therapeutic genes; at the same time, the advantages of enhancer such as tissue specificity, short sequence (50-100 bp) and pathological microenvironment response (hypoxia / inflammation) are utilized to realize tissue-specific and disease-responsive gene expression. Examples prove the universality of the system in driving large genes (Cas9) to realize tissue-specific expression, which has broad application prospects.

[0009] The application adopts the following technical solutions:

[0010] In a first aspect, the application provides a combination of regulatory elements for driving nucleic acid expression based on ITR-enhancer, which comprises:

[0011] at least one inverted terminal repeat sequence ITR;

[0012] at least one enhancer;

[0013] The combination of regulatory elements is delivered by an AAV vector.

[0014] In some embodiments of the application, in the combination of regulatory elements for driving nucleic acid expression based on ITR-enhancer, the ITR is an inverted and symmetric repeat sequence at both ends of the AAV genome, which can form a highly stable secondary structure and plays an essential role in the replication and packaging process of the virus. In the present application, ITR is also a core element for driving expression of exogenous target genes.

[0015] Further, the ITR includes one or more of ITR1, ITR2, ITR3, ITR5, ITR4, ITR6, and ITR7. Preferably, the ITR sequence of the present application is an ITR2 sequence. The ITR2 sequence includes a common wild-type ITR2 sequence (145 bp) and a truncated ITR2 sequence (deleting part of the palindromic sequence, the sequence length is reduced from 145 bp to 130 bp). More preferably, the ITR2 sequence used in the present application is a truncated ITR2 sequence.

[0016] In some embodiments of the present application, in the ITR-enhancer driven nucleic acid expression regulatory element, the enhancer sequence is a non-coding DNA sequence that can enhance the transcription efficiency of the target gene, including one or more of a broad-spectrum enhancer, a specific enhancer, and a pathological response enhancer. Among them, the specific enhancer includes an enhancer that has a specific enhancing effect on the heart, liver, skeletal muscle, lung, spleen, kidney and / or brain. In the present application, the enhancer is combined with the ITR to drive the efficient and specific expression of the target gene.

[0017] Preferably, the above-mentioned enhancer includes at least one of a broad-spectrum enhancer, a tissue-specific enhancer, and an inducible enhancer.

[0018] Preferably, the broad-spectrum enhancer includes a CMV enhancer; the tissue-specific enhancer includes at least one of a Myl7 atrial-specific enhancer, a Myl3 ventricular-specific enhancer, a liver-specific enhancer, a lung-specific enhancer, a skeletal muscle-specific enhancer, and a neuron-specific enhancer; and the inducible enhancer includes at least one of a corresponding tissue damage enhancer, a corresponding pathological microenvironment enhancer, and a corresponding environmental stress enhancer.

[0019] More preferably, the enhancer is a Myl7 enhancer or a Myl3 enhancer.

[0020] In some embodiments of the present application, in the ITR-enhancer driven nucleic acid expression regulatory element, the length of the enhancer sequence is 50-500 bp, and preferably, the length of the enhancer of the present application is 100 bp.

[0021] In some embodiments of the present application, 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, the 3' end of the PloyA, or the 5' end of the 3' ITR sequence. Preferably, the enhancer of the present application is located at the 3' end of the PloyA of the genome of the AAV, or the 5' end of the 3' ITR sequence.

[0022] Further, the above also includes: (iii) a target sequence of miR122.

[0023] Among them, the target sequence of miR122 (miR122 target sequence, abbreviated as miR122TS) is one of the earliest discovered tissue-specific microRNAs, which has high liver specificity and is almost undetectable in other tissues, and the miR122 sequence is highly conserved between different species. By inserting the target sequence of miR122 in the above-mentioned regulatory element, it can be ensured that the therapeutic gene is mainly expressed in non-liver cells (such as other targeted tissues) or only under specific conditions, thereby improving the safety and specificity of the treatment.

[0024] In a second aspect, the present application provides a recombinant nucleic acid molecule for improving target gene specific delivery, comprising: the above-mentioned combination of regulatory elements for ITR-enhancer-driven nucleic acid expression, an exogenous target gene, and a poly A.

[0025] Further, the above-mentioned exogenous target gene comprises 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 can also be various target genes for biological or medical research delivered by AAV. Preferably, the exogenous target gene of the present application is a Cre and Cas9 gene.

[0026] Further, the above-mentioned recombinant nucleic acid molecule is composed of at least ITR, enhancer, exogenous target gene, and poly A (PolyA, pA), wherein the pA sequence includes but is not limited to various poly A sequences of mRNA, and preferably, the pA sequence of the present application is SV40 PolyA and a short sequence PolyA.

[0027] Further, the above-mentioned recombinant nucleic acid molecule comprises, in order from 5' end to 3' end: inverted terminal repeat sequence ITR-5' UTR-exogenous target gene-3' UTR-3' terminal poly A tail-enhancer-inverted terminal repeat sequence ITR.

[0028] In a third aspect, the present application provides a recombinant adeno-associated virus for improving target gene specific delivery, comprising:

[0029] an AAV protein capsid;

[0030] a recombinant nucleic acid molecule, which is the above-mentioned recombinant nucleic acid molecule for improving target gene specific delivery.

[0031] Further, the above-mentioned 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, and preferably, the AAV protein capsid of the present application is AAV9.

[0032] Preferably, the above-mentioned ITR sequence comprises one or more of ITR1, ITR2, truncated ITR2, ITR3, ITR5, ITR4, ITR6, and ITR7.

[0033] In a fourth aspect, the present application provides a pharmaceutical composition comprising the above-mentioned combination of regulatory elements, or the above-mentioned recombinant nucleic acid molecule, or the above-mentioned recombinant adeno-associated virus, and a pharmaceutically acceptable excipient. In some preferred embodiments, the pharmaceutical composition can be formulated for intravenous injection, oral administration, inhalation, intranasal administration, intratracheal administration, intraarterial administration, intraocular administration, intramuscular administration, intraabdominal administration, and / or other parenteral routes of administration.

[0034] 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:

[0035] (i) preparing a DNA molecule comprising at least one ITR and an enhancer; and

[0036] (ii) packaging the DNA molecule using an AAV virus.

[0037] Further, the method for constructing an adeno-associated virus vector comprises the following steps:

[0038] Preparation of plasmid DNA containing an AAV genome: the AAV plasmid DNA comprises all the DNA sequences described in the present application, and the enhancer sequence, the exogenous target gene sequence, and the PolyA sequence are located between the ITR sequences.

[0039] Packaging of AAV vectors in HEK293 cells using a triple-plasmid method: the triple-plasmid method refers to the generation of AAV particles after co-transfecting HEK293 cells with plasmid DNA containing an AAV genome and two additional helper plasmids required for packaging AAV (Rep-Cap plasmid, Ad Helper plasmid);

[0040] Purification of AAV vectors: the method for purifying AAV vectors comprises collecting the cell pellet of HEK293 cells transfected with triple plasmids using PEG8000, releasing AAV from the cell pellet by repeated freeze-thawing, preliminarily purifying AAV using iodixanol gradient centrifugation, and further purifying and concentrating AAV using ultrafiltration centrifugation with an ultrafiltration tube.

[0041] 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, which comprises one or more of the following: 1) driving expression of an exogenous target gene; 2) driving expression of an exogenous target gene in a specific tissue; 3) driving expression of an exogenous target gene at a specific time period (e.g., expression at a disease period).

[0042] In some embodiments of the present application, the specific tissue comprises one or more of the following tissues: heart, liver, skeletal muscle, lung, spleen, kidney, and / or brain.

[0043] In some embodiments of the present application, the specific time includes, but is not limited to, a specific time expression of a disease onset period (such as when heart failure occurs).

[0044] In summary, the present application has the following beneficial effects:

[0045] (1) The present application provides an element combination for regulating nucleic acid expression based on ITR-enhancer, constructs a novel gene expression regulation system without exogenous promoter, releases 600-2000 bp vector capacity by deleting exogenous promoter, greatly improves the loading capacity of large therapeutic genes, and is particularly suitable for application scenarios of large gene expression, such as application of ITR-enhancer driven in a single AAV system to express a classic CRISPR / Cas9 system for gene knockout, to realize the application that needs a double AAV system in the traditional way.

[0046] (2) The present application provides an element combination for regulating nucleic acid expression based on ITR-enhancer, utilizes ITR-enhancer to drive exogenous target gene expression, and compared with the long sequence (1-2 kb) and low tissue specificity of the traditional tissue-specific promoter, utilizes the characteristics of high tissue specificity and short sequence of the enhancer to realize specific expression of the exogenous target gene.

[0047] (3) The present application provides an element combination for regulating nucleic acid expression based on ITR-enhancer, utilizes ITR-enhancer to drive exogenous target gene expression, and compared with the safety problems such as cytotoxicity caused by traditional AAV continuous expression, utilizes the pathological responsiveness characteristics of the enhancer to realize dynamic expression of the exogenous target gene and improve the safety of AAV vector for gene therapy. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 The experimental results of Example 1 show that AAV9-Cre-pA-CMVEnh can drive high-efficiency expression of target gene Cre: Figure A shows that AAV9-Cre-pA and AAV9-Cre-pA-CMVEnh were injected into the heart of a mouse, and the expression of TOM (red) reporter gene fluorescence signal in the frozen sections of the heart, liver, lung, spleen, kidney, brain and skeletal muscle of the mouse was detected on the 7th day after injection. Rosa fsCas9-tdTomato Figure B shows the expression results of TOM (red) reporter gene fluorescence signal in the frozen sections of the heart, liver, lung, spleen, kidney, brain and skeletal muscle of the mouse after injection of AAV9-Cre-pA and AAV9-Cre-pA-CMVEnh into the mouse body, and Figure C shows the expression results of TOM (red) reporter gene fluorescence signal in the frozen sections of the heart, liver, lung, spleen, kidney, brain and skeletal muscle of the mouse after injection of AAV9-Cre-pA and AAV9-Cre-pA-CMVEnh into the mouse body. Rosa26 fsCas9-tdTomatoQuantitative analysis results of various different tissues (atrium, ventricle, liver, spleen, lung, kidney, brain, skeletal muscle) of reporter mice (n=3); Fig. D is the results of two cell experiments confirming that the ITR transcriptional activity of AAV9-Cre-pA-CMVEnh depends on RNA polymerase II (Pol II); wherein, the red dots in A, B, C, D represent AAV9-Cre-pA (control group), and the green dots represent AAV9-Cre-pA-CMVEnh (experimental group); the number of points in the scatter column chart represents n value; TOM: tdTomato. Student's t test, *P<0.05, **P<0.01.

[0049] Figure 2 Figures showing the experimental results of Example 2 that AAV9-Cre-pA-miR122TS- Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh can drive the target gene Cre to express specifically in atrium and ventricle, respectively: Fig. A is the expression results of TOM (red) reporter gene fluorescence signal in the atrium of reporter mice injected with AAV9-Cre-pA-miR122TS-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh subcutaneously Rosa fsCas9-tdTomato Figures showing the experimental results of Example 2 that AAV9-Cre-pA-miR122TS- Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh can drive the target gene Cre to express specifically in atrium and ventricle, respectively: Fig. A is the expression results of TOM (red) reporter gene fluorescence signal in the atrium of reporter mice injected with AAV9-Cre-pA-miR122TS-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh subcutaneously Rosa fsCas9-tdTomato Figures showing the experimental results of Example 2 that AAV9-Cre-pA-miR122TS- Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh can drive the target gene Cre to express specifically in atrium and ventricle, respectively: Fig. A is the expression results of TOM (red) reporter gene fluorescence signal in the atrium of reporter mice injected with AAV9-Cre-pA-miR122TS-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh subcutaneously Rosa fsCas9-tdTomato Figures showing the experimental results of Example 2 that AAV9-Cre-pA-miR122TS- Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh can drive the target gene Cre to express specifically in atrium and ventricle, respectively: Fig. A is the expression results of TOM (red) reporter gene fluorescence signal in the atrium of reporter mice injected with AAV9-Cre-pA-miR122TS-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh subcutaneously Rosa fsCas9 -tdTomato Figures showing the experimental results of Example 2 that AAV9-Cre-pA-miR122TS- Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh can drive the target gene Cre to express specifically in atrium and ventricle, respectively: Fig. A is the expression results of TOM (red) reporter gene fluorescence signal in the atrium of reporter mice injected with AAV9-Cre-pA-miR122TS-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh subcutaneously Rosa fsCas9-tdTomato Figures showing the experimental results of Example 2 that AAV9-Cre-pA-miR122TS- Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh can drive the target gene Cre to express specifically in atrium and ventricle, respectively: Fig. A is the expression results of TOM (red) reporter gene fluorescence signal in the atrium of reporter mice injected with AAV9-Cre-pA-miR122TS-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh subcutaneously Rosa26 fsCas9-tdTomato(n=3) Quantitative analysis results of frozen sections of various tissues (atrium, ventricle, liver, spleen, lung, kidney, brain, and skeletal muscle) from newborn mice with reporter genes; Figure E shows the quantitative analysis results of two AAV9 variants, AAV9-Cre-pA-miR122TS-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh, in... Rosa26 fsCas9-tdTomato (n=3) Quantitative analysis results of frozen sections of various tissues (atrium, ventricle, liver, spleen, lung, kidney, brain, and skeletal muscle) from adult mice with reporter genes; where 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 plot represents the n value; TOM: tdTomato.

[0050] Figure 3 The following is a flowchart of the experimental design for the AAV9-Cas9-miR122TS-pA-Myl7Enh-U6-Scn5a.gRNA injection into C57 wild-type mice, as described in Example 3: Figure A shows the electrocardiogram results of mice injected with AAV9-Cas9-miR122TS-pA-Myl7Enh-U6-Scn5a.gRNA; Figure C shows the gene knockout results of mice injected with AAV9-Cas9-miR122TS-pA-Myl7Enh-U6-Scn5a.gRNA in the atrium, ventricle, liver, lung, spleen, kidney, brain, and skeletal muscle; the number of points in the scatter bar chart represents the n value; Figure D shows the immunofluorescence image of Scn5a atrial-specific knockout; Figure E shows the WB image of Scn5a atrial-specific knockout. Detailed Implementation

[0051] 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.

[0052] 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.

[0053] The use of all technical solutions of the present application can be for the purpose of prevention or treatment or for the purpose of non-prevention or non-treatment.

[0054] The term "ITR" used in the present application refers to an inverted terminal repeat sequence, which is a basic sequence of the AAV genome, can form a highly stable secondary structure, and plays an essential role in the replication and packaging process of the virus. The overall structure of ITR is highly conserved in different serotypes, but there are still some subtypes or variants, mainly divided into two categories of natural serotype related ITR and artificially modified ITR. There are slight differences in the ITR sequences of different AAV serotypes, but the core structure (such as palindromic sequence, Rep binding site, etc.) remains conservative.

[0055] Among them, ITR1, ITR2, ITR3, ITR4, ITR5 and ITR6 are natural serotype related ITRs, and truncated ITR2 and ITR7 are artificially modified ITRs, specifically:

[0056] ITR1 and ITR2 refer to ITRs derived from AAV serotype 2 (AAV2), and a typical AAV2 genome has one ITR at each end. The sequences of the two ITRs are not completely identical, so they are named ITR1 and ITR2 respectively.

[0057] ITR3 refers to ITR derived from AAV serotype 3 (AAV3);

[0058] ITR4 refers to ITR derived from AAV serotype 4 (AAV4);

[0059] ITR5 refers to ITR derived from AAV serotype 5 (AAV5);

[0060] ITR6 refers to ITR derived from AAV serotype 6 (AAV6).

[0061] Truncated ITR2 refers to an artificial variant obtained by deleting part of the sequence based on natural AAV2 ITR2;

[0062] ITR7 refers to a synthetic or significantly modified ITR.

[0063] The term "Enh" used in the present application refers to an enhancer, which is a non-coding DNA sequence that can enhance the transcription efficiency of a target gene. The term "CMV Enh" refers to the enhancer sequence of the universal promoter CMV, which is a broad-spectrum enhancer without tissue and time-space specificity. The term "Myl7Enh" refers to an atrial-specific enhancer, which is a non-coding sequence that enhances the atrial-specific expression of an exogenous target gene. The term "Myl3Enh" refers to a ventricular-specific enhancer, which is a non-coding sequence that enhances the ventricular-specific expression of an exogenous target gene.

[0064] The term "exogenous target gene" used in the present application refers to a gene of interest expressed driven by ITR-enhancer, which 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 for biological or medical research delivered by AAV.

[0065] The term "pA" used in the present application refers to a polyadenylation sequence, which is a termination signal sequence for mRNA transcription.

[0066] The term "AAV9" used in the present application refers to a recombinant AAV9 (rAAV9) based on wild-type AAV9 (human 9 type adeno-associated virus) and capable of delivering a gene of interest, and any complete or partial variant thereof obtained by various engineering, recombination, modification, and still retaining the affinity for the heart. In the AAV vector including AAV9 in the present application, the DNA sequence of the AAV vector includes the following parts in order from the 5' end to the 3' end: inverted terminal repeat (ITR)-5' untranslated region (5'UTR)-gene of interest-3' untranslated region (3'UTR)-3' terminal poly A tail (PolyA)-enhancer-inverted terminal repeat (ITR).

[0067] The following table lists the nucleic acid sequence numbers used in the description in the present application and the corresponding sequences (where the index marked by underlining and italic indicates a "tag" used to distinguish samples in the primer used for high-throughput sequencing, also known as a barcode or barcode, which can be freely selected by a technician skilled in high-throughput sequencing as needed).

[0068] Table 1 Nucleic acid sequence numbers used in the present application and their corresponding sequences

[0069]

[0070]

[0071] The specific embodiments of the present application are described in detail below. It should be understood that the specific embodiments described herein are only for illustration and explanation of the present application, and are not intended to limit the present application.

[0072] Example 1

[0073] This embodiment provides a nucleic acid regulatory element combination of ITR-enhancer, which can achieve efficient expression of AAV9 in vivo gene delivery in a Cre-Loxp system.

[0074] In this embodiment, for the Cre-Loxp system, the inventors first constructed an AAV9-Cre-pA adeno-associated virus for evaluating the expression of AAV9-Cre-pA driven genes in vivo in mice; and further constructed an AAV9-Cre-pA-CMVEnh for evaluating whether the combination of ITR-enhancer nucleic acid regulatory elements enhances the efficient expression of AAV9 in vivo gene delivery.

[0075] The specific process is described as follows:

[0076] 1. Construction of AAV9-Cre-pA and AAV9-Cre-pA-CMVEnh plasmids

[0077] Based on the AAV-U6sgRNA-U6sgRNA-Tnnt2-Cre plasmid vector, the plasmid vector is disclosed in the literature (Guo Y et al., Analysis of Cardiac Myocyte Maturation Using CASAAV, a Platform for Rapid Dissection of Cardiac Myocyte Gene Function In Vivo. Circ Res., 120(12): 1874-1888, 2017).

[0078] Preparation of AAV9-Cre-pA plasmid:

[0079] In the AAV-U6sgRNA-U6sgRNA-Tnnt2-Cre plasmid vector, restriction enzymes Kpnl, Nhel and Rsrl were selected as the enzyme digestion sites, and the following components were added and mixed: AAV-U6sgRNA-U6sgRNA-Tnnt2-Cre plasmid 1 μg; Kpnl 1 μL; Nhel 1 μL; Rsrl 1 μL; 10×CutSmart buffer 3 μL; nuclease-free water, added to 30 μL. The resulting mixture was placed in a 37°C water bath for 60 min. The enzyme digestion product was subjected to agarose gel electrophoresis, and the enzyme digestion product was recovered.

[0080] The Cre sequence was amplified by PCR, and the sequence of the upstream primer F of the primer pair used is shown in Table 1 as SEQ ID NO: 1 and the sequence of the downstream primer R is shown in Table 1 as SEQ ID NO: 2; the pA sequence was amplified by PCR, and the sequence of the primer pair used is shown in Table 1 as SEQ ID NO: 3 (F) and SEQ ID NO: 4 (R).

[0081] The PCR product was recovered by gel recovery, and the PCR product was cloned into the enzyme-digested plasmid vector using seamless cloning technology. The enzymes used in the preparation process and the seamless cloning kit can be purchased (NEB, R3142S, R3131S, R0501S; Nanjing Novozyme Biotech Co., Ltd., C117-01). The ligation product was transformed into Stable3 competent cells, plated on LB medium plates containing ampicillin, and single colonies were picked for Sanger sequencing to obtain the successfully cloned AAV9-Cre-pA plasmid for standby.

[0082] (2) Preparation of AAV9-Cre-pA-CMVEnh plasmid

[0083] The AAV-U6sgRNA-U6sgRNA-Tnnt2-Cre plasmid was digested and gel recovered as above.

[0084] The Cre sequence was amplified by PCR, and the primer pair sequence is shown in Table 1 as SEQ ID NO: 1 (F) and SEQ ID NO: 2 (R);

[0085] The pA sequence was amplified by PCR, and the primer pair sequence is shown in Table 1 as SEQ ID NO: 3 (F) and SEQ ID NO: 5 (R);

[0086] The CMVEnh sequence was amplified by PCR, and the primer pair sequence is shown in Table 1 as SEQ ID NO: 6 (F) and SEQ ID NO: 7 (R).

[0087] The PCR product was recovered by gel recovery, and the PCR product was cloned into the enzyme-digested plasmid vector using seamless cloning technology. The ligation product was transformed into Stable3 competent cells, plated on LB medium plates containing ampicillin, and single colonies were picked for Sanger sequencing to obtain the successfully cloned AAV9-Cre-pA-CMVEnh plasmid for standby.

[0088] 2. Packaging of AAV9 virus AAV9-Cre-pA and AAV9-Cre-pA-CMVEnh

[0089] In this step, the constructed AAV9-Cre-pA plasmid and AAV9-Cre-pA-CMVEnh plasmid were packaged to prepare AAV9 virus, respectively. HEK293T cells were used as host cells to prepare AAV9. For the plasmid required for packaging AAV9, a non-endotoxin plasmid large extraction kit (Tiangen Biotech (Beijing) Co., Ltd., DP117) was used for high-quality extraction of the plasmid.

[0090] The specific steps of AAV packaging are as follows:

[0091] ①Take the cryopreserved HEK293T cells out of liquid nitrogen for recovery, and place them in a 15 cm cell culture dish for culture. When the cells grow to ~90% confluence, they are subcultured. 10 dishes of 15 cm cell culture dish cultured HEK293T cells are needed for each AAV packaging. When the cell density reaches ~90% confluence, add 1.8 mL of transfection reagent PEI (1 mg / mL), 7 μg of AAV plasmid, 7 μg of Rep2 / Cap9 plasmid and 20 μg of pHelper plasmid to each dish of cells, and incubate at 37°C for 8-12 hours. Then replace the medium with serum-free, 1% double-antibody-containing high-glucose DMEM medium, and continue to culture at 37°C for 66-72 hours. Then collect the cells;

[0092] ②Centrifuge the collected cell pellets at 3000 rpm for 5 min, discard the supernatant, and resuspend the cell pellets in AAV lysis buffer (20 mM Tris pH 8.0, 1 mM MgCl2, 150 mM NaCl) at -80°C;

[0093] ③Add 1 / 4 volume of 2.5 M NaCl-containing 40% PEG8000 solution to the cell culture medium collected in step ②, and let it stand at 4°C for 2 h. Centrifuge at 4000 rpm for 30 min to precipitate the pellet, discard the supernatant, and resuspend the obtained AAV lysis buffer with the cell suspension. Add 50 μg / ml of omnipolynuclease to the cell suspension, and then place it at 37°C and -80°C for repeated freezing and thawing for 3 times to fully lyse the cells.

[0094] ④Centrifuge at 4000 rpm for 30 min at 4°C, collect the supernatant, and then apply the supernatant to a gradient of iodixanol (OptiPrep) for AAV purification by density gradient ultracentrifugation;

[0095] ⑤Wash and concentrate the AAV in a centrifugal filter tube with a molecular cut-off of 100 kDa using PBS solution containing 0.001% Pluronic F-68. Determine the AAV titer by real-time fluorescent quantitative PCR (RT-qPCR), and the obtained AAV titer is above 1E+13 vg / mL.

[0096] 3. Packaging AAV-DJ virus AAV-DJ-Cre-pA-CMVEnh

[0097] According to the same method for preparing AAV9 virus as described in step (2), only replace the Rep2 / Cap9 plasmid therein with Rep2 / Cap-DJ plasmid, thereby completing the plasmid AAV-DJ virus packaging (AAV-DJ is an artificially modified chimeric serotype with broad tropism and high transduction efficiency), and obtaining AAV-DJ-Cre-pA-CMVEnh.

[0098] ITR drives low expression of exogenous target genes.

[0099] Fluorescent reporter mice (Strain No. T002249) with the CAG promoter-Loxp-stop-Loxp-Cas9-2A-tdTomato safely knocked into the Rosa26 site were selected. Rosa26 fsCas9-tsTomato (This indicates that) the mouse contains a fluorescent reporter gene that can be activated by Cre.

[0100] AAV9-Cre-pA was processed according to 5×10 10 vg / g (vg / g is an abbreviation for vector genome per gram body weight, referring to the number of viral vector genomes per gram of body weight, the same below) was subcutaneously injected into Rosa26fsCas9-tdTomato (n=3) newborn mice. Seven days later, the mice were sacrificed, and the heart, liver, spleen, lungs, kidneys, brain, and skeletal muscle were harvested for fixation, dehydration, embedding, and frozen sectioning (section thickness 8 μm). The tissue sections were stained with 4',6-diamidinyl-2-phenylindole (DAPI) and imaged using confocal microscopy. Cells positive for the tdTomato (TOM) reporter gene fluorescent protein were counted and statistically analyzed using ImageJ and Graphpad Prism software.

[0101] The results are as follows Figure 1 As shown in Figure C, the positive cell rate of TOM in the heart is over 5%, while the positive cell rate in liver tissue sections reaches about 40%. In other organs such as the spleen, lungs, kidneys, skeletal muscle, and brain, the positive cell rate is almost 0%. This indicates that ITR can drive the expression of the target gene Cre, suggesting that ITR has weak promoter transcriptional activity.

[0102] 4. ITR-enhancers significantly increase the expression of exogenous target genes.

[0103] Prepare using the same method described above. Rosa26 fsCas9-tdTomato Fluorescent reporter gene mice.

[0104] AAV9-Cre-pA and AAV9-Cre-pA-CMVEnh were subcutaneously injected into two groups of Rosa26fsCas9-tdTomato neonatal mice (n=3) at a dose of 5×10¹⁰ vg / g. Figure 1A), and the mice were sacrificed 7 days later. The heart, liver, spleen, lung, kidney, brain, and skeletal muscle of the mice in the two groups were taken out, fixed, dehydrated, embedded, and frozen sectioned. The tissue sections were stained with 4', 6-diamidino-2-phenylindole (DAPI), and confocal microscopy imaging was performed. The fluorescent protein-positive cells of the tdTomato (TOM) reporter gene were counted and statistically analyzed using Image J and Graphpad Prism software.

[0105] As shown in Figure 1 B and Figure 1 C, compared with the mice injected with AAV9-Cre-pA, the mice injected with AAV9-Cre-pA-CMVEnh had almost 100% TOM-positive cells in the heart and liver tissue sections, ~25% TOM-positive cells in the lung, and ~10% TOM-positive cells in the spleen.

[0106] As shown in Figure 1 D, AAV-DJ-Cre-pA-CMVEnh infected Neuro2a and 393T cells, respectively, and RNA polymerase I (Pol I), RNA polymerase II (Pol II), and RNA polymerase III (Pol III) small molecule inhibitors were added, respectively. The results showed that only the Pol II inhibitor could significantly inhibit the transcriptional activity of the ITR-enhancer.

[0107] Conclusion: The ITR-enhancer can significantly enhance the expression of the target gene Cre, and the ITR transcriptional activity depends on Pol II.

[0108] Example 2

[0109] This example provides high-efficiency expression of an exogenous target gene in the heart driven by the nucleic acid regulatory element of the ITR-enhancer

[0110] The inventors selected atrial and ventricular specific enhancers, and used the Cre-Loxp system to select the atrial specific enhancer Myl7 enhancer (Myl7 Enh for short) and the ventricular specific enhancer Myl3 enhancer (Myl3 Enh for short). The miR122TS was also selected to inhibit the weak transcriptional activity of the ITR in the liver, thereby constructing AAV9-Cre-miR122TS-pA-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh adeno-associated viruses for evaluating whether the combination of the nucleic acid regulatory elements of the ITR-enhancer drives the tissue-specific high expression of AAV9 in vivo gene delivery.

[0111] The specific process is as follows:

[0112] AAV9-Cre-miR122TS-pA-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh plasmids were constructed.

[0113] pA-Myl3Enh plasmid

[0114] The AAV9-Cre-pA-CMVEnh plasmid constructed in Example 1 was selected, and restriction enzyme BamHI (NEB, R0136S) was selected as the enzyme digestion site. The following components were added and mixed: AAV9-Cre-pA-CMVEnh plasmid 1 μg; BamHI 1 μL; 10×CutSmart buffer 3 μL; nuclease-free water, added to 30 μL. The resulting mixture was placed in a 37°C water bath for 60 min. The enzyme digestion product was subjected to agarose gel electrophoresis, and the enzyme digestion product was gel recovered.

[0115] The chemically synthesized DNA fragment of the 3×miR122TS gene sequence (the sequence is shown as SEQ ID NO: 8 in Table 1, and was synthesized by Hangzhou Yikang Biological Technology 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 selected as the enzyme digestion site with restriction enzymes AscI (NEB, R0558S) and RsrII. The following components were added and mixed: AAV9-Cre-miR122TS-pA-CMVEnh plasmid 1 μg; AscI 1 μL; RsrII 1 μL; 10×CutSmart buffer 3 μL; nuclease-free water, added to 30 μL. The resulting mixture was placed in a 37°C water bath for 60 min. The enzyme digestion product was subjected to agarose gel electrophoresis, and the enzyme digestion product was gel recovered.

[0116] The chemically synthesized DNA fragment of the Myl7Enh (as shown in SEQ ID NO: 9 in Table 1) and the DNA fragment of the Myl3Enh (as shown in SEQ ID NO: 10 in Table 1) were respectively cloned into the AAV9-Cre-pA-CMVEnh vector, i.e., the AV9-Cre-miR122TS-pA-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh plasmids were successfully constructed.

[0117] (2) AAV9 virus packaging

[0118] Following the same method used to prepare AAV9 virus in Example 1, only the AAV plasmid was replaced with the plasmid in step (1) of this example, thereby completing the AAV9 virus packaging of two plasmids and obtaining AAV9-Cre-miR122TS-pA-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh.

[0119] (3) Assess the expression of Cre, the target gene driven by ITR-Myl7Enh and ITR-Myl3Enh.

[0120] AAV9-Cre-miR122TS-pA-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh are processed according to 5×10 10 vg / g were subcutaneously injected into Rosa26fsCas9-tdTomato (n=3) neonatal mice (e.g., vg / g). Figure 2 (As shown in A), mice were sacrificed 7 days later, and their hearts, livers, spleens, lungs, kidneys, brains, and skeletal muscles were collected for fixation, dehydration, embedding, and frozen sectioning (section thickness 8 μm). The tissue sections were stained with 4',6-diamidinyl-2-phenylindole (DAPI) and imaged using confocal microscopy. Cells positive for the tdTomato (TOM) reporter gene fluorescent protein were counted and statistically analyzed using ImageJ and Graphpad Prism software.

[0121] The results are as follows Figure 2 As shown in B, 2C, and 2D, mice injected with AAV9-Cre-miR122TS-pA-Myl7Enh exhibited a TOM-positive cell rate of almost 95% in the atria, while the positive cell rate in ventricular tissue sections was only about 5%. In other organs such as the spleen, lungs, kidneys, skeletal muscle, and brain, the positive cell rate was almost 0%. Mice injected with AAV9-Cre-miR122TS-pA-Myl3Enh showed a TOM-positive cell rate of only 5% in the atria, while the positive cell rate in ventricular tissue sections reached approximately 90%. In other organs such as the spleen, lungs, kidneys, skeletal muscle, and brain, the positive cell rate was almost 0%.

[0122] In addition, to evaluate whether the nucleic acid regulatory element of the ITR-enhancer driving the expression of the exogenous target gene is affected by the age of the subject and the administration method, the inventors performed tail vein injection of the viruses AAV9-Cre-miR122TS-pA-Myl3Enh and AAV9-Cre-miR122TS-pA-Myl7Enh on adult Rosa26fsCas9-tdTomato mice (4 weeks old, adult group, n = 3), and sacrificed the mice after 1 week, and took the heart, liver, spleen, lung, kidney, brain, and skeletal muscle tissue for frozen section and DAPI staining.

[0123] The results are shown in Figure 2 As shown in B, 2C, and 2E, the adult mice injected with AAV9-Cre-miR122TS-pA-Myl7Enh had a TOM-positive cell rate of almost 90% in the atrium, and a positive cell rate of only about 5% in the ventricular tissue section, and the positive cell rate in the remaining organs such as the spleen, lung, kidney, skeletal muscle, and brain was almost 0%. The adult mice injected with AAV9-Cre-miR122TS-pA-Myl3Enh had a TOM-positive cell rate of only about 5% in the atrium, and a positive cell rate of about 80% in the ventricular tissue section, and the positive cell rate in the remaining organs such as the spleen, lung, kidney, skeletal muscle, and brain was almost 0%.

[0124] Conclusion: The nucleic acid regulatory element of the ITR-enhancer can drive the expression of an exogenous target gene in a specific tissue by selecting different tissue-specific enhancers, and this regulation of gene expression is not affected by the administration route and the age of the animal.

[0125] Example 3

[0126] This example provides a nucleic acid regulatory element of an ITR-enhancer for realizing single- AAV delivery of a CRISPR / spCas9 system

[0127] In a conventional AAV delivery system, the nucleic acid regulatory element is an exogenous promoter. Since the sequence of the promoter is generally more than 500 bp, it occupies more than 10% of the AAV genome (~4.7 kb), resulting in a limitation on the gene load of AAV, which cannot deliver large exogenous target genes. The conventional AAV delivery CRISPR / spCas9 system requires two AAVs to achieve this, one AAV expresses spCas9 alone, and the other AAV delivers gRNA, and the use of double AAVs can cause safety problems.

[0128] In this application, the nucleic acid regulatory element of ITR-enhancer can maximize the DNA load 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 (referred to as Myl7ENh mini) combined with ITR to form ITR-Myl7Enh mini to achieve gene editing by single AAV delivery of CRISPR / spCas9 system.

[0129] To this end, the inventors designed a single-guide RNA (sgRNA) targeting the first exon of Scn5a driven by a U6 promoter. Scn5a encodes the alpha subunit of the cardiac sodium channel Nav1.5, which mediates rapid sodium ion influx (INa) and triggers myocardial cell depolarization. Mutations in this gene can cause a series of genetic arrhythmias, such as long QT syndrome type 3 (LQT3), Brugada syndrome, progressive cardiac conduction block (PCCD), etc.

[0130] The specific implementation steps are as follows:

[0131] Construct AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-Scn5a.sgRNA plasmid.

[0132] Select the AAV9-Cre-miR122TS-pA-Myl7Enh plasmid constructed in the embodiment, use restriction enzymes KpnI and HindIII (NEB, R0104S) as the enzyme digestion site, and add and mix the following components: AAV9-Cre-miR122TS-pA-Myl7Enh plasmid 1 μg; KpnI 1 μL; HindIII 1 μL; 10×CutSmart buffer 3 μL; nuclease-free water, add to 30 μL. Place the resulting mixture in a 37°C water bath for 60 min. Perform agarose gel electrophoresis on the enzyme digestion product, and gel recover the enzyme digestion product.

[0133] PCR amplify the spCas9 sequence, the primer pair sequence is shown in Table 1 SEQ ID NO: 11 (F) and SEQ ID NO: 12 (R), gel recover the PCR product, and use the Seamless Cloning Kit (Nanjing Novozyme Biotech Co., Ltd., C117-01) to clone the PCR product into the enzyme-digested plasmid vector. Transform the ligation product into Stable3 competent cells, and plate on LB medium plates containing ampicillin. Pick single colonies for Sanger sequencing to obtain the successfully cloned AAV9-spCas9-miR122TS-pA-Myl7Enh plasmid for standby use.

[0134] Using the AAV9-spCas9-miR122TS-pA-Myl7Enh above, select NheI and RsrII as enzyme cutting sites, add and mix the following components: AAV9-spCas9-miR122TS-pA-Myl7Enh plasmid 1 μg; NheI 1 μL; RsrII 1 μL; 10×CutSmart buffer 3 μL; nuclease-free water, add to 30 μL. The resulting mixed components are placed in a 37°C water bath for 60 min. The enzyme cutting product is subjected to agarose gel electrophoresis, and the enzyme cutting product is recovered by gel. The DNA fragment of the chemically synthesized pA-Myl7Enh mini-U6-sgRNA (as shown in SEQ ID NO: 13 in Table 1, entrusted to Hangzhou Yikang Biological Technology Co., Ltd.) is cloned into the AAV9-spCas9-miR122TS-pA-Myl7Enh vector, i.e. AAV9-spCas9-miR122TS-pA-Myl7Enh mini-U6-sgRNA plasmid is successfully constructed.

[0135] Using the AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-sgRNA plasmid above, select PaqCI as the enzyme cutting site, add and mix the following components: AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-sgRNA plasmid 1 μg; PaqCI 1 μL; 10×CutSmart buffer 3 μL; nuclease-free water, add to 30 μL. The resulting mixed components are placed in a 37°C water bath for 60 min. The enzyme cutting product is subjected to agarose gel electrophoresis, and the enzyme cutting product is recovered by gel.

[0136] Using the software CRISPick to design sgRNA for the mouse Scn5a gene, the optimal sgRNA located on the first exon is selected, as shown in SEQ ID NO: 14 in Table 1. From the above successfully constructed AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-sgRNA enzyme cutting site preferred PaqCI, the primer for synthesizing Scn5a.sgRNA is synthesized, and the primer sequences are shown in SEQ ID NO: 15 (F) and SEQ ID NO: 16 (R) in Table 1, respectively. T4 ligation can successfully clone the AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-Scn5a.sgRNA plasmid.

[0137] (2) AAV9 virus packaging

[0138] According to the same method for preparing AAV9 virus in Example 1, only the AAV plasmid therein is replaced by the plasmid in Step (1) of the present example, thereby packaging the AAV9 virus of the complete plasmid, to obtain AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-Scn5a.sgRNA.

[0139] (3) ITR-Myl7Enhmini drives spCas9 atrial-specific expression, and successful construction of a mouse atrial fibrillation model by knocking out Scn5a

[0140] The AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-Scn5a.sgRNA was subcutaneously injected into C57 newborn mice (n = 3) at a dose of 1 x 1011 11 vg / g (as shown in FIG. A), and after 30 days, the mice were anesthetized with tri- bromoethanol, and the mouse body surface ECG was detected using a Power Lab electrocardiograph. The results are shown in FIG. B. Figure 3 A, and FIG. B shows the ECG results, which show that the mice injected with AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-Scn5a.sgRNA exhibited obvious atrial fibrillation. Figure 3 A, and FIG. B shows the ECG results, which show that the mice injected with AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-Scn5a.sgRNA exhibited obvious atrial fibrillation.

[0141] After ECG detection, the mice were sacrificed, and the atrium, ventricle, liver, spleen, lung, kidney, brain, and skeletal muscle tissues were taken for Amplicon-Seq. The specific operation process is as follows. After the atrium, ventricle, liver, spleen, lung, kidney, brain, and skeletal muscle tissues of the mice (n = 3) were taken, the gDNA extraction kit (Shanghai Genechem Co., Ltd.) was used to extract the gDNA of the heart and liver tissues.

[0142] The specific operation process is: the primers for high-throughput sequencing library construction of target gene site are designed, and the primer sequences are shown in Table 1 as SEQ ID NO: 17 (F) and SEQ ID NO: 18 (R). The gDNA is used as a template for PCR amplification of the target fragment, and the following components are added: gDNA, 2 μg; upstream primer (F), 1 μL; downstream primer (R), 1 μL; 2x KeyPo Master Mix, 10 μL; nuclease-free water, added to 20 μL. The prepared sample is placed in a PCR instrument, and the PCR reaction program is as follows: 98℃ for 30s; 98℃ for 10s, 70℃ for 30s, 72℃ for 60s, Touchdown PCR cycle for 15 times; 98℃ for 10s, 70℃ for 30s, 72℃ for 60s, cycle for 15 times, 72℃ for 5min; 12℃ holding, and the PCR product is purified. The P5 primer and P7 primer provided by Mingma (Shanghai) Biotechnology Co., Ltd. are selected for the second round of PCR amplification, and the primer sequences are shown in Table 1 as SEQ ID NO: 19 (F) and SEQ ID NO: 20 (R). The first round of PCR product is used as a template for PCR amplification of the target fragment, and the following components are added: PCR product, 2 μg; upstream primer (F), 1 μL; downstream primer (R), 1 μL; 2x KeyPo Master Mix, 10 μL; nuclease-free water, added to 20 μL. The prepared sample is placed in a PCR instrument, and the PCR reaction program is as follows: 98℃ for 30s; 98℃ for 10s, 55℃ for 30s, 72℃ for 60s, cycle for 20 times; 72℃ for 5min; 12℃ holding. The PCR product is subjected to agarose gel electrophoresis, and the target fragment is cut and subjected to gel recovery and purification. The purified fragment is subjected to second-generation sequencing (Beijing Novogene Bioinformatics Technology Co., Ltd.), and the sequencing results are analyzed using CRISPResso2, and the statistical analysis and drawing are performed using Graphpad Prism software.

[0143] As Figure 3 C shows that the results show that the mice mainly produce gene editing (insertion / deletion mutation, represented by indels) in the atrium, and the indel production rate in the atrial tissue is ~23%, and the indel production rate in the remaining tissues is less than ~5%.

[0144] As Figure 3 D and 3E show that immunofluorescence and WB results suggest that Scn5a is only knocked out in the atrium, and the expression amount of ventricular Scn5a does not change.

[0145] The above merely describes the preferred embodiments of the present application and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A recombinant nucleic acid molecule for improved target gene specific delivery, characterized in that, The recombinant nucleic acid molecule comprises an inverted terminal repeat sequence ITR, an enhancer, an exogenous target gene, and a poly A, is delivered by an AAV9 vector, and has no exogenous promoter; The recombinant nucleic acid molecule comprises, from 5' end to 3' end, an inverted terminal repeat sequence ITR-5' UTR-exogenous target gene-3' UTR-3' terminal poly A tail-enhancer-inverted terminal repeat sequence ITR. The enhancer is an atrium-specific enhancer Myl7 enhancer or a ventricle-specific enhancer Myl3 enhancer; the ITR sequence is ITR2 or a truncated ITR2.

2. The recombinant nucleic acid molecule for improving target gene-specific delivery according to claim 1, wherein, The exogenous target gene is a Cre gene of a Cre / Loxp system.

3. The recombinant nucleic acid molecule for improving target gene-specific delivery according to claim 2, wherein, Also included is a target sequence of miR122.

4. The recombinant nucleic acid molecule for improving target gene-specific delivery according to claim 3, wherein, The recombinant nucleic acid molecule is any of the following: (i) Cre-miR122TS-pA-Myl7Enh; (ii) Cre-miR122TS-pA-Myl3Enh; (iii) Cre-miR122TS-pA-Myl7Enhmini; (iv) Cre-miR122TS-pA-Myl3Enhmini.

5. A recombinant adeno-associated virus with enhanced target gene specificity delivery, characterized in that, It comprises: (i) an AAV9 protein coat; (ii) a recombinant nucleic acid molecule, which is the recombinant nucleic acid molecule for improving target gene-specific delivery according to any one of claims 1-4.

6. The recombinant adeno-associated virus for improving target gene-specific delivery according to claim 5, wherein, The exogenous target gene comprises at least one of a therapeutic gene for gene therapy, a Cas gene in a gene editing system, and a Cre gene of a Cre / Loxp system.

7. The recombinant adeno-associated virus for improving target gene-specific delivery according to claim 6, wherein, The recombinant adeno-associated virus is any of the following: (i) AAV9-Cre-miR122TS-pA-Myl7Enh; (ii) AAV9-Cre-miR122TS-pA-Myl3Enh.

8. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the recombinant nucleic acid molecule according to any one of claims 1-4, or the recombinant adeno-associated virus according to any one of claims 5-7, and a pharmaceutically acceptable excipient.

Images