CN120818522A - A regulatory element combination based on ITR-enhancer driven nucleic acid expression and its application - Google Patents
A regulatory element combination based on ITR-enhancer driven nucleic acid expression and its applicationInfo
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Abstract
本申请涉及生物医药领域，具体公开了一种基于ITR‑增强子驱动核酸表达的调控元件组合及其应用。该基于ITR‑增强子驱动核酸表达的调控元件组合包含：至少一种反向末端重复序列ITR，和至少一种增强子；该调控元件组合通过AAV载体递送。本申请还提供一种提高靶基因特异性递送的重组核酸分子，其包括上述调控元件组合、外源靶基因以及多聚腺苷酸。利用本发明提供的AAV载体可在没有传统启动子的作用下驱动目标靶基因的表达，用增强子组织特异性、短序列（50‑100 bp）及病理微环境响应等优势，突破传统AAV载体容量、靶向性与动态性限制，提高AAV载体的DNA载量的同时，实现靶基因的组织和时空表达特异性。
The present application relates to the field of biomedicine, and specifically discloses a regulatory element combination based on ITR-enhancer driven nucleic acid expression and its application. The regulatory element combination based on ITR-enhancer driven nucleic acid expression comprises: at least one inverted terminal repeat sequence ITR, and at least one enhancer; the regulatory element combination is delivered via an AAV vector. The present application also provides a recombinant nucleic acid molecule for improving target gene specific delivery, which comprises the above-mentioned regulatory element combination, an exogenous target gene, and polyadenylic acid. The AAV vector provided by the present invention can drive the expression of the target gene without the action of a traditional promoter, and uses the advantages of enhancer tissue specificity, short sequence (50-100 bp) and pathological microenvironment response to break through the limitations of traditional AAV vector capacity, targeting and dynamics, while increasing the DNA loading capacity of the AAV vector, achieving tissue and spatiotemporal expression specificity of the target gene.
Description
Technical Field
The application relates to the field of biological medicine, in particular to a regulatory element combination based on ITR-enhancer-driven nucleic acid expression and application thereof.
Background
The gene therapy provides a potential solution for genetic diseases (such as diseases caused by single gene mutation), and the core concept mainly realizes the treatment of the genetic diseases by three modes, namely 1) gene supplementation, delivery of normal genes to cells of patients to make up for the defects of the original genes, 2) gene silencing, inhibition of abnormal gene expression by RNA interference and other technologies to block the harmful effects, 3) gene editing, accurate correction of pathogenic mutation (such as base substitution or fragment repair), and radical recovery of gene sequences. For example, 2018, us FDA approved Zolgensma gene therapy for treating spinal muscular atrophy (spinal muscular atrophy, SMA), delivered SMN1 genes to spinal neurons using AAV vectors, supplemented with in vivo gene therapy in which SMN is not normally expressed due to gene mutation, thereby increasing SMN protein expression with complete function, and thus curing the disease. The application of gene therapy is expected to realize the radical treatment of hereditary diseases and even achieve the effect of 'one-time cure'.
Gene therapy is achieved by delivering biological macromolecules such as DNA, RNA or protein to intervene in diseases, wherein DNA delivery is the most widely used strategy in clinic due to the characteristics of long-acting property, strong stability and capability of encoding complex functional genes. The core mechanism is to deliver functional gene fragments in the form of exogenous DNA into cells of a patient, thereby achieving gene repair or compensation. Among the various gene therapy vectors, adeno-associated virus (AAV) is widely used for its unique advantages.
AAV is a defective single stranded DNA virus, naturally occurring in humans. The AAV has the characteristics of 1) non-pathogenicity, AAV does not cause human diseases, is a naturally occurring virus vector with extremely high biological safety, 2) low immunogenicity, low immunogenicity of AAV, capability of avoiding treatment failure or serious side effects caused by excessive reaction of a host immune system even under the condition of multiple delivery, and 3) diversified tissue targeting, wherein the tissue targeting of the AAV vector can be regulated and controlled by modification of capsid proteins of the AAV vector. A number of AAV serotypes (e.g., AAV1, AAV2, AAV 9) have been identified and developed, each with a highly specific infectious capacity for different tissues (e.g., heart, liver, central nervous system, muscle tissue, etc.), and 4) long-lasting gene expression profiles, genes delivered by AAV can be expressed in target cells for long periods. Although the genome of AAV is typically present in the host cell in the form of a circular epigenetic chromosome (rather than integrated into the host genome), its stability is sufficient to support gene expression for up to months or even years. The unique biological properties of the gene are dominant in the field of DNA delivery, and are called "gold standard vectors" for gene therapy.
The wild type AAV genome packet is about 4.7kb in size and comprises three promoters (P5, P19, P40) consisting of the inverted terminal repeats (INVERTED TERMINAL REPEAT, ITR) at both ends 145 nt and the middle Rep, cap genes. Based on the genomic structure of AAV, engineered recombinant AAV vectors replace endogenous Rep and Cap genes with an expression cassette consisting of a promoter-driven target gene of interest and a polyA tail. However, the design of traditional AAV vectors (promoter+target gene) still faces three major core challenges in clinical applications, 1) the restriction of gene load, AAV packaging capacity is only about 4.7kb, whereas classical strong promoters (e.g., CMV promoters, 600-800 bp in length) occupy more capacity, limiting large therapeutic gene loads, 2) tissue-specific deficiencies, non-targeted expression driven by broad-spectrum promoters may trigger toxic responses, whereas tissue-specific promoters improve targeting, but their weak transcriptional activity and larger size (1-2 kb) further exacerbate the "load-specific" imbalance. 3) Long-term sustained expression of genes in AAV vectors may result in cytotoxicity, affecting therapeutic efficacy and safety.
Therefore, the novel gene delivery system with high-efficiency expression, accurate targeting and dynamic regulation capability is designed to break through the limitation of gene load and insufficient tissue specificity of AAV vectors and solve the toxicity problem caused by continuous expression, and is a key problem to be solved urgently in the current AAV gene therapy field.
Disclosure of Invention
In order to solve the problems, the application provides a regulatory element combination based on ITR-enhancer driving nucleic acid expression and application thereof.
Through experiments and researches, the inventor creatively invents an ITR-enhancer combination-based nucleic acid expression regulation and control adeno-associated virus (AAV) expression vector. Compared with the traditional AAV expression vector, the system can delete the exogenous promoter to release 600-2000 bp vector capacity, obviously improve the loading capacity of large therapeutic genes, and simultaneously realize the tissue specificity and disease responsive gene expression by utilizing the advantages of enhancer tissue specificity, short sequence (50-100 bp), pathological microenvironment response (hypoxia/inflammation) and the like. The example proves that the gene has wide application prospect in driving the universality of the tissue-specific expression of a large-scale gene (Cas 9).
The application adopts the following technical scheme:
In a first aspect, the application provides a combination of regulatory elements for driving expression of a nucleic acid based on an ITR-enhancer, comprising:
at least one inverted terminal repeat ITR;
at least one enhancer;
The regulatory element combination is delivered by an AAV vector.
In some embodiments of the invention, the ITR-enhancer-based expression regulatory element of the nucleic acid is driven by an inverted symmetry 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 invention, ITR is also a core element driving expression of exogenous target genes.
Further, the ITR comprises one or more of ITR1, ITR2, ITR3, ITR5, ITR4, ITR6, ITR 7. Preferably, the ITR sequence of the present invention is an ITR2 sequence. The ITR2 sequence comprises a normal wild-type ITR2 sequence (145 bp) and a truncated ITR2 sequence (deleted part of the palindromic sequence, the sequence length is reduced from 145bp to 130 bp). More preferably, the ITR2 sequences used in the present invention are truncated ITR2 sequences.
In some embodiments of the invention, the ITR-enhancer-based driver nucleic acid expression regulatory element, the enhancer sequence is a DNA sequence that is non-coding and capable of enhancing the transcriptional efficiency of the target gene, including one or more of a broad-spectrum enhancer, a specific enhancer, and a pathologically responsive enhancer. Wherein the specific enhancer comprises an enhancer having a specific enhancing effect on the heart, liver, skeletal muscle, lung, spleen, kidney and/or brain. In the invention, the combination of the enhancer and the ITR drives the target gene to be expressed efficiently and specifically.
Preferably, the enhancer comprises at least one of a broad-spectrum enhancer, a tissue-specific enhancer, and an inducible enhancer.
Preferably, the broad-spectrum enhancers comprise CMV enhancers, the tissue-specific enhancers comprise at least one of Myl7 atrial-specific enhancers, myl3 ventricular-specific enhancers, liver-specific enhancers, lung-specific enhancers, skeletal muscle-specific enhancers, and neuronal-specific enhancers, and the inducible enhancers comprise at least one of corresponding tissue injury enhancers, corresponding pathological microenvironment enhancers, and corresponding environmental pressure enhancers.
More preferably, the enhancer is the Myl7 enhancer or the Myl3 enhancer.
In some embodiments of the invention, the length of the enhancer sequence is 50-500 bp in the ITR-enhancer-based driving nucleic acid expression control element, and preferably the length of the enhancer is 100bp.
In some embodiments of the invention, the enhancer is located 3 'to the 5' ITR sequence of the AAV genome, 5 'to the start codon of the target gene or 3' to the PloyA, 5 'to the 3' ITR sequence in an ITR-enhancer-based driver nucleic acid expression regulatory element. Preferably, the enhancer of the invention is located 3' to the genomic PloyA of the AAV, 5' to the 3' itr sequence.
Further, the above also includes (iii) a target sequence of miR 122.
The target sequence of miR122 (miR 122 target sequence, miR122TS for short) is one of the earliest discovered tissue-specific microRNAs, has high liver specificity, can not be detected in other tissues, and miR122 sequences of different species are highly conserved. By inserting the target sequence of miR122 into the regulatory element, the therapeutic gene can be ensured to be mainly expressed in non-hepatic cells (such as targeted other tissues) or only under specific conditions, so that the safety and specificity of treatment are improved.
In a second aspect, the application provides a recombinant nucleic acid molecule for improving specific delivery of a target gene, the recombinant nucleic acid molecule comprising the above-described combination of regulatory elements for ITR-enhancer-based driving nucleic acid expression, an exogenous target gene, and polyadenylation.
Further, 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 of a Cre/Loxp system. Exogenous target genes can also be a variety of target genes delivered via AAV for biological or medical research. Preferably, the exogenous target genes described in the present invention are Cre and Cas9 genes.
Further, the recombinant nucleic acid molecule at least comprises four parts of ITR, enhancer, exogenous target gene and poly-A (PolyA, pA), wherein pA sequence includes but is not limited to poly-A sequence of various mRNA, preferably, the pA sequence is SV40 PolyA and a short-sequence PolyA.
Further, the recombinant nucleic acid molecule comprises an inverted terminal repeat ITR-5' UTR-exogenous target gene-3 ' UTR-3' terminal poly A tail-enhancer-inverted terminal repeat ITR from 5' end to 3' end.
In a third aspect, the present application provides a recombinant adeno-associated virus for enhancing the specific delivery of a target gene, comprising:
AAV protein capsids;
A recombinant nucleic acid molecule which is a recombinant nucleic acid molecule as described above that enhances the specific delivery of a target gene.
Further, 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.
Preferably, the above-mentioned ITR sequences comprise one or more of ITR1, ITR2, truncated ITR2, ITR3, ITR5, ITR4, ITR6, ITR 7.
In a fourth aspect, the application provides a pharmaceutical composition comprising a combination of regulatory elements as described above, or a recombinant nucleic acid molecule as described above, or a recombinant adeno-associated virus as described above, and a pharmaceutically acceptable excipient. In some preferred embodiments, the pharmaceutical compositions may be formulated for administration by intravenous injection, oral, inhalation, intranasal, intratracheal, intraarterial, intraocular, intramuscular, intraabdominal and/or other parenteral routes.
In a fifth aspect, the present application provides a method for constructing an adeno-associated viral vector for modulating expression of a nucleic acid 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.
Further, the construction method of the adeno-associated virus vector comprises the following steps:
plasmid DNA containing AAV genome is prepared, wherein the AAV plasmid DNA comprises all DNA sequences disclosed by the invention, and the enhancer sequence, the exogenous target gene sequence and the PolyA sequence are positioned between ITR sequences.
Packaging the AAV vector in HEK293 cells by using a three-plasmid method, wherein the three-plasmid method is characterized in that plasmid DNA containing AAV genome and two auxiliary packaging plasmids (Rep-Cap plasmid and Ad Helper plasmid) required for additionally packaging the AAV are transfected together to generate AAV particles;
The method for purifying the AAV vector comprises the steps of collecting cell sediment of three plasmid transfected HEK293 cells by using PEG8000, repeatedly freezing and thawing to release AAV from the cell sediment, primarily purifying the AAV by using iodixanol gradient centrifugation, and further purifying and concentrating the AAV by using ultrafiltration tube ultrafiltration centrifugation.
In a sixth aspect, the application provides the use of an adeno-associated viral vector for modulating nucleic acid expression based on an ITR-enhancer combination, the use comprising one or more of 1) driving expression of an exogenous target gene, 2) driving expression of the exogenous target gene in a specific tissue, and 3) driving expression of the exogenous target gene in a specific time period (e.g., during a disease period).
In some embodiments of the invention, the specific tissue includes, but is not limited to, one or more of heart, liver, skeletal muscle, lung, spleen, kidney, and/or brain, among others.
In some embodiments of the invention, the specific time includes, but is not limited to, a disease onset period (e.g., a specific time expression when heart failure occurs).
In summary, the application has the following beneficial effects:
(1) The invention provides an element combination for regulating nucleic acid expression based on an ITR-enhancer, and constructs a novel gene expression regulation system without an exogenous promoter, and the capacity of a vector released by the exogenous promoter is deleted to be 600-2000 bp, so that the loading capacity of a large therapeutic gene is greatly improved, and the novel gene expression regulation system is particularly suitable for application scenes of large gene expression, for example, gene knockout is carried out by using the ITR-enhancer to drive a classical CRISPR/Cas9 system to be expressed in a single AAV system, and the application which can be realized only by using a double AAV system in the prior art is realized.
(2) The invention provides an element combination for regulating and controlling nucleic acid expression based on an ITR-enhancer, which utilizes the ITR-enhancer to drive exogenous target gene expression, and can realize exogenous target gene specific expression by utilizing the characteristics of high tissue specificity, short sequence and the like of the enhancer compared with the defects of long sequence (1-2 kb) and low tissue specificity of the traditional tissue specific promoter.
(3) The invention provides an element combination for regulating and controlling nucleic acid expression based on an ITR-enhancer, which utilizes the ITR-enhancer to drive exogenous target genes to express, and compared with the safety problems such as cytotoxicity and the like possibly caused by the continuous expression of traditional AAV, the invention can realize the dynamic expression of exogenous target genes by utilizing the pathological responsiveness characteristic of the enhancer and improve the safety of the AAV vector in gene therapy.
Drawings
FIG. 1 is a graph showing experimental results of AAV9-Cre-pA-CMVEnh in example 1, wherein AAV9-Cre-pA and AAV9-Cre-pA-CMVEnh are injected into Rosa fsCas9-tdTomato reporter mice, FIG. B shows AAV9-Cre-pA and AAV9-Cre-pA-CMVEnh, AAV9 is obtained on day 7 after injection into mice, TOM (red) reporter fluorescent signal expression results are shown in frozen sections of heart, liver, lung, spleen, kidney, brain and skeletal muscle of the mice, C is quantitative analysis results of TOM fluorescent signals in frozen sections of various tissues of Rosa26 fsCas9-tdTomato (n=3) reporter mice (atrial, ventricular, liver, spleen, lung, kidney, brain and skeletal muscle) of the AAV9-Cre-pA-CMVEnh, wherein red dots represent the red dot numbers of the AAV9-Cre-pA in the AAV9-Cre-pA-CMVEnh, and red dots represent the red dot numbers of the AAV 9-Totdo-42 in the AAV-35 (red dot numbers of the AAV-4-6-35). Student's t test, < P <0.05, < P <0.01.
FIG. 2 is a diagram showing experimental results of AAV9-Cre-pA-miR122TS-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh in example 2 for driving specific expression of target gene Cre in atrium and ventricle, respectively, wherein FIG. A is a flow chart showing experimental design of AAV9-Cre-pA-miR122TS-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh injected into Rosa fsCas9-tdTomato reporter mice, and FIG. B is a flow chart showing AAV9-Cre-pA-miR122TS-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh injected subcutaneously into Rosa fsCas9-tdTomato reporter mice and tail vein injected into Rosa fsCas9-tdTomato reporter adult mice, respectively, showing that the mice are atrial, The results of fluorescence signal expression of ventricular TOM (red) reporter gene, and FIG. C shows AAV9-Cre-pA-miR122TS-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh injected subcutaneously in Rosa fsCas9 -tdTomato reporter gene neonatal mouse and caudal vein in Rosa fsCas9-tdTomato reporter gene adult mouse, liver, The results of fluorescent signal expression of TOM (red) reporter gene are shown in spleen, lung, kidney, brain and skeletal muscle frozen sections, and figure D shows frozen sections of various tissues (atrium, ventricle, liver, spleen, lung, kidney, etc.) of AAV9-Cre-pA-miR122TS-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh two AAV9 in Rosa26 fsCas9-tdTomato (n=3) reporter gene neonatal mice, Brain, skeletal muscle), graph E shows quantitative analysis results of two AAV9 in various tissue frozen sections (atrial, ventricular, liver, spleen, lung, kidney, brain, skeletal muscle) of Rosa26 fsCas9-tdTomato (n=3) reporter adult mice of AAV9-Cre-pA-miR122 TS-myc 7Enh and AAV9-Cre-miR122 TS-pA-myc 3Enh, wherein red dots in C, D, E represent AAV9-Cre-miR122 TS-myc 7Enh (Myl 7Enh group), green dots represent AAV9-Cre-pA-miR122 TS-myc 3Enh (Myl 3Enh group), points in a scatter bar graph represent n values, TOM: totdtomato.
FIG. 3 is a graph showing experimental design flow of mice injected with AAV9-Cas9-miR122TS-pA-Myl7Enh-U6-Scn5a.gRNA, C57 wild-type mice, B shows electrocardiogram results of mice injected with AAV9-Cas9-miR122TS-pA-Myl7Enh-U6-Scn5a.gRNA, C shows results of gene knockout of AAV9-Cas9-miR122TS-pA-Myl7Enh-U6-Scn5a.gRNA mice in atrial, ventricular, hepatic, pulmonary, splenic, renal, brain and skeletal muscle amplicon sequencing detection, n values in scattered-spot histogram, scn5a atrial-specific knockout immunofluorescence map, and Scn5a atrial-specific knockout map.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the following examples, which are to be construed as merely illustrative and not limitative of the scope of the invention, but are not intended to limit the scope of the invention to the specific conditions set forth in the examples, either as conventional or manufacturer-suggested, nor are reagents or apparatus employed to identify manufacturers as conventional products available for commercial purchase.
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 within the skill of the art.
The use of all technical solutions according to the invention can be used for preventive or therapeutic purposes or for non-preventive or non-therapeutic purposes.
The term "ITR" as used herein refers to an inverted symmetrical repeat sequence, which is the basic constitutive sequence of the AAV genome, capable of forming a highly stable secondary structure, which plays an essential role in the replication and packaging process of viruses. The overall structure of ITRs is highly conserved among the different serotypes, but there are still some subtypes or variants, largely divided into two classes, natural serotype-related ITRs and engineered ITRs. There are minor differences in ITR sequences from AAV serotype, but the core structure (e.g., palindromic sequences, rep binding sites, etc.) remains conserved.
Wherein, ITR1, ITR2, ITR3, ITR4, ITR5 and ITR6 are natural serotype related ITRs, and truncated ITR2 and ITR7 are artificially modified ITRs, in particular:
ITR1 and ITR2 refer to ITRs derived from AAV serotype 2 (AAV 2), one ITR at each end of a typical AAV2 genome, and the sequences of these two ITRs are not identical and are therefore designated ITR1 and ITR2, respectively.
ITR3 refers to ITR derived from AAV serotype 3 (AAV 3);
ITR4 refers to ITR derived from AAV serotype 4 (AAV 4);
ITR5 refers to ITR derived from AAV serotype 5 (AAV 5);
ITR6 refers to ITR derived from AAV serotype 6 (AAV 6).
Truncated ITR2 refers to an artificial variant obtained by deleting part of the sequence on the basis of natural AAV2 ITR 2;
ITR7 refers to a synthetic or significantly engineered ITR.
The term "Enh" as used herein refers to an enhancer, a DNA sequence that is non-coding and capable of enhancing 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 without tissue and space-time specificity. The term "Myl7Enh" refers to non-coding sequences that enhance atrial-specific expression of exogenous target genes. The term "Myl3Enh" refers to a non-coding sequence that enhances ventricular-specific expression of an exogenous target gene.
The term "exogenous target gene" as used herein refers to a target gene whose ITR-enhancer drives expression, which can be a therapeutic gene for gene therapy, a Cas gene in a gene editing system, a Cre gene of a Cre/Loxp system, or various target genes delivered by AAV for biological or medical research.
The term "pA" as used herein refers to a polyadenylation sequence, a termination signal sequence for mRNA transcription.
The term "AAV9" as used herein refers to recombinant AAV9 (rAAV 9) engineered based on wild-type AAV9 (human type 9 adeno-associated virus) capable of delivering a gene of interest and any complete or partial variants thereof that have been engineered, recombined, engineered, modified, or altered to retain affinity for the heart. In AAV vectors of the invention, including AAV9, the DNA sequence of the AAV vector comprises, in order from the 5' end to the 3' end, an inverted terminal repeat (INVERTEDTERMINAL 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).
The following table lists the nucleic acid sequence numbers and corresponding sequences used in the description of the invention (where underlined and italicized index indicates the "tag" used to distinguish samples in primers used in high throughput sequencing, also known as barcode or barcode, which can be freely selected by the skilled person of high throughput sequencing as desired).
TABLE 1 nucleic acid sequence numbers and corresponding sequences used in the invention
The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
Example 1
The present embodiments provide a nucleic acid regulatory element combination of an ITR-enhancer that enables efficient expression of gene delivery in AAV9 in the Cre-Loxp system.
In this example, for the Cre-Loxp system, the inventors first constructed AAV9-Cre-pA adeno-associated virus for evaluating the expression of AAV9-Cre-pA driver gene in mice, and further constructed AAV9-Cre-pA-CMVEnh for evaluating whether the combination of nucleic acid regulatory elements of the ITR-enhancer enhances efficient expression of gene delivery in AAV 9.
The specific process is as follows:
1. Construction of AAV9-Cre-pA and AAV9-Cre-pA-CMVEnh plasmids
Construction based on AAV-U6sgRNA-U6 sgRNA-Tnnt-Cre plasmid vector, see publications (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）.
AAV9-Cre-pA plasmid was prepared:
Restriction enzymes KpnI, nheI and RsrII are selected as cleavage sites in an AAV-U6sgRNA-U6 sgRNA-Tnnt-Cre plasmid vector, 1 μg of AAV-U6sgRNA-U6 sgRNA-Tnnt-Cre plasmid, 1 μl of KpnI, 1 μl of NheI, 1 μl of RsrII, 3 μl of 10× CutSmart buffer, and no nuclease water are added to 30 μl. The resulting mixed components were placed in a 37 ℃ water bath for reaction 60 min. And (3) carrying out agarose gel electrophoresis on the enzyme digestion product, and recovering the enzyme digestion product by gel.
PCR amplification of Cre sequence is performed using the primer pair having the upstream primer F sequence shown in SEQ ID NO.1 and the downstream primer R sequence shown in SEQ ID NO. 2 of Table 1, and PCR amplification of pA sequence using the primer pair having the sequences shown in SEQ ID NO. 3 (F) and SEQ ID NO. 4 (R) of Table 1.
The PCR product was gel recovered and cloned into the digested plasmid vector by a seamless cloning technique, and the enzyme and the seamless cloning kit used in the preparation process were commercially available (NEB, R3142S, R3131S, R0501S; nanjinouzan Biotechnology Co., ltd., C117-01). The connection product is transformed into Stable3 competent cells, the cells are coated on an LB culture medium plate containing ampicillin, and monoclonal is selected to carry out Sanger sequencing, thus obtaining AAV9-Cre-pA plasmid with successful cloning for standby.
(2) Preparation of AAV9-Cre-pA-CMVEnh plasmid
AAV-U6sgRNA-U6sgRNA-Tnnt2-Cre plasmid cleavage and gel recovery procedures were as described above.
PCR amplification of Cre sequence, the primer pair sequences are shown as SEQ ID NO.1 (F) and SEQ ID NO. 2 (R) in Table 1;
PCR amplification of pA sequence is performed, and the primer pair sequences are shown as SEQ ID NO. 3 (F) and SEQ ID NO. 5 (R) in Table 1;
the CMVEnh sequences were PCR amplified using primer pairs of the sequences shown in Table 1 as SEQ ID NO. 6 (F) and SEQ ID NO. 7 (R).
And (3) performing gel recovery on the PCR product, and cloning the PCR product into the plasmid vector after enzyme digestion by using a seamless cloning technology. The connection product is transformed into Stable3 competent cells, the cells are coated on an LB culture medium plate containing ampicillin, and monoclonal is selected to carry out Sanger sequencing, thus obtaining AAV9-Cre-pA-CMVEnh plasmid which is successfully cloned for standby.
2. Packaging AAV9 virus AAV9-Cre-pA and AAV9-Cre-pA-CMVEnh
The AAV9 virus packaging preparation is carried out on the constructed AAV9-Cre-pA plasmid and AAV9-Cre-pA-CMVEnh plasmid respectively. AAV9 was prepared using HEK293T cells as host cells. For the plasmid required for packaging AAV9, high quality extraction of the plasmid can be performed using an endotoxin-free plasmid mass extraction kit (Tiangen Biochemical technology (Beijing), inc., DP 117).
The AAV packaging method comprises the following specific steps:
① And (3) taking out the frozen HEK293T cells from the liquid nitrogen for resuscitation, placing the frozen HEK293T cells in a 15cm cell culture dish for culture, and carrying out cell passage when the cells grow to the fusion degree of 90%. Each AAV package required 10 discs of 15cm cell culture dishes of HEK293T cells. When the cell density reaches to 90% of fusion degree, 1.8mL of transfection reagent PEI (1 mg/mL) is added into each cell, 7 mug of AAV plasmid, 7 mug of Rep2/Cap9 plasmid and 20 mug of pHelper plasmid are added, the cells are cultured for 8-12 hours at 37 ℃ and then are replaced by serum-free high-sugar DMEM medium containing 1% double antibody, and the cells are collected after the cells are continuously cultured for 66-72 hours at 37 ℃;
② Centrifuging the collected cell pellet at 3000rpm for 5min, discarding supernatant, and re-suspending the cell pellet in AAV lysis buffer (20mM Tris pH8.0, 1mM MgCl2, 150mM NaCl), and temporarily storing at-80deg.C;
③ Adding 1/4 volume of 40% PEG8000 solution containing 2.5M NaCl to the cell culture medium collected in the ② th step, standing at 4 ℃ for 2h, centrifuging at 4000rpm for 30min to separate out precipitate, discarding supernatant, re-suspending the obtained precipitate AAV lysis buffer, mixing with ② th cell suspension, adding 50 μg/ml totipotent nuclease to the cell suspension, and repeatedly freezing and thawing at 37 ℃ and-80 ℃ for 3 times to fully lyse the cells.
④ Centrifuging at 4000rpm for 30min at 4deg.C, collecting supernatant, spreading the supernatant on gradient iodixanol (OptiPrep), and performing AAV purification by density gradient ultracentrifugation;
⑤ AAV titers were determined by real-time fluorescent quantitative PCR (RT-qPCR) in 100kDa molecular cut-off centrifuge tubes by washing and concentrating AAV with PBS containing 0.001% Pluronic F-68, and the AAV titers were above 1E+13vg/mL.
3. Packaging AAV-DJ virus AAV-DJ-Cre-pA-CMVEnh
According to the same method for preparing AAV9 virus as described in the step (2), only Rep2/Cap9 plasmid is replaced by Rep2/Cap-DJ plasmid, thereby completing AAV-DJ virus packaging of the plasmid (AAV-DJ is artificially modified chimeric serotype, has wide tropism and higher transduction efficiency), and AAV-DJ-Cre-pA-CMVEnh is obtained.
ITR-driven low expression of exogenous target genes
A fluorescent reporter mouse (cuvettes, strain No. T002249, denoted Rosa26 fsCas9-tsTomato) was selected with the Rosa26 safety site knocked into the CAG promoter-Loxp-stop-Loxp-Cas 9-2A-tdtomao, and a fluorescent reporter gene activatable with Cre was present in the mouse.
AAV9-Cre-pA was subcutaneously injected into Rosa26 fsCas-tdTomato (n=3) neonatal mice at a rate of 5X 10 10 vg/g (vg/g is an abbreviation of vector genome per gram body weight, hereinafter the same applies) and, after 7 days, the mice were sacrificed and hearts, livers, spleens, lungs, kidneys, brains, skeletal muscles were fixed, dehydrated, embedded, and frozen in sections (slice thickness 8 μm). Tissue sections were stained with 4', 6-diamidino-2-phenylindole (DAPI) and confocal microscopy imaged. The TdTomato (TOM) reporter gene was counted and statistically analyzed for fluorescent protein positive cells using Image J and GRAPHPAD PRISM software.
As shown in FIG. 1C, the TOM positive cell rate was about 5% or more in the heart, and about 40% in the liver tissue sections, and almost 0% in the other organs such as spleen, lung, kidney, skeletal muscle, brain. Therefore, the ITR can drive the expression of the target gene Cre, which shows that the ITR has weak promoter transcriptional activity.
ITR-enhancers significantly increase exogenous target gene expression
A Rosa26 fsCas9-tdTomato fluorescent reporter mouse was prepared in the same manner as described above.
AAV9-Cre-pA and AAV9-Cre-pA-CMVEnh were injected subcutaneously into two groups of Rosa26fsCas9-tdTomato neonatal mice (n=3) (shown in fig. 1A) at a dose of 5 x 1010vg/g, and after 7 days the mice were sacrificed and the heart, liver, spleen, lung, kidney, brain, skeletal muscle of the two groups were harvested for fixation, dehydration, embedding, and frozen sections. Tissue sections were stained with 4', 6-diamidino-2-phenylindole (DAPI) and confocal microscopy imaged. The TdTomato (TOM) reporter gene was counted and statistically analyzed for fluorescent protein positive cells using Image J and GRAPHPAD PRISM software.
As shown in FIGS. 1B and 1C, compared with AAV 9-Cre-pA-injected mice, AAV 9-Cre-pA-CMVEnh-injected mice had almost 100% TOM positive cell rate, 25% TOM positive cell rate of lung and 10% TOM positive cell rate of spleen in heart and liver tissue sections.
As shown in FIG. 1D, 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, which showed that only Pol II inhibitors significantly inhibited the transcriptional activity of the ITR-enhancers.
It was concluded that the ITR-enhancer significantly enhances the expression of the target gene Cre, and that ITR transcriptional activity is Pol II dependent.
Example 2
This example provides nucleic acid regulatory elements of ITR-enhancers driving efficient expression of exogenous target genes in the heart
The inventor selects an atrium and a ventricle specific Enhancer, utilizes a Cre-Loxp system, selects an atrium specific Enhancer Myl7 Enhancer (Myl 7 Enhancer, myl7Enh for short) and a ventricle specific Enhancer Myl3 Enhancer (Myl 3 Enhancer, myl3Enh for short), and simultaneously selects miR122TS to inhibit weak transcriptional activity of ITR in liver, thereby constructing AAV9-Cre-miR122TS-pA-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh adeno-associated virus, and is used for evaluating whether a combination of nucleic acid regulating elements of the ITR-enhancers drives tissue-specific high expression of gene delivery in AAV 9.
The specific process is as follows:
Construction of AAV9-Cre-miR122TS-pA-Myl7Enh and AAV9-Cre-miR122TS-
PA-Myl3Enh plasmid
AAV9-Cre-pA-CMVEnh plasmid constructed in example 1 was selected, and the restriction enzyme BamHI (NEB, R0136S) was selected as the cleavage site, and 1. Mu.g of AAV9-Cre-pA-CMVEnh plasmid, 1. Mu.L of BamHI, 3. Mu.L of 10X CutSmart buffer, and nuclease-free water were added to 30. Mu.L of the mixture. The resulting mixed components were placed in a 37 ℃ water bath for reaction 60 min. And (3) carrying out agarose gel electrophoresis on the enzyme digestion product, and recovering the enzyme digestion product by gel.
The DNA fragment of the chemically synthesized 3 xmiR 122TS gene sequence (the sequence is shown as SEQ ID NO:8 in Table 1, entrusted to the limited synthesis of Kangshen biotechnology in Hangzhou) is cloned on an AAV9-Cre-pA-CMVEnh vector to construct an AAV9-Cre-miR122TS-pA-CMVEnh plasmid. The AAV9-Cre-miR122TS-pA-CMVEnh plasmid further selects the restriction enzymes AscI (NEB, R0558S) and RsrII as cleavage sites, and 1. Mu.g of AAV9-Cre-miR122TS-pA-CMVEnh plasmid, 1. Mu.L of AscI, 1. Mu.L of RsrII, 3. Mu.L of 10X CutSmart buffer solution and no nuclease water are added and mixed to 30. Mu.L. The resulting mixed components were placed in a 37 ℃ water bath for reaction 60 min. And (3) carrying out agarose gel electrophoresis on the enzyme digestion product, and recovering the enzyme digestion product by gel.
The DNA fragments of Myl7Enh (shown as SEQ ID NO:9 in Table 1) and Myl3Enh (shown as SEQ ID NO:10 in Table 1) which are chemically synthesized are cloned to AAV9-Cre-pA-CMVEnh vector respectively, so that AV9-Cre-miR122TS-pA-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl3Enh plasmids can be successfully constructed.
(2) AAV9 virus packaging
According to the same manner as that for preparing AAV9 virus in example 1, only the AAV plasmid therein was replaced with the plasmid in the step (1) of this example, thereby completing AAV9 virus packaging of the two plasmids, and AAV9-Cre-miR122TS-pA-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl 3Enh were obtained.
(3) Evaluation of expression of ITR-Myl7Enh and ITR-Myl3Enh driven target Gene Cre
AAV9-Cre-miR122TS-pA-Myl7Enh and AAV9-Cre-miR122TS-pA-Myl 3Enh were subcutaneously injected into Rosa26fsCas 9-tdtomao (n=3) neonatal mice (as shown in fig. 2A) at 5×10 10 vg/g, respectively, and after 7 days the mice were sacrificed and hearts, livers, spleens, lungs, kidneys, brains, skeletal muscles were fixed, dehydrated, embedded, and frozen into sections (slice thickness 8 μm). Tissue sections were stained with 4', 6-diamidino-2-phenylindole (DAPI) and confocal microscopy imaged. The TdTomato (TOM) reporter gene was counted and statistically analyzed for fluorescent protein positive cells using Image J and GRAPHPAD PRISM software.
As shown in FIGS. 2B, 2C and 2D, the mice injected with AAV9-Cre-miR122TS-pA-Myl7Enh had almost up to 95% TOM positive cell rate in the atrium, but only about 5% positive cell rate in the ventricular tissue section, and almost 0% positive cell rate in the other organs such as spleen, lung, kidney, skeletal muscle and brain. The mice injected with AAV9-Cre-miR122TS-pA-Myl3Enh have TOM positive cell rate of only 5% in the atrium, but positive cell rate of about 90% in the ventricular tissue section, and almost 0% in other organs such as spleen, lung, kidney, skeletal muscle and brain.
In addition, to assess whether expression of the exogenous target gene driven by the nucleic acid regulatory element of the TR-enhancer is affected by age and mode of administration of the subject, the inventors performed tail vein injection of viruses AAV9-Cre-miR122 TS-pA-myc 3Enh and AAV9-Cre-miR122 TS-pA-myc 7Enh in adult Rosa26 fsCas-tdhamano mice (4 weeks old, adult group, n=3), sacrificed the mice after 1 week, frozen sections were taken from heart, liver, spleen, lung, kidney, brain, skeletal muscle tissues and DAPI stained.
As shown in FIGS. 2B, 2C and 2E, adult mice injected with AAV9-Cre-miR122TS-pA-Myl7Enh had almost 90% TOM positive cell rate in the atria, but only about 5% positive cell rate in ventricular tissue sections, and almost 0% positive cell rate in other organs such as spleen, lung, kidney, skeletal muscle and brain. Adult mice injected with AAV9-Cre-miR122TS-pA-Myl3Enh have only about 5% of TOM positive cell rate in the atrium, but about 80% of positive cell rate in ventricular tissue sections, and almost 0% of positive cell rate in other organs such as spleen, lung, kidney, skeletal muscle and brain.
It was concluded that the nucleic acid regulatory elements of the ITR-enhancers drive the expression of exogenous target genes in specific tissues by selecting different tissue-specific enhancers, and that such regulatory gene expression is not affected by the route of administration and the age of the animal.
Example 3
This example provides nucleic acid regulatory elements of the ITR-enhancer to achieve single AAV delivery of CRISPR/spCas9 systems
In a traditional AAV delivery system, the nucleic acid regulatory element is an exogenous promoter, and the sequence of the promoter is generally more than 500bp, so that more than 10% of the AAV genome (4.7 kb) is occupied, the gene load of AAV is limited, and a large exogenous target gene cannot be delivered. Traditional AAV delivery CRISPR/spCas9 systems require dual AAV to achieve, where one AAV expresses spCas9 alone and the other AAV delivers gRNA, and the use of dual AAV presents safety issues.
In the application, the nucleic acid regulatory element of the ITR-enhancer can maximally increase the DNA load of AAV by deleting an exogenous promoter and selecting a tissue-specific and short-sequence enhancer. Therefore, in this embodiment, the inventors select short-sequence Myl7Enh (abbreviated as Myl7ENh mini) to combine with ITR to form ITR-Myl7Enh mini to deliver CRISPR/spCas9 system via single AAV to implement gene editing.
To this end, the inventors designed a spCas9 single-stranded guide RNA (sgRNA) targeting exon 1 of Scn5a, driven expression by the U6 promoter. Scn5a encodes the alpha subunit of the cardiac sodium ion channel nav1.5, mediating rapid sodium influx (INa), triggering depolarization of the cardiomyocytes. The gene mutation can lead to a series of genetic arrhythmias, such as long QT syndrome type 3 (LQT 3), brugada syndrome, progressive heart block (PCCD), and the like.
The specific implementation steps are as follows:
Construction of AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-Scn5a.sgRNA plasmid.
AAV9-Cre-miR122TS-pA-Myl7Enh plasmid constructed in the example is selected, restriction enzymes KpnI and HindIII (NEB, R0104S) are selected as enzyme cutting sites, and 1 mug of AAV9-Cre-miR122TS-pA-Myl7Enh plasmid, 1 mug of KpnI, 1 mug of HindIII, 3 mug of 10 multiplied by CutSmart buffer solution and 30 mug of nuclease-free water are added and mixed. The resulting mixed components were placed in a 37 ℃ water bath for reaction 60min. And (3) carrying out agarose gel electrophoresis on the enzyme digestion product, and recovering the enzyme digestion product by gel.
The spCas9 sequence was amplified by PCR using the primer pair sequences shown as SEQ ID NO. 11 (F) and SEQ ID NO. 12 (R) in Table 1, the PCR product was gel recovered and cloned into the digested plasmid vector using a seamless cloning kit (Nanjinouzan Biotechnology Co., ltd., C117-01). The connection product is transformed into Stable3 competent cells, the cells are coated on an LB culture medium plate containing ampicillin, and monoclonal is selected to carry out Sanger sequencing, thus obtaining AAV9-spCas9-miR122TS-pA-Myl7Enh plasmid with successful cloning for standby.
Using the AAV9-spCas9-miR122TS-pA-Myl7Enh, nheI and RsrII were selected as cleavage sites, 1. Mu.g of AAV9-spCas9-miR122TS-pA-Myl7Enh plasmid, 1. Mu.L of NheI, 1. Mu.L of RsrII, 3. Mu.L of 10X CutSmart buffer solution and no nuclease water were added and mixed. The resulting mixed components were placed in a 37 ℃ water bath for reaction 60 min. And (3) carrying out agarose gel electrophoresis on the enzyme digestion product, and recovering the enzyme digestion product by gel. The DNA fragment of the chemically synthesized pA-Myl7Enhmini-U6-sgRNA (shown as SEQ ID NO:13 in Table 1, entrusted with the limited synthesis of Kangshen biotechnology in Hangzhou) is cloned onto an AAV9-spCas9-miR122TS-pA-Myl7Enh vector, and then the AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-sgRNA plasmid can be successfully constructed.
By using the AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-sgRNA plasmid, paqCI is selected as a cleavage site, and 1 mug of AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-sgRNA plasmid, paqCI mu L of 10X CutSmart buffer solution 3 mu L of nuclease-free water and 30 mu L of nuclease-free water are added and mixed. The resulting mixed components were placed in a 37 ℃ water bath for reaction 60 min. And (3) carrying out agarose gel electrophoresis on the enzyme digestion product, and recovering the enzyme digestion product by gel.
The mice Scn5a gene was designed for sgRNA using software CRISPick and the optimal sgRNA located on one exon was selected as shown in Table 1 under SEQ ID NO. 14. From the constructed AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-sgRNA, preferably PaqCI cleavage site, a primer of Scn5a.sgRNA is synthesized, the primer sequences are respectively shown as SEQ ID NO. 15 (F) and SEQ ID NO. 16 (R) in table 1, and the T4 connection can successfully clone the AAV9-spCas9-miR122TS-pA-Myl 7Enhmini-U6-Scn5a.sgRNA plasmid.
(2) AAV9 virus packaging
According to the same manner as that for preparing AAV9 virus in example 1, only the AAV plasmid therein was replaced with the plasmid in the step (1) of this example, whereby AAV9 virus packaging of the plasmid was completed to obtain AAV9-spCas 9-miR 122TS-pA-Myl7Enhmini-U6-Scn5a.sgRNA.
(3) ITR-Myl7Enhmini drives spCas9 atrial specific expression, and Scn5a is knocked out to successfully construct mouse atrial fibrillation model
AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-Scn5a.sgRNA was subcutaneously injected into C57 neonatal mice (n=3) at 1× 11 vg/g (as shown in FIG. 3A), and after 30 days, the mice were anesthetized with tribromoethanol, and the Power Lab electrocardiograph was used to detect the body surface electrocardiograms of the mice. Results as shown in fig. 3B, the electrocardiographic results showed that the mice injected with AAV9-spCas9-miR122TS-pA-Myl7Enhmini-U6-scn5a. Sgrna exhibited significant atrial fibrillation.
Mice were sacrificed after completion of the electrocardiographic examination and amplicon sequencing was performed on atrial, ventricular, liver, spleen, lung, kidney, brain, skeletal muscle tissue (Amplicon-Seq). The specific operation flow is as follows. After taking out the atrial, ventricular, hepatic, spleen, lung, kidney, brain, skeletal muscle tissues of the mice (n=3), gDNA extraction of cardiac and hepatic tissues was performed using a tissue genomic DNA (gDNA) extraction kit (division of bioengineering (shanghai)).
The specific operation flow is that the primer design of the high-flux sequencing library construction is carried out on the target gene locus, and the primer sequences are respectively shown as SEQ ID NO. 17 (F) and SEQ ID NO. 18 (R) in the table 1. The target fragment was amplified by PCR using gDNA as a template, and the following components were added, gDNA, 2. Mu.g, 1. Mu.L of the upstream primer (F), 1. Mu.L of the downstream primer (R), 2X KeyPo Master Mix, 10. Mu.L, and no nuclease water, to 20. Mu.L. The added samples were placed in a PCR apparatus and the PCR reaction was performed by 15 cycles of 98℃30s, 98℃10s,70℃30s,72℃ 60s,Touchdown PCR s, 98℃10s,70℃30s,72℃60s, 15 cycles, 72℃5min, and 12℃holding, and the PCR product was purified. The second round of PCR amplification was performed using the P5 primer and the P7 primer provided by Shanghai Biotechnology Co., ltd. With primer sequences shown in SEQ ID NO:19 (F) and SEQ ID NO:20 (R) in Table 1, respectively. The first round of PCR products were used as templates for PCR amplification of the target fragment, and the following components were added, namely, 2. Mu.g of PCR products, 1. Mu.L of the upstream primer (F), 1. Mu.L of the downstream primer (R), 2X KeyPo Master Mix, 10. Mu.L of nuclease-free water, and 20. Mu.L of the primer (R). The added sample was placed in a PCR apparatus, and the PCR reaction was performed at 98℃for 30s, 98℃for 10s,55℃for 30s,72℃for 60s, and 20 cycles, 72℃for 5min, and 12 ℃. After agarose gel electrophoresis of the PCR product, the target fragment was excised, gel recovered and purified, and the purified fragment was subjected to second generation sequencing (Beijing norelsen technologies Co., ltd.) and the sequencing result was analyzed using CRISPResso and GRAPHPAD PRISM software was used for statistical analysis and mapping.
As shown in FIG. 3C, the results show that mice mainly produce gene edits (insertion/deletion mutations, indicated by indexes) in the atrium, the index production rate in the atrial tissue is 23%, and the index production rate in the rest of the tissues is lower than 5%.
As shown in FIGS. 3D and 3E, immunofluorescence and WB results suggested that Scn5a was knocked out only in the atrium, while the expression level of the ventricle Scn5a was unchanged.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, and various modifications and variations may be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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