WO2025251192A1 - 一种新型重组腺相关病毒双载体系统 - Google Patents

一种新型重组腺相关病毒双载体系统

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WO2025251192A1
WO2025251192A1 PCT/CN2024/097307 CN2024097307W WO2025251192A1 WO 2025251192 A1 WO2025251192 A1 WO 2025251192A1 CN 2024097307 W CN2024097307 W CN 2024097307W WO 2025251192 A1 WO2025251192 A1 WO 2025251192A1
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raav
recombinase
sequence
recombinase recognition
recognition sequence
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French (fr)
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邱宇翔
刘荇
徐富强
龚静茹
黄天晓
闫苏
文鹏杰
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Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus

Definitions

  • This invention belongs to the field of biotechnology, specifically relating to a novel recombinant adeno-associated virus dual-vector system.
  • Recombinant adeno-associated virus is a widely used tool virus and one of the most promising viral vectors in gene therapy.
  • rAAV Recombinant adeno-associated virus
  • the maximum genome capacity that a commonly used rAAV capsid can pack is approximately 4.7 kb.
  • rAAVs To increase the loading capacity of rAAVs, researchers have developed methods such as homologous recombination, mRNA trans-splicing, and intronomer methods to express target genes using combinations of two rAAVs. These methods are mainly used for expressing large target gene fragments, requiring specific splitting designs for the target gene. Furthermore, some methods require the addition of extra helper sequences, leaving limited options for promoters, enhancers, post-transcriptional regulatory elements, and reporter genes. However, as researchers delve deeper into the structural and functional analysis of various tissues and organs, the need for more detailed cell subtype analysis and precise structural and functional resolution increases, making it particularly important to screen for more specific functional sequences over a wider range of lengths. Therefore, it is necessary to develop an rAAV expression vector that is easier to design and improve, and can simultaneously expand the loading capacity of various auxiliary elements and target genes.
  • homologous recombination at the pretranscriptional level utilizes the cell's own homologous recombination mechanism, resulting in the ligation of two rAAV genomes into a long linear DNA expression cassette
  • mRNA trans-splicing at the posttranscriptional level utilizes intron splicing, resulting in the ligation of two mRNA precursors into a complete linear mRNA
  • intron peptide methods at the posttranslational level utilize self-splicing of peptides, resulting in the ligation of two peptide chains into a complete protein.
  • this invention aims to provide a novel recombinant adeno-associated virus dual-vector system.
  • This system constructs the target gene and regulatory sequence on two recombinant adeno-associated viruses, respectively, and uses recombinase to mediate the combination of the two rAAVs into a complete linear or circular expression cassette to express the exogenous gene.
  • This method is more convenient and flexible and can be used to load large gene fragments and regulatory sequences. More importantly, it uses a novel dual rAAV recombination method, which can be used to form special expression cassette structures.
  • This invention provides a novel recombinant adeno-associated virus (AAV) dual-vector system, comprising a recombinant AAV vector rAAV-T and a recombinant AAV vector rAAV-C;
  • the rAAV-T genome includes a recombinase recognition sequence I, a recombinase recognition sequence II, a promoter sequence, a regulatory sequence, and a reporter gene sequence, wherein the recombinase recognition sequence I and the recombinase recognition sequence II are different;
  • the rAAV-C genome includes a recombinase recognition sequence I', a recombinase recognition sequence II', and a target gene sequence, wherein the recombinase recognition sequence I' and the recombinase recognition sequence II' are located upstream and downstream of the target gene sequence, respectively; ...
  • Recombinase recognition sequence I and recombinase recognition sequence I' are recognition sequences of the recombinase, and recombination occurs under the mediation of the recombinase; recombinase recognition sequence II and recombinase recognition sequence II' are another pair of recognition sequences of the same recombinase, and recombination occurs under the mediation of the recombinase; only I and I', II and II' of the recombinase recognition sequences can react with each other, and the reaction is irreversible, and any other combination cannot react; after recombinase recognition sequences I and I' and recombinase recognition sequences II and II' react, the functional sequence on rAAV-T and the target gene on rAAV-C form the correct expression cassette structure, thereby expressing the target gene.
  • the recombinase is a tyrosine recombinase Cre
  • the combination of the recombinase recognizing sequences I and I' includes, but is not limited to, lox66 and lox71, JT15 and JTZ17, lox2272/71 and lox66/2272, and lox5171/71 and lox66/5171.
  • the combination of the recombinase recognition sequences II and II' is selected from one of lox66 and lox71, JT15 and JTZ17, lox2272/71 and lox66/2272, and lox5171/71 and lox66/5171.
  • the promoter sequence is selected from one or more of CAG, CaMKII ⁇ , CAR, CBA, CD68, c-fos, ChAT, CMV, CR, Ef1 ⁇ , E-SARE, GAD67, GFAP, GFAP104, gfaABC1D, Grm6, hGRK1, hSyn, hUbC, LP1B, L7/Pcp2, MBP, MCK, mDlX, mOXT, mTH, nEF, Nestin, NPY, Nrl, PGK, PV, RAM, RK, ROH, RPE65, SFRP2, SST, TBG, TCAP, TH, Thy1, TPH2, TRE, TRPV1, UAS, and Vgat.
  • the target gene sequence is selected from genes that are nucleotide-coding sequences and/or functional RNA products used to study the structure and function of tissues and organs, overexpression of gene products, manipulation of the nervous system, and gene therapy-related proteins;
  • the protein is preferably one or more of fluorescent proteins, neuronal activating proteins, neuronal inhibiting proteins, calcium ion signaling probe proteins, small molecule signaling probe proteins, apoptosis-mediating proteins, disease-related mutant proteins, normal proteins under physiological conditions, cytokines, antiviral factors, viral infection co-receptors, recombinases, and gene editing tool proteins;
  • the functional RNA is selected from one or more of small RNA, small interfering RNA, small hairpin RNA, small guide RNA, organelle localization RNA, and barcode RNA used for RNA sequencing or in situ hybridization analysis.
  • regulatory sequences include transcriptional and/or post-transcriptional regulatory sequences
  • the regulatory sequence is selected from one or more of WPRE, oPRE, cw3sl, SV40 polyA, hGH polyA, bGH polyA, rbGlob polyA, etc.
  • the reporter gene sequence includes a marker protein, an enzyme reaction chromogenic protein, and other molecular tag sequences for indication.
  • the recombinant adeno-associated virus vector rAAV-T and the recombinant adeno-associated virus vector rAAV-C have the same or different serotypes and have the ability to infect the same cells in tissues or organs;
  • the serotypes include AAV1, AAV2, AAV2-Retro, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9-Retro, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV-PHP.eB, AAV-myo, and their derived serotypes.
  • the rAAV-T genome also contains other auxiliary functional sequences.
  • the correct expression cassette structure formed by the functional sequence on rAAV-T and the target gene on rAAV-C is either a linear expression cassette structure or a circular expression cassette structure;
  • the positions of recombinase recognition sites I and II on rAAV-T are changed by means of (1) or (2) to achieve different expression cassette structures of rAAV-T and rAAV-C under the action of recombinase:
  • rAAV-T and rAAV-C form a linear expression cassette structure under the action of recombinase.
  • the rAAV-T genome was designed to contain elements in the following order: ITR-promoter-reporter gene-recombinase recognition sequence I-recombinase recognition sequence II-regulatory sequence-ITR;
  • the designed rAAV-C genome contains the following elements in the following order: ITR - recombinase recognition sequence I’ - target gene - recombinase recognition sequence II’ - ITR;
  • Recombinases can mediate recombination between I and I’, and between II and II’, and the sequence orientations of I and I’, and II and II’ satisfy the requirement that a linear expression cassette structure can be formed after recombination: (Continued from ITR) - promoter - reporter gene - target gene - regulatory sequence - (Continued from ITR).
  • the rAAV-T genome was designed to contain elements in the following order: ITR - recombinase recognition sequence II - regulatory sequence - promoter - reporter gene - recombinase recognition sequence I - ITR;
  • the designed rAAV-C genome contains the following elements in the following order: ITR - recombinase recognition sequence I’ - target gene - recombinase recognition sequence II’ - ITR;
  • Recombinases can mediate recombination between I and I’, and between II and II’, and the sequence orientations of I and I’, and II and II’ satisfy the requirement that a circular expression cassette structure can be formed after recombination: (regulatory sequence above) - promoter - reporter gene - target gene - regulatory sequence - (promoter below).
  • the present invention also provides a plasmid for packaging the novel recombinant adeno-associated virus dual-vector system, characterized in that the plasmid comprises plasmid T for packaging the recombinant adeno-associated virus vector rAAV-T and plasmid C for packaging the recombinant adeno-associated virus vector rAAV-C; plasmid T contains a recombinase recognition sequence I, a recombinase recognition sequence II, a promoter sequence, a regulatory sequence, and a reporter gene sequence, wherein the recombinase recognition sequence I and the recombinase recognition sequence II are different; plasmid C contains a recombinase recognition sequence I’, a recombinase recognition sequence II’, and a target gene sequence, wherein the recombinase recognition sequence I’ and the recombinase recognition sequence II’ are respectively located within the target gene sequence.
  • the upstream and downstream of the recombinase; the recombinase recognition sequence I and recombinase recognition sequence I’ are the recognition sequences of the recombinase, and recombination occurs under the recombinase-mediated mechanism; the recombinase recognition sequence II and recombinase recognition sequence II’ are another pair of recognition sequences of the same recombinase, and recombination occurs under the recombinase-mediated mechanism; only I and I’, II and II’ of the recombinase recognition sequences can react with each other, and the reaction is irreversible, and any other combination cannot react; after the recombinase recognition sequences I and I’ and recombinase recognition sequences II and II’ react, the functional sequence on rAAV-T and the target gene on rAAV-C form the correct expression cassette structure to express the target gene.
  • the recombinase corresponding to the recombinase recognition sequence is a tyrosine recombinase Cre
  • the combination of the recombinase recognition sequences I and I' is selected from one of lox66 and lox71, JT15 and JTZ17, lox2272/71 and lox66/2272, lox5171/71 and lox66/5171;
  • the combination of the recombinase recognition sequences II and II' is selected from one of lox66 and lox71, JT15 and JTZ17, lox2272/71 and lox66/2272, and lox5171/71 and lox66/5171.
  • the order of the elements contained in plasmid T for packaging the recombinant adeno-associated virus vector rAAV-T is: ITR-promoter-reporter gene-recombinase recognition sequence I-recombinase recognition sequence II-regulatory sequence-ITR;
  • the order of the elements contained in plasmid C for packaging the recombinant adeno-associated virus vector rAAV-C is: ITR-recombinase recognition sequence I’-target gene-recombinase recognition sequence II’-ITR;
  • the order of elements contained in plasmid T for packaging recombinant adeno-associated virus vector rAAV-T is: ITR - recombinase recognition sequence II - regulatory sequence - promoter - reporter gene - recombinase recognition sequence I - ITR;
  • the order of elements contained in plasmid C for packaging recombinant adeno-associated virus vector rAAV-C is: ITR - recombinase recognition sequence I’ - target gene - recombinase recognition sequence II’ - ITR.
  • the present invention also provides a method for expressing a target gene in specific cells or animal tissues and organs using the novel recombinant adeno-associated virus dual vector system, the method comprising: simultaneously injecting rAAV-T and rAAV-C into specific cells or animal tissues and organs, and introducing a recombinase into the cells or animals via transgenes or vectors, wherein rAAV-T and rAAV-C form a complete linear or circular expression cassette under the mediation of the recombinase to express the target gene.
  • the recombinase is introduced into cells or animals via a vector selected from one or more of the following: an expression plasmid carrying the recombinase gene, a viral vector carrying the recombinase gene, or other chemical or biological vectors for introducing the recombinase gene or protein.
  • the tissue is selected from nerve tissue, muscle, epithelial tissue, or connective tissue.
  • the nerve tissue is selected from the central nervous system and related tissues of its neural circuits.
  • the organs are selected from the bladder, eye, ear, nose, mouth, tongue, pharynx, larynx, vomeronasal organ, salivary gland, liver, kidney, spleen, heart, intestine, stomach, pancreas, lung, trachea, blood vessels, lymphatic vessels, lymph nodes, limbs, pituitary gland, thyroid gland, parathyroid gland, pancreatic islets, adrenal gland, or reproductive organs.
  • rAAV-T and rAAV-C can be used in conventional applications such as physiological mechanism research, disease modeling, cell regulation, and neural circuit labeling.
  • Specific applications of this invention include long promoter screening, enhancer screening, post-transcriptional regulatory sequence screening, circular DNA expression cassette delivery, and special circuit labeling.
  • This invention provides a method for expressing a foreign gene by constructing a target gene and a regulatory sequence separately on two recombinant adeno-associated viruses (rAAVs), and then using recombinase to mediate the formation of complete expression cassettes with different structures from the two rAAVs.
  • the two rAAVs in this invention each contain only the target gene or auxiliary functional sequences that regulate or report the expression of the target gene. Furthermore, they contain two pairs of recombinase recognition sites that mediate recombination between the two rAAVs. In the presence of recombinase, the two rAAVs can recombine into a complete expression cassette, which can form a linear or circular structure.
  • this invention has the following advantages:
  • This invention completely separates the target gene and other functional sequences onto two rAAVs, which can be easily and flexibly modified and combined. It also increases the range of selectable gene and functional sequence lengths, expanding the loading capacity of the target gene and other functional elements.
  • the rAAV in this invention can be combined to generate different DNA expression cassette structures, which can be used to explore and compare the expression differences between linear and circular DNA structures.
  • This invention utilizes exogenous enzyme-mediated recombination, which is more efficient than intracellular mechanisms. It is suitable for research that requires rapid expression of target genes, such as disease modeling, loop labeling, and gene therapy, and can shorten the experimental cycle.
  • Figure 1 shows a plasmid map used to package recombinant adeno-associated virus AAV-T1.
  • Figure 2 shows a plasmid map used to package recombinant adeno-associated virus rAAV-T2.
  • Figure 3 shows a plasmid map used to package recombinant adeno-associated virus rAAV-C.
  • Figure 4 shows the plasmid construction method for packaging recombinant adeno-associated viruses AAV-T1 and rAAV-T2.
  • Figure 5 shows the plasmid construction method for packaging recombinant adeno-associated virus rAAV-C.
  • Figure 6 shows a schematic diagram and results of injecting rAAV-T1 and rAAV-C, and rAAV-T2 and rAAV-C, respectively, with rAAV-Cre expressing Cre enzyme, into the LH brain region of C57BL/6 mice. Scale bars: 250 ⁇ m.
  • Example 1 Design and preparation of recombinant adeno-associated virus rAAV-T and rAAV-C vectors
  • T1 pAAV-CAG-NLS-eGFP-JT15-lox2272/71-WPRE-polyA, nucleotide sequence as shown in SEQ ID NO.1, plasmid map as shown in Figure 1, promoter is CAG, reporter gene is NLS-eGFP, regulatory sequence is WPRE-polyA, recombinase recognition sites I and II are JT15 and lox2272/71, respectively.
  • T2 pAAV-lox2272/71-WPRE-polyA-CAG-NLS-eGFP-JT15, nucleotide sequence as shown in SEQ ID NO.2, plasmid map as shown in Figure 2, promoter is CAG, reporter gene is NLS-eGFP, regulatory sequence is WPRE-polyA, recombinase recognition sites I and II are JT15 and lox2272/71, respectively;
  • Recombinant adeno-associated viruses rAAV-T1, rAAV-T2, and rAAV-C were prepared using a three-plasmid packaging system.
  • HEK-293T cells were co-transfected with the T1, T2, and C core plasmids and the helper plasmid pAd-Helper and the AAV capsid plasmid pAAV-RC2/9, respectively, at the same number of plasmid molecules.
  • Cell cultures were collected 72 hours after transfection, concentrated and purified using iodixanol density gradient centrifugation, and rAAV titers were detected using SYBR Green qPCR. Three rAAV strains were ultimately obtained.
  • rAAV-T1 rAAV-CAG-NLS-eGFP-JT15-lox2272/71-WPRE-polyA, with a titer of 2.8 ⁇ 1013 VG/mL.
  • rAAV-T2 rAAV-lox2272/71-WPRE-polyA-CAG-NLS-eGFP-JT15, with a titer of 1 ⁇ 1013 VG/mL.
  • rAAV-C rAAV-JTZ17-H2B-tdTomato-lox66/2272, with a titer of 3.2 ⁇ 1013 VG/mL.
  • Example 2 Combination of recombinant adeno-associated virus rAAV-T and rAAV-C to express the target gene
  • Figure 6 illustrates the combined use of recombinant adeno-associated viruses rAAV-T1, rAAV-T2, and rAAV-C.
  • Figure 6A shows rAAV-T1 + rAAV-C
  • Figure 6C shows rAAV-T2 + rAAV-C.
  • rAAV and rAAV-Cre expressing Cre enzyme purchased from Shenzhen Brinkes Biotechnology Co., Ltd.
  • rAAV-T immediately expressed the NLS-eGFP fusion protein upon entering the cells.
  • mouse brains were harvested via cardiac perfusion. After fixation with paraformaldehyde and dehydration with sucrose solution, the brains were cut into 40 ⁇ m thick sections using a cryostat for imaging.

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Abstract

本发明公开一种新型重组腺相关病毒双载体系统，其中包括重组腺相关病毒载体rAAV-T与重组腺相关病毒载体rAAV-C，其中rAAV-T基因组包含两个不同的重组酶识别序列I和II，还包含辅助功能序列；rAAV-C基因组包含重组酶识别序列I'和II'，还包含目的基因序列。通过改变重组酶识别位点I和II在rAAV-T上的位置，rAAV-T和rAAV-C在重组酶作用下可以形成线性或环形的表达盒结构。本发明将目的基因与其他功能序列完全分离在两个rAAV上，可以方便灵活改造与组合使用，扩大了目的基因和其他功能元件的装载容量。

Description

一种新型重组腺相关病毒双载体系统 技术领域
本发明属于生物技术领域，具体涉及一种新型重组腺相关病毒双载体系统。
背景技术
重组腺相关病毒(recombinant adeno-associated virus,rAAV)是一种应用广泛的工具病毒，也是基因治疗领域最有前景的病毒载体之一，通过改造rAAV的衣壳、基因组和开发新的给药途径，可以丰富不同层面上的rAAV应用方式，帮助研究人员解决实际问题。常用的rAAV衣壳能包装的基因组最大容量约为4.7kb，当使用单个rAAV表达目的基因时，往往需要在目的基因长度与启动子、增强子、转录后调控元件、报告基因的长度之间做出平衡，以匹配衣壳容量。为了扩大rAAV的装载量，研究人员已经开发了同源重组法、mRNA反式剪接法、内含肽法等利用两个rAAV组合表达目的基因的方法。上述方法主要用于表达大片段目的基因，需针对目的基因进行拆分设计，且有的方法需要增加额外的辅助序列，留给启动子、增强子、转录后调控元件和报告基因的选择空间较少。然而，随着研究人员对各组织器官结构功能解析的深入，细分细胞亚型、解析精细的结构功能的需求增加，在更大的长度范围上筛选更特异的功能序列也变得尤其重要。因此，有必要开发出一种更加便于设计和改进，且可以同时扩大各种辅助元件和目的基因装载容量的rAAV表达载体。
已有的双rAAV组合表达目的基因的方法利用了多种细胞内外机制：同源重组法在转录前层面，利用细胞自身同源重组机制，结果是将两个rAAV基因组连接成一段长线性DNA表达盒；mRNA反式剪接法在转录后层面，利用内含子剪接机制，结果是将两个mRNA前体连接成为完整线性mRNA；内含肽法在翻译后层面，利用自剪接肽段，结果是连接两部分肽链形成完整蛋白。在不同层面可以利用不同的机制或开发新的辅助方法，以产生不同结构的表达盒和表达中间产物。鉴于转录前基因层面是最基础、易于操控的层面，尝试在转录前DNA上利用外源蛋白辅助产生不同结构的表达盒，是一个值得尝试的方向。
发明内容
为了解决现有技术中的不足，本发明旨在提供一种新型重组腺相关病毒双载体系统，其将目的基因与调控序列分别构建在两个重组腺相关病毒上，并通过重组酶介导两个rAAV组合成完整的线性或环形表达盒以表达外源基因的方法，该方法一方面更加方便灵活且可用于装载大片段基因与调控序列，更重要的是使用了新的双rAAV重组方式，可用于形成特殊的表达盒结构。
本发明的具体技术方案如下：
本发明提供一种新型重组腺相关病毒双载体系统，其中包括重组腺相关病毒载体rAAV-T与重组腺相关病毒载体rAAV-C；所述rAAV-T基因组包含重组酶识别序列I，重组酶识别序列II，启动子序列，调控序列和报告基因序列，所述重组酶识别序列I和重组酶识别序列II不相同；所述rAAV-C基因组包含重组酶识别序列I’，重组酶识别序列II’和目的基因序列，重组酶识别序列I’和重组酶识别序列II’分别位于目的基因序列的上下游；所述重组酶识别序列I和重组酶识别序列I’为重组酶的识别序列，在重组酶介导下发生重组；所述重组酶识别序列II和重组酶识别序列II’为同一种所述重组酶的另一对识别序列，在重组酶介导下发生重组；所述重组酶识别序列仅I和I’、II和II’可以互相发生反应，且反应不可逆，任意其他组合均不可发生反应；重组酶识别序列I和I’以及重组酶识别序列II和II’发生反应后，rAAV-T上的功能序列与rAAV-C上的目的基因形成正确的表达盒结构从而表达目的基因。
进一步地，所述重组酶为酪氨酸重组酶Cre，所述重组酶识别序列I与I’的组合包括但不限于lox66与lox71、JT15与JTZ17、lox2272/71与lox66/2272、lox5171/71与lox66/5171；
进一步地，所述重组酶识别序列II与II’的组合选自lox66与lox71、JT15与JTZ17、lox2272/71与lox66/2272、lox5171/71与lox66/5171中的一种。
进一步地，所述启动子序列选自CAG、CaMKIIα、CAR、CBA、CD68、c-fos、ChAT、CMV、CR、Ef1α、E-SARE、GAD67、GFAP、GFAP104、gfaABC1D、Grm6、hGRK1、hSyn、hUbC、LP1B、L7/Pcp2、MBP、MCK、mDlX、mOXT、mTH、nEF、Nestin、NPY、Nrl、PGK、PV、RAM、RK、ROH、RPE65、SFRP2、SST、TBG、TCAP、TH、Thy1、TPH2、TRE、TRPV1、UAS和Vgat中的一种或以上。
进一步地，所述目的基因序列选自用于研究组织器官结构与功能、过表达基因产物、操控神经系统、基因治疗相关的蛋白的核苷酸编码序列和/或功能RNA产物的基因等；所述蛋白优选为荧光蛋白、激活神经元蛋白、抑制神经元蛋白、钙离子信号探针蛋白、小分子信号探针蛋白、介导凋亡蛋白、疾病相关突变蛋白、生理条件下的正常蛋白、细胞因子、抗病毒因子、病毒感染辅助受体、重组酶和基因编辑工具蛋白中一种或多种；所述功能性RNA选自小RNA、小干扰RNA、小发卡RNA、小向导RNA、细胞器定位RNA和用于RNA测序或原位杂交分析的Barcode RNA中的一种或多种。
进一步地，所述调控序列包括转录和/或转录后调控序列；
优选地，所述调控序列选自WPRE、oPRE、cw3sl、SV40 polyA、hGH polyA、bGH polyA、rbGlob polyA等中的一或多种。
进一步地，所述报告基因序列包括标记蛋白、酶反应显色蛋白、其他用于指示的分子标签序列。
进一步地，所述重组腺相关病毒载体rAAV-T与重组腺相关病毒载体rAAV-C具有相同或不同的血清型，且具有感染组织或器官中同一细胞的能力；
进一步地，所述血清型包括AAV1、AAV2、AAV2-Retro、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV9-Retro、AAV10、AAV11、AAV12、AAV13、AAV-DJ、AAV-PHP.eB、AAV-myo及其衍生血清型。
进一步地，所述rAAV-T基因组还包含其他辅助功能序列
进一步地，rAAV-T上的功能序列与rAAV-C上的目的基因形成的正确的表达盒结构为线性表达盒结构或环形表达盒结构；
优选地，通过如下方式(1)或(2)改变重组酶识别位点I和II在rAAV-T上的位置，实现rAAV-T和rAAV-C在重组酶作用下形成不同的表达盒结构：
(1)rAAV-T和rAAV-C在重组酶作用下形成线性表达盒结构
设计rAAV-T基因组包含元件的顺序为：ITR-启动子-报告基因-重组酶识别序列I-重组酶识别序列II-调控序列-ITR；
设计rAAV-C基因组包含元件的顺序为：ITR-重组酶识别序列I’-目的基因-重组酶识别序列II’-ITR；
重组酶可介导I与I’、II与II’重组，且I与I’、II与II’的序列方向满足重组后可形成线性的表达盒结构：(上接ITR)-启动子-报告基因-目的基因-调控序列-(下接ITR)。
(2)rAAV-T和rAAV-C在重组酶作用下形成环形表达盒结构
设计rAAV-T基因组包含元件的顺序为：ITR-重组酶识别序列II-调控序列-启动子-报告基因-重组酶识别序列I-ITR；
设计rAAV-C基因组包含元件的顺序为：ITR-重组酶识别序列I’-目的基因-重组酶识别序列II’-ITR；
重组酶可介导I与I’、II与II’重组，且I与I’、II与II’的序列方向满足重组后可形成环形的表达盒结构：(上接调控序列)-启动子-报告基因-目的基因-调控序列-(下接启动子)。
本发明还提供一种用于包装所述的新型重组腺相关病毒双载体系统的质粒，其特征在于，所述质粒包括用于包装重组腺相关病毒载体rAAV-T的质粒T与用于包装重组腺相关病毒载体rAAV-C的质粒C；所述质粒T包含重组酶识别序列I，重组酶识别序列II，启动子序列，调控序列和报告基因序列，所述重组酶识别序列I和重组酶识别序列II不相同；所述质粒C包含重组酶识别序列I’，重组酶识别序列II’和目的基因序列，重组酶识别序列I’和重组酶识别序列II’分别位于目的基因序列的上下游；所述重组酶识别序列I和重组酶识别序列I’为重组酶的识别序列，在重组酶介导下发生重组；所述重组酶识别序列II和重组酶识别序列II’为同一种所述重组酶的另一对识别序列，在重组酶介导下发生重组；所述重组酶识别序列仅I和I’、II和II’可以互相发生反应，且反应不可逆，任意其他组合均不可发生反应；重组酶识别序列I和I’以及重组酶识别序列II和II’发生反应后，rAAV-T上的功能序列与rAAV-C上的目的基因形成正确的表达盒结构从而表达目的基因。
进一步地，所述的质粒中，所述重组酶识别序列对应的重组酶为酪氨酸重组酶Cre，所述重组酶识别序列I与I’的组合选自lox66与lox71、JT15与JTZ17、lox2272/71与lox66/2272、lox5171/71与lox66/5171中的一种；
所述重组酶识别序列II与II’的组合选自lox66与lox71、JT15与JTZ17、lox2272/71与lox66/2272、lox5171/71与lox66/5171中的一种。
进一步地，所述的质粒中，所述用于包装重组腺相关病毒载体rAAV-T的质粒T包含元件的顺序为：ITR-启动子-报告基因-重组酶识别序列I-重组酶识别序列II-调控序列-ITR；所述用于包装重组腺相关病毒载体rAAV-C的质粒C包含元件的顺序为：ITR-重组酶识别序列I’-目的基因-重组酶识别序列II’-ITR；
或者，所述用于包装重组腺相关病毒载体rAAV-T的质粒T包含元件的顺序为：ITR-重组酶识别序列II-调控序列-启动子-报告基因-重组酶识别序列I-ITR；所述用于包装重组腺相关病毒载体rAAV-C的质粒C包含元件的顺序为：ITR-重组酶识别序列I’-目的基因-重组酶识别序列II’-ITR。
本发明还提供所述的新型重组腺相关病毒双载体系统在特定的细胞或动物组织器官中表达目的基因的方法，所述方法包括：在特定的细胞或动物组织器官中同时注射rAAV-T与rAAV-C，并将重组酶通过转基因或载体导入细胞或动物，rAAV-T与rAAV-C在重组酶介导下形成完整线性或环形表达盒以表达目的基因。
进一步地，重组酶通过载体导入细胞或动物中载体选自携带重组酶基因的表达质粒、携带重组酶基因的病毒载体、其他导入重组酶基因或蛋白的化学或生物载体中的一种或多种。
进一步地，所述组织选自神经组织、肌肉、上皮组织或结缔组织。
优选地，神经组织选自中枢神经系统及其神经环路相关组织。
进一步地，所述器官选自膀胱、眼、耳、鼻、口腔、舌、咽、喉、犁鼻器、唾液腺、肝、肾、脾、心、肠道、胃、胰腺、肺、气管、血管、淋巴管、淋巴结、四肢、垂体、甲状腺、甲状旁腺、胰岛、肾上腺或生殖器。
本发明中rAAV-T与rAAV-C在常规的应用场景下可以用于生理机制研究、疾病造模、细胞调控或神经环路标记等具体的研究。本发明特殊的应用场景包括长启动子筛选、增强子筛选、转录后调控序列筛选、环状DNA表达盒递送、特殊环路标记等。
本发明提供一种将目的基因与调控序列分别构建在两个重组腺相关病毒上，并通过重组酶介导两个rAAV形成不同结构的完整表达盒以表达外源基因的方法。不同于已有的双rAAV表达方法，本发明中的两个rAAV上分别只有目的基因或调控、报告该目的基因表达的辅助功能序列，此外还含有两对介导两个rAAV之间重组的重组酶识别位点，重组酶存在时，两个rAAV可以重组为完整表达盒，且表达盒可以形成线性或环形结构。与现有方法相比，本发明具有以下优点：
1.本发明将目的基因与其他功能序列完全分离在两个rAAV上，可以方便灵活改造与组合使用，且分别增加了可选择的基因与功能序列长度范围，扩大了目的基因和其他功能元件的装载容量。
2.本发明中的rAAV可以组合产生不同的DNA表达盒结构，可用于探究和比较线性和环形DNA结构的表达差异。
3.本发明利用外源酶介导重组，效率高于细胞内机制，适用于需要快速表达目的基因的研究如疾病造模、环路标记、基因治疗等，并能够缩短实验周期。
附图说明
图1为用于包装重组腺相关病毒AAV-T1的质粒图谱。
图2为用于包装重组腺相关病毒rAAV-T2的质粒图谱。
图3为用于包装重组腺相关病毒rAAV-C的质粒图谱。
图4为用于包装重组腺相关病毒AAV-T1、rAAV-T2的质粒构建方法。
图5为用于包装重组腺相关病毒rAAV-C的质粒构建方法。
图6为rAAV-T1与rAAV-C、rAAV-T2与rAAV-C分别与表达Cre酶的rAAV-Cre混合，并注射至C57BL/6品系小鼠的LH脑区的模式图及结果展示图。Scale bars:250μm。
具体实施方式
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。本发明所述技术方案，如未特别说明，均为rAAV载体领域的常规技术。
实施例1：重组腺相关病毒rAAV-T与rAAV-C载体的设计与制备
构建用于包装重组腺相关病毒rAAV-T1、rAAV-T2、rAAV-C的质粒T1与T2与C：
T1：pAAV-CAG-NLS-eGFP-JT15-lox2272/71-WPRE-polyA，核苷酸序列如SEQ ID NO.1所示，质粒图谱如图1，启动子为CAG，报告基因为NLS-eGFP，调控序列为WPRE-polyA，重组酶识别位点I和II分别为JT15和lox2272/71。
T2：pAAV-lox2272/71-WPRE-polyA-CAG-NLS-eGFP-JT15，核苷酸序列如SEQ ID NO.2所示，质粒图谱如图2，启动子为CAG，报告基因为NLS-eGFP，调控序列为WPRE-polyA，重组酶识别位点I和II分别为JT15和lox2272/71；
C：pAAV-JTZ17-H2B-tdTomato-lox66/2272，核苷酸序列如SEQ ID NO.3所示，质粒图谱如图3，目的基因为H2B-tdTomato，重组酶识别位点I’和II’分别为JTZ17和lox66/2272；
本发明中的各质粒的构建流程如图4-5所示。
采用三质粒包装系统制备重组腺相关病毒rAAV-T1、rAAV-T2与rAAV-C。分别将T1、T2、C核心质粒与辅助质粒pAd-Helper、AAV衣壳质粒pAAV-RC2/9三种质粒按相同质粒分子数共转染HEK-293T细胞。转染72小时后收集细胞培养物，用碘克沙醇密度梯度离心法进行浓缩和纯化，最后用SYBR Green qPCR法检测rAAV滴度，最终获得三种rAAV：
rAAV-T1：rAAV-CAG-NLS-eGFP-JT15-lox2272/71-WPRE-polyA，滴度为2.8×1013VG/mL。
rAAV-T2：rAAV-lox2272/71-WPRE-polyA-CAG-NLS-eGFP-JT15，滴度为1×1013VG/mL。
rAAV-C：rAAV-JTZ17-H2B-tdTomato-lox66/2272，滴度为3.2×1013VG/mL。
实施例2：重组腺相关病毒rAAV-T与rAAV-C组合使用以表达目的基因
图6为重组腺相关病毒rAAV-T1、rAAV-T2与rAAV-C的组合使用的方式示意图。图6A为rAAV-T1+rAAV-C，图6C为rAAV-T2+rAAV-C。
针对每一组病毒，将两种rAAV与表达Cre酶的rAAV-Cre(购买自深圳布林凯斯生物技术有限公司)按毒粒数10:10:1混合后(总滴度为2.15×1013VG/mL)，通过脑立体定位注射法注射200nL病毒混合溶液于野生型C57/BL6小鼠的LH脑区。rAAV-T进入细胞后即可正常表达NLS-eGFP融合蛋白。rAAV-T与rAAV-C组合在Cre的作用下重组，从而表达H2B-tdTomato融合蛋白。
三周后通过心脏灌流取小鼠脑，经过多聚甲醛固定、蔗糖溶液脱水后，使用冷冻切片机切成40μm厚度的切片用于成像。
活体检测结果如图6B、6D所示。针对rAAV-T1与rAAV-C的组合，在LH脑区有绿色核定位eGFP信号表达，证明该处有rAAV-T1感染细胞并表达，同时部分含有eGFP的细胞同时含有红色核定位tdTomato信号，证明rAAV-C在该细胞中与rAAVT1发生重组并表达目的基因tdTomato，不存在单独表达tdTomato但不表达eGFP的细胞，证明tdTomato不会单独表达，具有严谨性。rAAV-T2与rAAV-C的组合标记结果近似rAAV-T1与rAAV-C，值得注意的是使用rAAV-T2时tdTomato信号略强，可能是由于rAAV-T2结构更利于rAAV-C发生重组，或重组形成的环形表达盒更稳定所致。
以上结果表明，作为示例的rAAV-T和rAAV-C可以在重组酶的作用下发生重组反应并且表达目的基因，且具有高效、表达严谨的特点。因此，利用两对loxP重组rAAV-T和rAAV-C为不同结构的表达盒并表达目的基因是一种可行的策略。
SEQ ID NO.1



SEQ ID NO.2



SEQ ID NO.3


显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (15)

  1. 一种新型重组腺相关病毒双载体系统，其特征在于，所述双载体系统包括重组腺相关病毒载体rAAV-T与重组腺相关病毒载体rAAV-C；所述rAAV-T基因组包含重组酶识别序列I，重组酶识别序列II，启动子序列，调控序列和报告基因序列，所述重组酶识别序列I和重组酶识别序列II不相同；所述rAAV-C基因组包含重组酶识别序列I’，重组酶识别序列II’和目的基因序列，重组酶识别序列I’和重组酶识别序列II’分别位于目的基因序列的上下游；所述重组酶识别序列I和重组酶识别序列I’为重组酶的识别序列，在重组酶介导下发生重组；所述重组酶识别序列II和重组酶识别序列II’为同一种所述重组酶的另一对识别序列，在重组酶介导下发生重组；所述重组酶识别序列仅I和I’、II和II’可以互相发生反应，且反应不可逆，任意其他组合均不可发生反应；重组酶识别序列I和I’以及重组酶识别序列II和II’发生反应后，rAAV-T上的功能序列与rAAV-C上的目的基因形成正确的表达盒结构从而表达目的基因。
  2. 根据权利要求1所述的双载体系统，其特征在于，所述重组酶识别序列对应的重组酶为酪氨酸重组酶Cre，所述重组酶识别序列I与I’的组合选自lox66与lox71、JT15与JTZ17、lox2272/71与lox66/2272、lox5171/71与lox66/5171中的一种；
    所述重组酶识别序列II与II’的组合选自lox66与lox71、JT15与JTZ17、lox2272/71与lox66/2272、lox5171/71与lox66/5171中的一种。
  3. 根据权利要求1所述的双载体系统，其特征在于，rAAV-T上的功能序列与rAAV-C上的目的基因形成的正确的表达盒结构为线性表达盒结构或环形表达盒结构。
  4. 根据权利要求3所述的双载体系统，其特征在于，通过如下方式(1)或(2)改变重组酶识别位点I和II在rAAV-T上的位置，实现rAAV-T和rAAV-C在重组酶作用下形成不同的表达盒结构：
    (1)rAAV-T和rAAV-C在重组酶作用下形成线性表达盒结构
    设计rAAV-T基因组包含元件的顺序为：ITR-启动子-报告基因-重组酶识别序列I-重组酶识别序列II-调控序列-ITR；
    设计rAAV-C基因组包含元件的顺序为：ITR-重组酶识别序列I’-目的基因-重组酶识别序列II’-ITR；
    重组酶可介导I与I’、II与II’重组，且I与I’、II与II’的序列方向满足重组后形成线性的表达盒结构：启动子-报告基因-目的基因-调控序列；
    (2)rAAV-T和rAAV-C在重组酶作用下形成环形表达盒结构
    设计rAAV-T基因组包含元件的顺序为：ITR-重组酶识别序列II-调控序列-启动子-报告基因-重组酶识别序列I-ITR；
    设计rAAV-C基因组包含元件的顺序为：ITR-重组酶识别序列I’-目的基因-重组酶识别序列II’-ITR；
    重组酶可介导I与I’、II与II’重组，且I与I’、II与II’的序列方向满足重组后形成环形的表达盒结构：(上接调控序列)-启动子-报告基因-目的基因-调控序列-(下接启动子)。
  5. 根据权利要求1所述的双载体系统，其特征在于，所述启动子序列选自CAG、CaMKIIα、CAR、CBA、CD68、c-fos、ChAT、CMV、CR、Ef1α、E-SARE、GAD67、GFAP、GFAP104、gfaABC1D、Grm6、hGRK1、hSyn、hUbC、LP1B、L7/Pcp2、MBP、MCK、mDlX、mOXT、mTH、nEF、Nestin、NPY、Nrl、PGK、PV、RAM、RK、ROH、RPE65、SFRP2、SST、TBG、TCAP、TH、Thy1、TPH2、TRE、TRPV1、UAS和Vgat中的一种或以上。
  6. 根据权利要求1所述的双载体系统，其特征在于，所述调控序列包括转录和/或转录后调控序列。
  7. 根据权利要求1所述的双载体系统，其特征在于，所述报告基因序列选自标记蛋白、酶反应显色蛋白、其他用于指示的分子标签序列中的一种或多种。
  8. 根据权利要求1所述的双载体系统，其特征在于，所述rAAV-T基因组还包含其他辅助功能序列。
  9. 根据权利要求1所述的双载体系统，其特征在于，所述重组腺相关病毒载体rAAV-T与重组腺相关病毒载体rAAV-C具有相同或不同的血清型，且具有感染组织或器官中同一细胞的能力。
  10. 根据权利要求9所述的双载体系统，其特征在于，所述血清型包括AAV1、AAV2、AAV2-Retro、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV9-Retro、AAV10、AAV11、AAV12、AAV13、AAV-DJ、AAV-PHP.eB、AAV-myo及其衍生血清型。
  11. 一种用于包装权利要求1所述的新型重组腺相关病毒双载体系统的质粒，其特征在于，所述质粒包括用于包装重组腺相关病毒载体rAAV-T的质粒T与用于包装重组腺相关病毒载体rAAV-C的质粒C；所述质粒T包含重组酶识别序列I，重组酶识别序列II，启动子序列，调控序列和报告基因序列，所述重组酶识别序列I和重组酶识别序列II不相同；所述质粒C包含重组酶识别序列I’，重组酶识别序列II’和目的基因序列，重组酶识别序列I’和重组酶识别序列II’分别位于目的基因序列的上下游；所述重组酶识别序列I和重组酶识别序列I’为重组酶的识别序列，在重组酶介导下发生重组；所述重组酶识别序列II和重组酶识别序列II’为同一种所述重组酶的另一对识别序列，在重组酶介导下发生重组；所述重组酶识别序列仅I和I’、II和II’可以互相发生反应，且反应不可逆，任意其他组合均不可发生反应；重组酶识别序列I和I’以及重组酶识别序列II和II’发生反应后，rAAV-T上的功能序列与rAAV-C上的目的基因形成正确的表达盒结构从而表达目的基因。
  12. 根据权利要求11所述的质粒，其特征在于，所述重组酶识别序列对应的重组酶为酪氨酸重组酶Cre，所述重组酶识别序列I与I’的组合选自lox66与lox71、JT15与JTZ17、lox2272/71与lox66/2272、lox5171/71与lox66/5171中的一种；
    所述重组酶识别序列II与II’的组合选自lox66与lox71、JT15与JTZ17、lox2272/71与lox66/2272、lox5171/71与lox66/5171中的一种。
  13. 根据权利要求11所述的质粒，其特征在于，所述用于包装重组腺相关病毒载体rAAV-T的质粒T包含元件的顺序为：ITR-启动子-报告基因-重组酶识别序列I-重组酶识别序列II-调控序列-ITR；所述用于包装重组腺相关病毒载体rAAV-C的质粒C包含元件的顺序为：ITR-重组酶识别序列I’-目的基因-重组酶识别序列II’-ITR；
    或者，所述用于包装重组腺相关病毒载体rAAV-T的质粒T包含元件的顺序为：ITR-重组酶识别序列II-调控序列-启动子-报告基因-重组酶识别序列I-ITR；所述用于包装重组腺相关病毒载体rAAV-C的质粒C包含元件的顺序为：ITR-重组酶识别序列I’-目的基因-重组酶识别序列II’-ITR。
  14. 权利要求1所述的新型重组腺相关病毒双载体系统在特定的细胞或动物组织器官中表达目的基因的方法，其特征在于，所述方法包括：在特定的细胞或动物组织器官中同时注射rAAV-T与rAAV-C，并将重组酶通过转基因或载体导入细胞或动物，rAAV-T与rAAV-C在重组酶介导下形成完整表达盒以表达目的基因。
  15. 根据权利要求14所述的方法，其特征在于，重组酶通过载体导入细胞或动物中载体选自携带重组酶基因的表达质粒、携带重组酶基因的病毒载体、其他导入重组酶基因或蛋白的化学或生物载体中的一种或多种。
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