WO2019228117A1 - 慢病毒载体及其递送外源rna的方法 - Google Patents

慢病毒载体及其递送外源rna的方法 Download PDF

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WO2019228117A1
WO2019228117A1 PCT/CN2019/084879 CN2019084879W WO2019228117A1 WO 2019228117 A1 WO2019228117 A1 WO 2019228117A1 CN 2019084879 W CN2019084879 W CN 2019084879W WO 2019228117 A1 WO2019228117 A1 WO 2019228117A1
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rna
protein
lentiviral vector
binding protein
plasmid
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蔡宇伽
凌思凯
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上海交通大学
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Publication of WO2019228117A1 publication Critical patent/WO2019228117A1/zh

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  • the invention belongs to the field of biotechnology, and particularly relates to a lentiviral vector and a method for delivering foreign RNA.
  • Lentiviral vectors are viral vectors that have been modified from HIV-1 and have lost their ability to replicate. It can efficiently infect cells and is a common carrier for biological research and gene therapy. At present, lentiviral vectors can be divided into first generation, second generation, and third generation. The higher the generation number, the better the security.
  • RNA is a linear long-chain molecule formed by ribonucleotides through phosphodiester bonds. RNA can be divided into coding and non-coding RNA. Encoding RNA functions by encoding proteins; non-coding RNA does not encode proteins and can directly perform biological functions.
  • RNA has important application potential in the fields of vaccines, gene therapy, gene editing, and cell reprogramming. However, its application is limited by the following factors: (1) RNA is poorly stable and is easily degraded by nucleases in the environment; (2) RNA cannot enter cells by itself and requires an effective vector system; (3) It is difficult to use RNA delivery technology directly in vivo.
  • RNA delivery solutions include electroporation, nanoparticles made of chemical materials, Sendai virus, and lentiviral particles modified from second-generation lentiviral vectors.
  • the second-generation lentiviral vectors are modified from lentiviral particles (Prel, A., et.al., Highly efficient, efficient, and effective in terms of functional RNAs) using new 2-versatile MS2-chimeric retrovirus-like particles.
  • the purpose of the present invention is to overcome the above-mentioned problems in the prior art, and to provide a lentiviral vector and a method for delivering foreign RNA.
  • the present invention addresses the problem of RNA delivery into cells, including in vitro and in vivo delivery of RNA.
  • the invention can deliver Cas9 mRNA and gRNA for gene editing and gene therapy; can deliver tumor or viral antigen mRNA for immunotherapy; can deliver cell reprogramming factor mRNA to make pluripotent stem cells and modify cell functions; can deliver chimeric antigen receptors Somatic mRNA is used in cellular immunotherapy.
  • the present invention packs the target RNA into a lentiviral vector, and uses the lentivirus to protect and deliver the RNA.
  • the principle of the invention is: using the interaction between the RNA binding protein and the RNA stem-loop structure it recognizes, packaging the foreign target RNA carrying the recognizable RNA sequence into a lentiviral particle.
  • the core features of the present invention are: integrating the RNA-binding protein to the N-terminus of the third-generation lentivirus GagPol long-chain protein; and placing the stem-loop structure recognized by the RNA-binding protein into the foreign target RNA expression frame.
  • the object of the present invention is achieved by the following technical solutions:
  • the present invention relates to a lentiviral vector obtained by transfecting a plasmid containing a genomic sequence of a lentiviral vector into a virus-producing cell; collecting a supernatant, concentrating, and preparing the same;
  • the genomic sequence of the lentiviral vector is located on a plasmid expressing a membrane protein, a plasmid expressing a lentivirus GagPol long chain protein containing an RNA binding protein, and a plasmid containing an RNA stem-loop structure recognized by the RNA binding protein, respectively.
  • the virus-producing cells include 293T, 293FT, and HEK293.
  • the membrane protein-expressing plasmid includes membrane proteins modified by VSV-G, CD4 recognition protein, CD8 recognition protein, RD114, and baboon endogenous retrovirus membrane protein.
  • the plasmid expressing the RNA-binding protein-containing lentivirus GagPol long-chain protein integrates the RNA-binding protein to the N-terminus of the third-generation lentivirus GagPol long-chain protein.
  • the codon sequence of the GagPol long chain protein is shown in SEQ ID NO.1.
  • the RNA binding protein is an MS2 capsid protein; the codon sequence of the MS2 capsid protein is shown in SEQ ID NO.2 .
  • the RNA binding protein is MS2 capsid protein; the RNA sequence recognized by the MS2 capsid protein is shown in SEQ ID NO.3 .
  • the concentration is selected by high-speed centrifugation or HPLC.
  • the present invention also relates to a method for delivering foreign RNA of interest using the lentiviral vector of the present invention.
  • the exogenous target RNA is one or more of mRNA, gRNA, and RNA with other functions.
  • the method includes the following steps:
  • the plasmid expressing the foreign target RNA with the RNA stem-loop structure recognized by the RNA binding protein is obtained by fusing the RNA sequence recognized by the RNA binding protein with the foreign target RNA sequence.
  • the plasmid expressing the foreign target RNA with the RNA stem-loop structure recognized by the RNA binding protein is obtained by fusing the RNA sequence recognized by the MS2 capsid protein and the target RNA sequence.
  • RNA sequence recognized by the MS2 capsid protein is shown in SEQ ID NO.3.
  • the concentration is selected by high-speed centrifugation or HPLC.
  • the virus-producing cells include 293T, 293FT, and HEK293.
  • the membrane protein-expressing plasmid includes membrane proteins modified by VSV-G, CD4 recognition protein, CD8 recognition protein, RD114, and baboon endogenous retrovirus membrane protein.
  • the plasmid expressing the RNA-binding protein-containing lentivirus GagPol long-chain protein is obtained by fusing the MS2 capsid protein with the lentivirus GagPol long-chain protein.
  • the codon sequence of the GagPol long chain protein is shown in SEQ ID NO.1; the codon sequence of the MS2 capsid protein is shown in SEQ ID NO.2.
  • the invention also relates to the use of a lentiviral vector of the invention for delivering Cas9 mRNA and gRNA for gene editing and gene therapy.
  • the present invention also relates to the use of a lentiviral vector of the present invention for carrying a mRNA expressing a tumor antigen and a viral antigen in a vaccine.
  • the present invention also relates to the use of the lentiviral vector of the present invention in expressing a cell reprogramming factor for manufacturing pluripotent stem cells and modifying cell functions.
  • the present invention also relates to the use of a lentiviral vector of the present invention in delivering a chimeric antigen receptor mRNA for cellular immunotherapy.
  • the present invention has the following beneficial effects:
  • HIV genes such as tat and rev are removed from the long-chain protein of GagPol, which reduces the possibility of viral genome recombination to produce HIV with replication ability during the packaging of virus particles. And greatly improve safety.
  • RNA-binding protein at the N-terminus of the long-chain protein of the lentivirus GagPol, reducing the negative effect of foreign proteins on the normal morphology of the virus particles.
  • the present invention has the ability to package and carry long-chain RNA, for example, it can carry Cas9 mRNA (about 4.2kb) and base editing enzyme (about 5.1kb), which greatly improves the application value.
  • the present invention can deliver Cas9 mRNA and gRNA for gene editing and gene therapy.
  • the present invention can carry mRNAs expressing tumor antigens and viral antigens for use in vaccines.
  • the present invention can be used for expressing cell reprogramming factors and chimeric antigen receptors, for producing pluripotent stem cells, and for modifying cell functions.
  • Figure 1 is a lentiviral vector delivery GFP mRNA experiment
  • Figure 2 shows the gene editing experiment of Cas9 mRNA delivery by lentiviral vector.
  • Step 1 Cotransfect virus-producing cells with a plasmid expressing a membrane protein, a plasmid expressing a long-chain protein GagPol containing an RNA-binding protein, and a plasmid expressing a foreign target RNA with an RNA stem-loop structure recognized by the RNA-binding protein. (293T). among them,
  • Plasmids expressing membrane proteins include membrane proteins modified by VSV-G, CD4 recognition protein, CD8 recognition protein, RD114, and baboon endogenous retrovirus membrane proteins.
  • VSV-G is selected in this embodiment.
  • Virus-producing cells include 293T, 293FT, HEK293 and so on.
  • 293T is selected.
  • the plasmid expressing the RNA-binding protein-containing lentivirus GagPol long chain protein in this example is obtained by fusing the MS2 capsid protein with the lentivirus GagPol long chain protein.
  • the codon sequence of GagPol long chain protein is as follows (SEQ ID NO.1):
  • the codons of the MS2 capsid protein are as follows (SEQ ID NO. 2), and the protein encoded by it can be linked to the GagPol long chain protein encoded by the above SEQ ID NO. 1, for example, placed at the N-terminus of the GagPol long chain protein.
  • the plasmid expressing the foreign target RNA with the RNA stem-loop structure recognized by the RNA binding protein is obtained by fusing the RNA sequence recognized by the MS2 capsid protein with the target RNA sequence.
  • RNA sequence recognized by MS2 capsid protein is as follows (SEQ ID NO. 3), which can be linked to the foreign target RNA in a single or multiple repetitions.
  • Step 2 Collect the supernatant containing virus particles and concentrate them by high-speed centrifugation or HPLC to obtain high titer lentiviral particles containing foreign target RNA.
  • Example 2 Delivery of GFP mRNA using a lentiviral vector.
  • GFP mRNA is used as the foreign target RNA.
  • GFP is a fluorescent reporter gene protein; this embodiment specifically uses a lentiviral vector to deliver GFP mRNA to 293T cells.
  • Cas9 mRNA and gRNA targeting AAVS1 site were selected as the foreign target RNA.
  • This example specifically describes the delivery of Cas9 mRNA and gRNA targeting the human genome AAVS1 site to 293T cells by a lentiviral vector.
  • RNA becomes more stable; meanwhile, with the ability of lentiviral particles to efficiently infect cells, the target RNA can efficiently enter cells to express gene editing enzymes, tumors or viral antigens, and cells Reprogramming factors, chimeric antigen receptors, etc. Therefore, the present invention can deliver Cas9 mRNA and gRNA for gene editing and gene therapy; can deliver tumor or viral antigen mRNA for immunotherapy; can deliver cell reprogramming factor mRNA to make pluripotent stem cells and modify cell functions; can deliver chimeric Antigen receptor mRNA is used in cellular immunotherapy.

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Abstract

本发明公开了一种慢病毒载体及其递送外源RNA的方法。具体地，利用RNA结合蛋白和其所识别的RNA序列相互结合的原理，将RNA结合蛋白整合至慢病毒GagPol长链蛋白的骨架，并将其所识别的RNA序列连接到外源目的RNA，从而使目的RNA在慢病毒颗粒的组装过程中被包装进慢病毒颗粒。外源目的RNA可以是mRNA、gRNA或者具有其它功能的RNA。本发明可用于基因编辑、基因治疗、细胞治疗、免疫治疗、再生医学以及基础生物学等领域。

Description

慢病毒载体及其递送外源RNA的方法 技术领域
本发明属于生物技术领域，具体涉及一种慢病毒载体及其递送外源RNA的方法。
背景技术
慢病毒载体是从HIV-1改造而来的、失去自我复制能力的病毒载体。可以高效感染细胞，是生物学研究和基因治疗常用的载体。目前，慢病毒载体可以分为第一代、第二代和第三代，代数越高，安全性越好。
RNA由核糖核苷酸通过磷酸二酯键形成的线性长链分子。RNA可以分为编码和非编码RNA。编码RNA通过编码蛋白质发挥功能；非编码RNA不编码蛋白质，可以直接发挥生物学功能。
RNA在疫苗、基因治疗、基因编辑、细胞重编程等领域具有重要的应用潜力。但其应用受到以下几个因素的限制，(1)RNA稳定较差，极易被环境中的核酸酶降解；(2)RNA自身无法进入细胞，需要一套有效的载体系统；(3)现有RNA递送技术很难直接用于体内。
目前，RNA的递送方案有电转、化学材料形成的纳米颗粒、仙台病毒以及第二代慢病毒载体改造而来的慢病毒颗粒。其中，第二代慢病毒载体改造而来的慢病毒颗粒(Prel,A.,et al.,Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles.Mol Ther Methods Clin Dev,2015.2:p.15039.)；该方案通过(1)将MS2衣壳蛋白(RNA结合蛋白)整合至慢病毒核衣壳(nucleocapsid,NC)蛋白内部，(2)将MS2衣壳蛋白所识别的茎环结构放至目的RNA的表达框中，实现将目的RNA包装进慢病毒颗粒。然而第二代慢病毒载体保留有较多的HIV基因,文献显示第二代慢病毒载体可以在体内生成具有复制能力的HIV病毒(Skrdlant,L.M.,et al.,Detection of Replication Competent Lentivirus Using a qPCR Assay for VSV-G.Mol Ther Methods Clin Dev,2018.8:p.1-7.)。因此，出于安全性考虑，第二代慢病毒载体已经不再被用于基因治疗。
发明内容
本发明的目的在于克服上述现有技术问题，提供一种慢病毒载体及其递送外源RNA 的方法。本发明解决的是RNA往细胞内的递送问题，包括RNA的体外和体内递送。本发明可以递送Cas9 mRNA和gRNA用于基因编辑和基因治疗；可以递送肿瘤或者病毒抗原mRNA用于免疫治疗；可以递送细胞重编程因子mRNA制造多能干细胞、改造细胞功能；可以递送嵌合抗原受体mRNA用于细胞免疫治疗。
本发明将目的RNA包装进慢病毒载体，利用慢病毒保护和递送RNA。本发明的原理为：利用RNA结合蛋白与其所识别的RNA茎环结构的相互作用，将携带该种可识别的RNA序列的外源目的RNA包装进慢病毒颗粒。
本发明的核心特点是：将RNA结合蛋白整合至第三代慢病毒GagPol长链蛋白的N-端；将RNA结合蛋白所识别的茎环结构置于外源目的RNA表达框。
具体而言，本发明的目的是通过以下技术方案来实现的：
第一方面，本发明涉及一种慢病毒载体，所述慢病毒载体是通过将含有慢病毒载体基因组序列的质粒转染入病毒生产细胞；收集上清，浓缩，制备而得；
所述慢病毒载体基因组序列分别位于表达膜蛋白的质粒、表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒和含RNA结合蛋白所识别的RNA茎环结构的质粒上。
优选的，所述病毒生产细胞包括293T、293FT、HEK293。
优选的，所述表达膜蛋白的质粒包括VSV-G、CD4识别蛋白、CD8识别蛋白、RD114、狒狒内源逆转录病毒(baboon endogenous retrovirus)膜蛋白所改造来的膜蛋白。
优选的，所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒是将RNA结合蛋白整合至第三代慢病毒GagPol长链蛋白的N-端。
优选的，所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒中，GagPol长链蛋白的密码子序列如SEQ ID NO.1所示。
优选的，所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒中，所述RNA结合蛋白为MS2衣壳蛋白；所述MS2衣壳蛋白的密码子序列如SEQ ID NO.2所示。
优选的，所述含RNA结合蛋白所识别的RNA茎环结构的质粒中，所述RNA结合蛋白为MS2衣壳蛋白；所述MS2衣壳蛋白所识别的RNA序列如SEQ ID NO.3所示。
优选的，所述浓缩选用高速离心或者HPLC方法进行浓缩。
第二方面，本发明还涉及一种利用本发明的慢病毒载体递送外源目的RNA的方法。
优选的，所述外源目的RNA为mRNA、gRNA、其它功能的RNA中的一种或几种。
优选的，所述方法包括如下步骤：
S1、将所述表达膜蛋白的质粒、所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒和表达带RNA结合蛋白所识别的RNA茎环结构的外源目的RNA的质粒共转染病毒生产细胞；
S2、收集含病毒颗粒的上清，浓缩，得到含外源目的RNA的慢病毒颗粒。
优选的，所述表达带RNA结合蛋白所识别的RNA茎环结构的外源目的RNA的质粒是通过将RNA结合蛋白所识别的RNA序列与外源目的RNA序列融合而得。
更优选的，所述表达带RNA结合蛋白所识别的RNA茎环结构的外源目的RNA的质粒是通过将MS2衣壳蛋白所识别的RNA序列与目的RNA序列融合而得到。
进一步优选的，所述MS2衣壳蛋白所识别的RNA序列如SEQ ID NO.3所示。
优选的，所述浓缩选用高速离心或者HPLC方法进行浓缩。
优选的，所述病毒生产细胞包括293T、293FT、HEK293。
优选的，所述表达膜蛋白的质粒包括VSV-G、CD4识别蛋白、CD8识别蛋白、RD114、狒狒内源逆转录病毒(baboon endogenous retrovirus)膜蛋白所改造来的膜蛋白。
优选的，所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒是通过将MS2衣壳蛋白与慢病毒GagPol长链蛋白融合而得到。
更优选的，所述GagPol长链蛋白的密码子序列如SEQ ID NO.1所示；所述MS2衣壳蛋白的密码子序列如SEQ ID NO.2所示。
第三方面，本发明还涉及一种本发明的慢病毒载体在递送Cas9 mRNA和gRNA用于实现基因编辑和基因治疗中的用途。
第四方面，本发明还涉及一种本发明的慢病毒载体在携带表达肿瘤抗原和病毒抗原的mRNA用于疫苗中的用途。
第五方面，本发明还涉及一种本发明的慢病毒载体在表达细胞重编程因子用于制造多能干细胞、改造细胞功能中的用途。
第六方面，本发明还涉及一种本发明的慢病毒载体在递送嵌合抗原受体(chimeric antigen receptor)mRNA用于细胞免疫治疗中的用途。
与现有技术相比，本发明具有如下有益效果：
(1)以第三代慢病毒技术为基础，在慢病毒GagPol长链蛋白中去掉了tat和rev等HIV基因，减小了病毒颗粒包装过程中，病毒基因组重组产生具有复制能力的HIV的可能性，极大提高了安全性。
(2)优化了慢病毒GagPol长链蛋白的密码子，使其可以在人源细胞系高效表达，不再需要表达HIV蛋白REV来提高慢病毒颗粒的产量。
(3)将RNA结合蛋白置于慢病毒GagPol长链蛋白的N-端，减小了外源蛋白对病毒颗粒正常形态的负面影响。
(4)本发明具有包装和携带长链RNA的能力，比如可以携带Cas9 mRNA(约4.2kb)和碱基编辑酶(约5.1kb)等，极大提高了应用价值。
(5)本发明可以递送Cas9 mRNA和gRNA用于实现基因编辑和基因治疗。
(6)本发明可以携带表达肿瘤抗原和病毒抗原的mRNA用于疫苗。
(7)本发明可以用于表达细胞重编程因子和嵌合抗原受体，用于制造多能干细胞、改造细胞功能。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1为慢病毒载体递送GFP mRNA实验；
图2为慢病毒载体递送Cas9 mRNA实现基因编辑实验。
具体实施方式
下面结合实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干调整和改进。这些都属于本发明的保护范围。
实施例1、慢病毒颗粒的制备
步骤1：将表达膜蛋白的质粒、表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒、表达带RNA结合蛋白所识别的RNA茎环结构的外源目的RNA的质粒共转染病毒生产细胞(293T)。其中，
1)表达膜蛋白的质粒包括VSV-G、CD4识别蛋白、CD8识别蛋白、RD114、狒狒内源逆转录病毒(baboon endogenous retrovirus)膜蛋白所改造来的膜蛋白。本实施例中选用VSV-G。
2)病毒生产细胞包括293T、293FT、HEK293等。本实施例中选用293T。
3)本实施例中表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒是通过将MS2衣壳蛋白与慢病毒GagPol长链蛋白融合而得到。
GagPol长链蛋白的密码子序列如下(SEQ ID NO.1)：
Figure PCTCN2019084879-appb-000001
Figure PCTCN2019084879-appb-000002
MS2衣壳蛋白的密码子如下(SEQ ID NO.2)，其编码的蛋白可与上述SEQ ID NO.1序列编码的GagPol长链蛋白相连，比如置于该GagPol长链蛋白的N-端。
Figure PCTCN2019084879-appb-000003
4)表达带RNA结合蛋白所识别的RNA茎环结构的外源目的RNA的质粒是通过将MS2衣壳蛋白所识别的RNA序列与目的RNA序列融合而得到。
其中，MS2衣壳蛋白所识别的RNA序列如下(SEQ ID NO.3)，该序列可以以单次或者多次重复的方式与外源目的RNA链接。
Figure PCTCN2019084879-appb-000004
5)上述3)和4)所涉及的用于制备携带外源目的RNA的慢病毒颗粒所需要的质粒通过分子克隆方法得到。
步骤2：收集含病毒颗粒的上清，通过高速离心或者HPLC等方法进行浓缩，得到高滴度的含外源目的RNA的慢病毒颗粒。
实施例2、利用慢病毒载体递送GFP mRNA。
具体步骤同实施例1，外源目的RNA选用GFP mRNA。GFP为一种发荧光的报告基因蛋白；本实施例具体为慢病毒载体递送GFP mRNA至293T细胞。
由图1可知，293T细胞感染携带GFP mRNA的慢病毒颗粒48小时后，流式细胞术分析GFP阳性细胞高达99.8％。
实施例3、利用慢病毒载体递送Cas9 mRNA以及靶向AAVS1位点的gRNA
具体步骤同实施例1，外源目的RNA选用Cas9 mRNA以及靶向AAVS1位点的gRNA。本实施例具体为慢病毒载体递送Cas9 mRNA和靶向人类基因组AAVS1位点的gRNA至293T细胞。
如图2所示，293T细胞感染携带Cas9 mRNA以及靶向AAVS1位点的gRNA的慢病毒颗粒72小时后，PCR扩增AAVS1位点并测序。TIDE分析基因敲除效率达34.1％。
综上，利用本发明的方法，通过慢病毒颗粒的保护，RNA变得更加稳定；同时借助慢病毒颗粒高效感染细胞的能力，目的RNA可高效进入细胞表达基因编辑酶、肿瘤或者病毒抗原、细胞重编程因子、嵌合抗原受体等。因此，本发明可以递送Cas9 mRNA和gRNA用于基因编辑和基因治疗；可以递送肿瘤或者病毒抗原mRNA用于免疫治疗；可以递送细胞重编程因子mRNA制造多能干细胞、改造细胞功能；可以递送嵌合抗原受体mRNA用于细胞免疫治疗。
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变形或修改，这并不影响本发明的实质内容。

Claims (10)

  1. 一种慢病毒载体，其特征在于，所述慢病毒载体是通过将含有慢病毒载体基因组序列的质粒转染入病毒生产细胞；收集上清，浓缩，制备而得；
    所述慢病毒载体基因组序列分别位于表达膜蛋白的质粒、表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒和含RNA结合蛋白所识别的RNA茎环结构的质粒上。
  2. 根据权利要求1所述的慢病毒载体，其特征在于，所述病毒生产细胞包括293T、293FT、HEK293。
  3. 根据权利要求1所述的慢病毒载体，其特征在于，所述表达膜蛋白的质粒包括VSV-G、CD4CD8识别蛋白、CD8识别蛋白、RD114、狒狒内源逆转录病毒膜蛋白所改造来的膜蛋白。
  4. 根据权利要求1所述的慢病毒载体，其特征在于，所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒是将RNA结合蛋白整合至第三代慢病毒GagPol长链蛋白的N-端。
  5. 根据权利要求1或4所述的慢病毒载体，其特征在于，所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒中，GagPol长链蛋白的密码子序列如SEQ ID NO.1所示。
  6. 根据权利要求5所述的慢病毒载体，其特征在于，所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒中，所述RNA结合蛋白为MS2衣壳蛋白；所述MS2衣壳蛋白的密码子序列如SEQ ID NO.2所示。
  7. 根据权利要求6所述的慢病毒载体，其特征在于，所述含RNA结合蛋白所识别的RNA茎环结构的质粒中，RNA结合蛋白所识别的RNA序列如SEQ ID NO.3所示。
  8. 一种利用如权利要求1～7中任一项所述的慢病毒载体递送外源目的RNA的方法。
  9. 根据权利要求8所述的利用慢病毒载体递送外源目的RNA的方法，其特征在于，所述方法包括如下步骤：
    S1、将所述表达膜蛋白的质粒、所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒和表达带RNA结合蛋白所识别的RNA茎环结构的外源目的RNA的质粒共转染病毒生产细胞；
    S2、收集含病毒颗粒的上清，浓缩，得到含外源目的RNA的慢病毒颗粒。
  10. 一种如权利要求1～7中任一项所述的慢病毒载体在递送Cas9 mRNA和gRNA用于实现基因编辑和基因治疗，或是在携带表达肿瘤抗原和病毒抗原的mRNA用于疫苗，或是在表达细胞重编程因子用于制造多能干细胞、改造细胞功能，或是在递送嵌合抗原受体mRNA用于细胞免疫治疗中的用途。
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