WO2024254903A1 - 一种体外制备的稳定的rna分子及其在体内原位car治疗产品制备中的应用 - Google Patents

一种体外制备的稳定的rna分子及其在体内原位car治疗产品制备中的应用 Download PDF

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WO2024254903A1
WO2024254903A1 PCT/CN2023/102811 CN2023102811W WO2024254903A1 WO 2024254903 A1 WO2024254903 A1 WO 2024254903A1 CN 2023102811 W CN2023102811 W CN 2023102811W WO 2024254903 A1 WO2024254903 A1 WO 2024254903A1
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virus
rna
sequence
rna molecule
mrna
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璩良
尹洁
王君华
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Fudan University
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Definitions

  • the present invention relates to a stable RNA molecule prepared in vitro and its application in the preparation of an in vivo in situ CAR therapeutic product, belonging to the technical field of biomedicine.
  • CAR Chimeric antigen receptor
  • Polycistronic refers to an mRNA molecule that can encode multiple polypeptide chains at the same time. It is mostly found in prokaryotes and less common in eukaryotes. Currently, most widely used vectors adopt polycistronic expression schemes, that is, using internal ribosome entry sites (Internal ribosome entry site, IRES) or self-cleaving 2A peptide (self-cleaving 2A peptide, 2A) and other elements to connect multiple genes to achieve the purpose of expressing multiple genes on a single vector. This technology uses DNA plasmids as vectors, and there is a risk of integration into the genome when introduced into the target cells.
  • IRES Internal ribosome entry site
  • 2A self-cleaving 2A peptide
  • RNA vaccine technology including mRNA vaccine technology and circular RNA vaccine technology
  • mRNA vaccine technology is a medical technology that has achieved major breakthroughs in recent years. Although this technology has shown broad application prospects in the prevention and treatment of infectious diseases, autoimmune diseases, genetic diseases and cancer, the technology still has huge room for optimization and improvement.
  • Current RNA technologies are all monocistronic RNA technologies, that is, one RNA molecule can only translate into one polypeptide (a single protein subunit or a single fusion protein). Therefore, in applications where multiple functional proteins need to be expressed simultaneously (such as multivalent vaccines, combined drug administration, etc.), the current monocistronic RNA technology encounters a technical application bottleneck.
  • ICB immune checkpoint inhibition
  • CAR-T chimeric antigen receptor T cell
  • the clinical response rate of ICB therapy is very low, only 20%-30%, and it is almost ineffective for most cancer patients; although CAR-T therapy has achieved great success in the treatment of B lymphoma, it still has three serious problems: 1 Time-consuming, resource-consuming, and extremely expensive personalized cell therapy 1. It is a treatment for cancer, not an off-the-shelf drug; 2. It is only effective for some blood tumors and can hardly kill solid tumors; 3. It has serious side effects, such as cytokine storm and killing of normal cells. Therefore, the development of cutting-edge biotechnology for cancer treatment is still urgently needed.
  • RNA therapy technology has the advantages of being reversible and controllable. RNA can directly translate proteins in the cytoplasm to function without being transported into the nucleus; and RNA does not have the safety risk of integrating into genomic DNA. Therefore, RNA therapy technology is an important opportunity to develop in situ CAR immunotherapy in vivo.
  • Matthias Stephan's team directly intravenously infused mRNA encoding Anti-CD19 CAR transmembrane protein into mice repeatedly, and the therapeutic effect achieved the efficacy of adoptive cell therapy in the mouse model (Parayath et al., Nature Communications, 2020). In January 2022, JOEL G. RURIK et al.
  • CAR therapy often improves its therapeutic effect by combining it with multiple functional proteins, such as cytokines, monoclonal antibodies, vaccines, small molecule targeted drugs, etc. If the CAR molecule and the above multiple functional proteins can be expressed in vivo and act synergistically on the same RNA molecule, it will help improve the targeting and tumor killing effect of CAR therapy and effectively improve the response rate of solid tumor treatment.
  • the cytokines and PDZ domains that promote inflammation and immune cell infiltration are transmembrane or secretory proteins, the current monocistronic RNA technology is technically limited to encode them on the same RNA molecule.
  • mRNA has a short half-life and poor stability. Naked mRNA directly entering the body is easily degraded by the RNA enzymes that are widely present in the body.
  • circular RNA is more stable than mRNA, it is easy to cause immunogenicity in the body, affecting the therapeutic effect and limiting its application in medical treatment.
  • RNA enzymes that are widely present in the body.
  • circular RNA is more stable than mRNA, it is easy to cause immunogenicity in the body, affecting the therapeutic effect and limiting its application in medical treatment.
  • it is necessary to add a 5' end cap structure and a 3' end polyA tail.
  • circular RNA does not need to be capped or tailed, it needs to undergo a cyclization reaction. The preparation process is cumbersome, which seriously restricts production efficiency and increases production costs.
  • the present invention provides a stable RNA molecule prepared in vitro and its application in the preparation of in situ CAR therapeutic products in vivo.
  • the first object of the present invention is to provide a stable RNA molecule prepared in vitro, the RNA molecule comprising:
  • An IRES sequence disposed upstream of the coding region of the target protein
  • the protective RNA secondary structure is a small nucleolar RNA (SnoRNA) sequence and/or a Triplex sequence.
  • the small nucleolar RNA sequence is selected from one or more of H/ACA box snoRNAs, C/D box snoRNAs, and small Cazar body RNAs (scaRNAs).
  • the Triplex sequence is an RNA sequence with a triple helix structure.
  • RNA molecule further comprises:
  • the target protein is one or more antibodies; or, one or more antigens; or, one or more gene editing enzymes; or, one or more regulatory factors; or, one or more fluorescent proteins.
  • the target proteins include: human coagulation factor IX (hFIX), pulmonary surfactant protein B antibody (SP-B), vascular endothelial growth factor A (VEGF-A), methylmalonyl-CoA mutase (hMUT), human erythropoietin (hEPO), cystic fibrosis transmembrane conductance regulator (CFTR), Cas9 endonuclease, Cpf1, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALENs), anti-HIV antibodies, fbr CD19 and CD22 bispecific antibodies, fbr CD3 and CLDN6 bispecific antibodies, Gaussia luciferase (Glue), Firefly luciferase (Flue), and enhanced green fluorescent protein (eGFP).
  • hFIX human coagulation factor IX
  • SP-B pulmonary surfactant protein B antibody
  • VEGF-A vascular endothelial growth factor
  • the IRES sequences upstream of the coding region of each target protein are different.
  • the 5'UTR sequence is selected from the RNA binding protein family or the YTHDF family; the 3'UTR sequence is selected from human ⁇ -globin 1 (HBA1), human ⁇ -globin 2 (HBA2), mouse ⁇ -globin, human ⁇ -globin, xrRNA or Apt-eIF4G.
  • HBA1 human ⁇ -globin 1
  • HBA2 human ⁇ -globin 2
  • mouse ⁇ -globin human ⁇ -globin
  • human ⁇ -globin xrRNA or Apt-eIF4G.
  • the second object of the present invention is to provide a method for preparing the stable RNA molecule prepared in vitro, comprising the following steps:
  • the method also includes encapsulating the RNA molecule using a delivery system.
  • the delivery system includes one or more of lipid nanoparticles, liposomes, lipid complexes, polymer materials, micelles, polypeptides, and protamine.
  • the third object of the present invention is to provide the application of the stable RNA molecule prepared in vitro, which includes the application in the preparation of vaccine products or the preparation of in situ CAR therapeutic products in vivo.
  • the present invention uses only one mRNA molecule to express multiple effector proteins, which is different from the multiple mRNA molecules required to express different proteins with monocistronic RNA. It avoids the problem of drug component homogeneity during the drug-making process, improves safety and drug batch parallelism, and provides a guarantee for later clinical applications.
  • the present invention redesigns mRNA, adds protective RNA secondary structures at both ends of mRNA to protect mRNA from being cut by RNA exonucleases, improves RNA stability, and uses IRES to replace the 5' end m7G cap structure to recruit ribosomes to start protein translation, so that mRNA retains translation function and increases its stability, without the need to add caps and tails at both ends, greatly reducing the cost of mRNA preparation and improving the efficiency of mRNA in vitro production.
  • the present invention constructs a multicistronic RNA platform, uses IRES to express multiple protein sequences respectively, and makes up for the deficiency that single cistron RNA technology cannot express multiple functional proteins at the same time.
  • Figure 1 is a flow cytometric analysis result 72 hours after transfection of linear RNA encoding green fluorescent protein into 293T cells;
  • FIG2 is a graph showing the number of green fluorescent protein positive cells analyzed by flow cytometry 48 hours after the linear RNA obtained after plasmid IVT constructed in Example 1 was transfected into 293T cells;
  • FIG3 is a bar chart analysis of the analysis results of FIG2 ;
  • FIG4 is an analysis diagram of the MFI results of the flow cytometric analysis of FIG2 ;
  • FIG5 is a conceptual diagram of CAR therapy according to Example 3.
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • the plasmid can be cut with restriction endonuclease PmeI to change it from a circular plasmid to a linear plasmid. Then, T7 RNA polymerase is used for in vitro transcription (IVT) reaction, and no additional capping and tailing reaction is required during mRNA preparation, nor is a series of cyclization and de-linear RNA steps required after the circular RNA in vitro transcription (IVT) reaction, and stable linear RNA can be generated in one step.
  • IVT in vitro transcription
  • the purified linear RNA was encapsulated in liposomes (lipo2000).
  • the linear RNA and lipo2000 were mixed in a ratio of 1:1.5 and transfected into 293T cells.
  • Flow cytometry analysis was performed 72 hours after transfection.
  • the experimental results are shown in Figures 2 to 4.
  • the results show that the linear RNA molecules constructed by the present invention can stably express green fluorescent protein in cells and have optimization space; among them, the linear RNA molecules with sno116-14 at the 5' end and Triplex at the 3' end have better expression effects.
  • the expression effects of linear RNA molecules with random sequences at both ends are poor.
  • T7 promoter, SnoRNA or/and Triplex sequence, 5'UTR sequence, IRES1 sequence, IRES2 sequence, IRES3 sequence, 3'UTR sequence, SnoRNA or/and Triplex sequence were amplified by PCR and connected to the vector containing the selection marker through Gibson Assmble to form the polycisRNA-EV empty vector. Then, the RBD antigen coding sequence for different SARS-CoV subtypes was amplified by PCR and cloned into the polycisRNA-EV backbone to construct the corresponding polycisRNA plasmid for the subsequent IVT reaction.
  • the plasmid can be cut with restriction endonuclease PmeI to change it from a circular plasmid to a linear plasmid. Then, T7 RNA polymerase is used for in vitro transcription (IVT) reaction, and no additional capping and tailing reaction is required during mRNA preparation, nor is a series of cyclization and de-linear RNA steps required after the circular RNA in vitro transcription (IVT) reaction, and stable linear polycisRNA can be generated in one step.
  • IVT in vitro transcription
  • Purified polycisRNA is encapsulated in lipid nanoparticles (LNPs).
  • LNPs lipid nanoparticles
  • the resulting polycisRNA/ The delivery nanoparticle components undergo concentration, buffer exchange, and sterile filtration, and are then finally evaluated and formulated into a vaccine in this sequence that can be directly injected into the human body to prevent disease.
  • T7 promoter SnoRNA or/and Triplex sequence, 5'UTR sequence, IRES1 sequence, IRES2 sequence, IRES3 sequence, 3'UTR sequence, SnoRNA or/and Triplex sequence were amplified by PCR and connected to the vector containing the selection marker through Gibson Assmble to form a polycisRNA-EV empty vector. Then, the coding sequences of lung cancer cell surface antigen polypeptide, programmed death receptor 1 (PD-1) antibody and epidermal growth factor receptor (EGFR) antibody were amplified by PCR and cloned into the downstream of IRES1 sequence, IRES2 sequence and IRES3 sequence in turn to construct the corresponding polycisRNA plasmid for the subsequent IVT reaction.
  • PD-1 programmed death receptor 1
  • EGFR epidermal growth factor receptor
  • the polycisRNA plasmid is digested with restriction endonuclease PmeI to convert the circular plasmid into a linear plasmid, and then T7 RNA polymerase is used for in vitro transcription (IVT) reaction to generate a stable linear polycisRNA in one step.
  • IVTT in vitro transcription
  • PolycisRNA can be delivered using commonly used delivery systems including lipid nanoparticles (LNP), liposomes, lipid complexes, polymer materials, micelles, peptides, protamine, electroporation, etc.
  • LNP lipid nanoparticles
  • This example uses lipid nanoparticles (L NP) to encapsulate the purified polycisRNA.
  • the resulting polycisRNA/delivery nanoparticle assembly is concentrated, buffer exchanged, and sterile filtered. It is then finally evaluated and packaged in this order to prepare a vaccine, which is injected into cancer patients to treat the disease.
  • CAR chimeric antigen receptor
  • IL2 cytokine
  • PD-1 programmed death receptor 1
  • the polycisRNA plasmid is digested with restriction endonuclease PmeI to convert the circular plasmid into a linear plasmid, and then T7 RNA polymerase is used for in vitro transcription (IVT) reaction to generate a stable linear polycisRNA in one step.
  • IVTT in vitro transcription
  • Lipid nanoparticles that can target T cells are used as a delivery system, in which the purified polycisRNA is encapsulated, and a certain dose of LNP-polycisRNA is injected into the body as needed for corresponding treatment.

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Abstract

一种体外制备的稳定的RNA分子及其在体内原位CAR治疗产品制备中的应用，重新设计了mRNA，在mRNA两端添加保护性RNA二级结构来保护mRNA不被RNA外切酶切割，提高RNA的稳定性，并使用IRES来替代5'端m7G帽子结构招募核糖体启动蛋白翻译，使mRNA保留翻译功能，增加其稳定性，而不需要在其两端分别加帽加尾，极大地降低mRNA制备的成本、并提高mRNA体外生产的效率。此外，在此基础上，构建了多顺反子RNA平台，使用IRES来分别表达多个蛋白序列，弥补了单顺反子RNA技术不能同时表达多个功能性蛋白的不足。

Description

一种体外制备的稳定的RNA分子及其在体内原位CAR治疗产品制备中的应用 技术领域
本发明涉及一种体外制备的稳定的RNA分子及其在体内原位CAR治疗产品制备中的应用，属于生物医药技术领域。
背景技术
CAR(Chimeric antigen receptor，嵌合抗原受体)的概念自1989年由科学家G Gross提出，发展至今，将CAR分子通过基因工程手段体外转导入T细胞的CAR-T(嵌合抗原受体T细胞)疗法已经成为了一种重要的免疫疗法并获批应用于临床上白血病和淋巴瘤的治疗。但该疗法目前仍存在许多问题，在应用上程序繁琐，成本昂贵，通常需要与其他医疗手段联合进行治疗以达到较好效果，且目前针对实体瘤的治疗几乎没有效果。
多顺反子指能够同时编码多个多肽链的一个mRNA分子，多存在于原核生物中，在真核生物中较少。目前广泛应用的载体多采用多顺反子表达方案，即利用内部核糖体进入位点(Internal ribosome entry site,IRES)或自剪切多肽2A(self-cleaving 2A peptide,2A)等元件来连接多个基因，实现在单个载体上表达多个基因的目的，该技术以DNA质粒为载体，导入目的细胞中存在整合到基因组的风险。
RNA疫苗技术，包括mRNA疫苗技术和circular RNA疫苗技术，是近几年取得重大突破的医学技术。虽然该项技术已经在感染性疾病、自身免疫病、遗传病以及癌症的预防和治疗中显示出广阔的应用前景，但是该技术仍然具有巨大的优化和提升空间。目前的RNA技术都属于单顺反子RNA技术，即一个RNA分子只能翻译出一种多肽(单个蛋白亚基或者单个融合蛋白)。因此，在需要同时表达多个功能性蛋白的应用情形下(比如多价疫苗、联合给药等)，当前的单顺反子RNA技术则遭遇技术应用瓶颈。
近几年，免疫检查点抑制(ICB)和嵌合抗原受体T细胞(CAR-T)免疫疗法已经成为重要的癌症治疗手段，但仍存在许多挑战。ICB疗法的临床反应率很低，只有20％—30％，对大多数癌症患者几乎无效；CAR-T疗法虽然在治疗B淋巴瘤方面取得了巨大成功，但其仍存在三个严重问题：①耗时、耗资源、极其昂贵的个体化细胞治 疗，而不是“现货型”药物；②仅对部分的血液肿瘤有效，对实体瘤几乎不能杀伤；③严重的副作用，比如细胞因子风暴和杀伤正常细胞等。因此，开发用于癌症治疗的前沿生物技术仍然是迫切所需的。
RNA治疗技术具备可逆、可调控的优势，RNA可以直接在细胞质中翻译蛋白发挥功能，不需要转运进细胞核内；且RNA没有整合进基因组DNA的安全风险。因此，RNA治疗技术是开发体内原位CAR免疫疗法的一个重要契机。2020年11月，Matthias Stephan团队将编码Anti-CD19 CAR跨膜蛋白的mRNA直接静脉重复输注进小鼠体内，治疗效果在小鼠模型上达到了过继性细胞疗法的疗效(Parayath et al.,Nature Communications,2020)。2022年1月，JOEL G.RURIK等报道了基于mRNA技术的体内重编程CAR-T疗法，通过向体内T细胞靶向递送Anti-FAP-CAR mRNA，清除了小鼠体内纤维化心肌细胞(Rurik et al.,Science，2022)。
目前，CAR疗法常通过与多种功能蛋白联合治疗来提高其治疗效果，如细胞因子、单克隆抗体、疫苗、小分子靶向药物等，如果能将CAR分子与以上多个功能性蛋白在同一个RNA分子上同时在体表达、协同作用，将有助于提高CAR疗法的靶向性、肿瘤杀伤效果，有效提高实体瘤治疗响应率。但由于促炎、促免疫细胞浸润的细胞因子与PDZ结构域都是属于跨膜或者分泌蛋白，采用当前的单顺反子RNA技术在同一个RNA分子上进行编码受到技术限制。当前的单顺反子RNA技术不能应用于同时表达多个功能性蛋白用于免疫治疗的主要原因在于：(1)脂纳米颗粒(LNP)对RNA分子进行包裹时是随机过程、且一个LNP颗粒包裹的RNA分子数目有限；因此，当用LNP对多种单顺反子RNA进行包封时，制备的LNP-RNA药物批次的平行性、稳定性和有效性都会收到严重制约。(2)由于哺乳动物细胞中分泌型蛋白和膜蛋白分选的特殊机制(信号肽的引导和切割)，用一个RNA分子翻译一种融合蛋白(各蛋白亚基由2A连接而成)的策略仅仅适用于胞质蛋白、核内蛋白或单个跨膜蛋白与其他胞质蛋白的组合，不能适用于多个分泌蛋白或跨膜蛋白的融合翻译；然而应用于疫苗的抗原、治疗性抗体、细胞因子、嵌合抗原受体(CAR)等大多数功能性蛋白都是分泌蛋白或膜蛋白。基于以上原因，开发多顺反子RNA技术，即利用一个RNA分子同时翻译出多个功能性分泌蛋白或膜蛋白，是当前底层RNA技术研究领域所迫切需要的，并且其未来在疾病的治疗中都极具应用开发前景。
此外，在体内环境中，mRNA半衰期短、稳定性差，裸露的mRNA直接进入体内容易被体内广泛存在的RNA酶降解；而环状RNA虽然比mRNA稳定，但容易引起机体产生免疫原性，影响治疗效果，限制了其在医学治疗中的应用。并且，mRNA在体 外制备时，需要额外添加5’端帽子结构以及3’端polyA尾，而环状RNA虽然不需要加帽加尾，但需要进行环化反应，制备工艺程序繁琐，在生产上严重制约了生产效率，并增加了生产成本。
发明内容
为了解决上述问题，本发明提供一种体外制备的稳定的RNA分子及其在体内原位CAR治疗产品制备中的应用。
本发明的第一个目的是提供一种体外制备的稳定的RNA分子，所述RNA分子包括：
编码目的蛋白的区域；以及，
设置在所述目的蛋白的编码区上游的IRES序列；以及，
设置在所述RNA分子两端的保护性RNA二级结构。
进一步地，所述保护性RNA二级结构为小核仁RNA(SnoRNA)序列或/和Triplex序列。
进一步地，所述小核仁RNA序列选自H/ACA box snoRNAs、C/D box snoRNAs、小卡扎尔体RNA(scaRNAs)中的一种或多种。
进一步地，所述Triplex序列为具有三螺旋结构的RNA序列。
进一步地，所述RNA分子还包括：
5’端设置在保护性RNA二级结构之后的5’UTR序列，以及，3’端设置在保护性RNA二级结构之前的3’UTR序列。
进一步地，所述目的蛋白为一个或多个。
进一步地，所述目的蛋白为一种或多种抗体；或，一种或多种抗原；或，一种或多种基因编辑酶；或，一种或多种调节因子；或，一种或多种荧光蛋白。
进一步地，所述目的蛋白包括：人凝血因子IX(hFIX)、肺表面活性蛋白B抗体(SP-B)、血管内皮生长因子A(VEGF-A)、甲基丙二酸单酰辅酶A变位酶(hMUT)、人促红细胞生成素(hEPO)、囊性纤维化跨膜传导调节因子(CFTR)、Cas9内切酶、Cpf1、锌指核酸酶(ZFN)、转录激活剂样效应子核酸酶(TALENs)、抗HIV抗体、fbr CD19和CD22双特异性抗体、fbr CD3和CLDN6双特异性抗体、Gaussia荧光素酶(Glue)、Firefly荧光素酶(Flue)、增强型绿色荧光蛋白(eGFP)。
进一步地，所述目的蛋白为多个时，每个目的蛋白的编码区上游的IRES序列均不相同。
进一步地，所述IRES来自以下病毒的IRES序列：陶拉综合征病毒、铁人三项瘤病毒、泰勒脑脊髓炎病毒、猿猴病毒40、红火火蚁病毒1、禾谷缢管蚜病毒、网状内皮组织增生症病毒、富曼脊髓灰质炎病毒1、克什米尔蜂病毒、人鼻病毒2、人免疫缺陷病毒1型、Himetobi  P病毒、丙型肝炎病毒、甲型肝炎病毒、GB肝炎病毒、口蹄疫病毒、人肠道病毒71、马鼻炎病毒、脑心肌炎病毒、果蝇C病毒、蟋蟀麻痹病毒、牛病毒性腹泻病毒1、黑蜂王细胞病毒、蚜虫致死性麻痹病毒、禽类脑脊髓炎病毒、急性蜜蜂麻痹病毒、木槿褪绿环斑病毒、经典猪瘟病毒、猿猴小核糖核酸病毒、芜菁皱缩病毒、柯萨奇病毒B3或柯萨奇病毒A。
进一步地，在目的蛋白为多个时，多个目的蛋白的编码区上游的IRES序列为不同序列。
进一步地，5’UTR序列选自RNA结合蛋白家族或YTHDF家族；3’UTR序列选自人α-珠蛋白1(HBA1)、人α-珠蛋白2(HBA2)小鼠α-珠蛋白、人β-珠蛋白、xrRNA或Apt-eIF4G。
本发明的第二个目的是提供所述体外制备的稳定的RNA分子的制备方法，包括如下步骤：
S1、分别扩增RNA聚合酶启动子、保护性RNA二级结构序列、5’UTR序列、3’UTR序列、IRES序列和目的蛋白的编码基因序列，连接到空载体上得到表达载体；
S2、将S1步骤得到的表达载体经过酶切线性化，经过体外转录合成所述RNA分子。
进一步地，所述方法还包括采用递送系统封装所述RNA分子。
进一步地，所述递送系统包括脂质纳米颗粒、脂质体、脂质复合物、高分子材料、胶束、多肽、鱼精蛋白中的一种或多种。
本发明的第三个目的是提供所述体外制备的稳定的RNA分子的应用，所述应用包括疫苗产品制备或体内原位CAR治疗产品制备中的应用。
本发明的polycisRNA有效解决了以下问题：
(1)目前个体化过继细胞治疗中因异体移植免疫排斥引发的耗时、耗资源、成本极其昂贵的制约问题：基于本发明的多顺反子RNA技术，可以开发出isCAR-T/isCAR-M“现货型”新型免疫疗法，以多顺反子RNA为载体，在体内原位直接生成CAR-免疫细胞，并通过多种效应蛋白协同作用，增强疗效；因mRNA的特性使开发的免疫疗法避免了个体化治疗复杂的过程，并具有剂量-时间依赖性疗效特性，临床安全性很高。
(2)本发明针对mRNA结构进行改造，去除5’帽子结构和3’polyA尾且保留mRNA蛋白翻译功能，并依赖SnoRNA序列或Triplex序列提高mRNA稳定性，在生产过程中仅需以DNA为模板进行体外转录即可得到产品，无需加帽加尾或环化环节，极大的节约了生产时间，降低了生产成本。
(3)本发明仅以一个mRNA分子表达多种效应蛋白，不同于以单顺反子RNA表达不同蛋白所需的多个mRNA分子，在成药过程中避免了药物成分均一性问题，提高了安全性和药物批次平行性，为后期临床应用提供了保障。
本发明的有益效果是：
本发明重新设计了mRNA，在mRNA两端添加保护性RNA二级结构来保护mRNA不被RNA外切酶切割，提高RNA的稳定性，并使用IRES来替代5’端m7G帽子结构招募核糖体启动蛋白翻译，使mRNA保留翻译功能，增加其稳定性，而不需要在其两端分别加帽加尾，极大地降低mRNA制备的成本、并提高mRNA体外生产的效率。此外，本发明在此基础上，构建了多顺反子RNA平台，使用IRES来分别表达多个蛋白序列，弥补了单顺反子RNA技术不能同时表达多个功能性蛋白的不足。
附图说明：
图1为向293T细胞中转染编码绿色荧光蛋白线性RNA 72小时后的流式细胞分析结果图；
图2为实施例1构建的质粒IVT后得到的线性RNA转染293T细胞48小时后进行流式细胞分析的绿色荧光蛋白阳性细胞数目结果图；
图3为图2分析结果的柱状图分析；
图4为图2流式细胞分析的MFI结果分析图；
图5为实施例3CAR疗法的概念图。
具体实施方式
下面结合具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
缩略语：
GFP Green fluorescent protein绿色荧光蛋白
ORF Open reading frame开放阅读框
IRES Internal ribosome entry site内部核糖体进入位点
UTR Untranslated region非翻译区
HEK Human embryonic kidney人胚胎肾脏
EMCV Encephalomyocarditis virus脑心肌炎病毒
表1常用保护性RNA二级结构序列

表2常用IRES序列

表3其他IRES序列




表4 5’UTR序列序列

表5 3’UTR序列
实施例1：
1)载体构建
PCR扩增T7启动子、SnoRNA116-14序列、5’UTR序列、CVB3IRES序列、编码绿 色荧光蛋白序列、3’UTR序列、SnoRNA116-13序列，将CVB3IRES序列、编码绿色荧光蛋白序列依次连入载体骨架，然后在两端添加随机生成的序列生成Random1-Random2线性RNA，两端分别添加snoRNA116-14、snoRNA116-13序列，生成snoRNA1-snoRNA2线性RNA；在IRES前添加5’UTR序列，编码绿色荧光蛋白序列后添加3’UTR序列，形成骨架载体，在两端保护结构位点分别添加snoRNA或/和Triplex序列或/和随机序列，形成snoRNA1-snoRNA2(U)Random1-snoRNA2(U)、Random3-snoRNA(U)、Triplex-snoRNA2(U)snoRNA1-Random2(U)、snoRNA1-Triplex110(U)线性RNA。
根据本发明所构建载体，可用限制性内切酶PmeI酶切质粒，使其由环状质粒变为线性质粒。再利用T7RNA聚合酶进行体外转录(IVT)反应，不需要mRNA制备过程中额外的加帽加尾反应,，也不需要环状RNA体外转录(IVT)反应后一系列的环化、去线性RNA等步骤，即可一步产生稳定的线性RNA。
2)制备递送系统
采用脂质体(lipo2000)将纯化的线性RNA封装在其中。将线性RNA与lipo2000以1：1.5的比例混合转染293T细胞，在转染72小时后进行流式细胞术分析，实验结果见图2～4。结果说明本发明所构建的线性RNA分子能够在细胞内稳定表达绿色荧光蛋白，并具有优化空间；其中，以5’端为sno116-14,3’端为Triplex的线性RNA分子表达效果更好。而两端都为随机序列的线性RNA分子的表达效果较差。
实施例2：传染病疫苗
本实施例以新冠疫苗为例：
1)载体构建
PCR扩增T7启动子、SnoRNA或/和Triplex序列、5’UTR序列、IRES1序列、IRES2序列、IRES3序列、3’UTR序列、SnoRNA或/和Triplex序列，并通过Gibson Assmble连接到含有筛选标记的载体上，构成polycisRNA-EV空载体。然后，PCR扩增针对不同SARS-CoV亚型的RBD抗原编码序列，克隆到polycisRNA-EV主干中，构建相应的polycisRNA质粒进行接下来的IVT反应。
根据本发明所构建载体，可用限制性内切酶PmeI酶切质粒，使其由环状质粒变为线性质粒。再利用T7RNA聚合酶进行体外转录(IVT)反应，不需要mRNA制备过程中额外的加帽加尾反应,，也不需要环状RNA体外转录(IVT)反应后一系列的环化、去线性RNA等步骤，即可一步产生稳定的线性polycisRNA。
2)制备递送系统
采用脂质的纳米颗粒(LNP)将纯化的polycisRNA封装在其中。由此产生的polycisRNA/ 递送纳米颗粒组件经过浓缩、缓冲液交换和无菌过滤。然后最终评估并按此顺序制备成疫苗，可直接向人体内注射预防疾病。
实施例3：癌症疫苗
本实施例以肺癌为例：
1)载体构建
PCR扩增T7启动子、SnoRNA或/和Triplex序列、5’UTR序列、IRES1序列、IRES2序列、IRES3序列、3’UTR序列、SnoRNA或/和Triplex序列，并通过Gibson Assmble连接到含有筛选标记的载体上，构成polycisRNA-EV空载体。然后，PCR扩增肺癌细胞表面抗原多肽、程序性死亡受体1(PD-1)抗体和表皮生长因子受体(EGFR)抗体编码序列，依次克隆到IRES1序列、IRES2序列、IRES3序列的下游，构建相应的polycisRNA质粒进行接下来的IVT反应。
将polycisRNA质粒用限制性内切酶PmeI酶切质粒，使其由环状质粒变为线性质粒。再利用T7RNA聚合酶进行体外转录(IVT)反应，一步产生稳定的线性polycisRNA。
2)制备递送系统
可以使用常用的递送系统包括脂质纳米颗粒(LNP)、脂质体、脂质复合物、高分子材料、胶束、多肽、鱼精蛋白、电穿孔等将polycisRNA进行递送。本实施例使用脂质的纳米颗粒(L NP)，将纯化的polycisRNA封装在其中。由此产生的polycisRNA/递送纳米颗粒组件经过浓缩、缓冲液交换和无菌过滤。然后最终评估并按此顺序包装制备成疫苗，向癌症患者体内注射以治疗疾病。
实施例4：CAR疗法
1)PCR扩增T7启动子、SnoRNA或/和Triplex序列、5’UTR序列、IRES1序列、IRES2序列、IRES3序列、3’UTR序列、SnoRNA或/和Triplex序列，并通过Gibson Assmble连接到含有筛选标记的载体上，构成polycisRNA-EV空载体。然后，PCR扩增嵌合抗原受体(CAR)、细胞因子(IL2等)和程序性死亡受体1(PD-1)抗体编码序列，克隆到polycisRNA-EV主干中，依次克隆到IRES1序列、IRES2序列、IRES3序列的下游，构建相应的polycisRNA质粒进行接下来的IVT反应。
将polycisRNA质粒用限制性内切酶PmeI酶切质粒，使其由环状质粒变为线性质粒。再利用T7RNA聚合酶进行体外转录(IVT)反应，一步产生稳定的线性polycisRNA。
2)制备递送系统
使用能够靶向T细胞的脂质纳米颗粒(LNP)作为递送系统，将纯化的polycisRNA封装在其中，根据需要将一定剂量的LNP-polycisRNA注入体内以进行相应的治疗。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (11)

  1. 一种体外制备的稳定的RNA分子，其特征在于，所述RNA分子包括：
    编码目的蛋白的区域；以及，
    设置在所述目的蛋白的编码区上游的IRES序列；以及，
    设置在所述RNA分子两端的保护性RNA二级结构。
  2. 根据权利要求1所述的RNA分子，其特征在于，所述保护性RNA二级结构为小核仁RNA序列或/和Triplex序列。
  3. 根据权利要求1所述的RNA分子，其特征在于，所述RNA分子还包括：
    5’端设置在保护性RNA二级结构之后的5’UTR序列，以及3’端设置在保护性RNA二级结构之前的3’UTR序列。
  4. 根据权利要求1所述的RNA分子，其特征在于，所述目的蛋白为一个或多个。
  5. 根据权利要求1所述的RNA分子，其特征在于，所述目的蛋白为一种或多种抗体；或，一种或多种抗原；或，一种或多种基因编辑酶；或，一种或多种调节因子；或，一种或多种荧光蛋白。
  6. 根据权利要求2所述的RNA分子，其特征在于，所述小核仁RNA序列选自H/ACA box snoRNAs、C/D box snoRNAs、小卡扎尔体RNA中的一种或多种。
  7. 根据权利要求1所述的RNA分子，其特征在于，所述IRES来自以下病毒的IRES序列：陶拉综合征病毒、铁人三项瘤病毒、泰勒脑脊髓炎病毒、猿猴病毒40、红火火蚁病毒1、禾谷缢管蚜病毒、网状内皮组织增生症病毒、富曼脊髓灰质炎病毒1、克什米尔蜂病毒、人鼻病毒2、人免疫缺陷病毒1型、Himetobi P病毒、丙型肝炎病毒、甲型肝炎病毒、GB肝炎病毒、口蹄疫病毒、人肠道病毒71、马鼻炎病毒、脑心肌炎病毒、果蝇C病毒、蟋蟀麻痹病毒、牛病毒性腹泻病毒1、黑蜂王细胞病毒、蚜虫致死性麻痹病毒、禽类脑脊髓炎病毒、急性蜜蜂麻痹病毒、木槿褪绿环斑病毒、经典猪瘟病毒、猿猴小核糖核酸病毒、芜菁皱缩病毒、柯萨奇病毒B3或柯萨奇病毒A。
  8. 根据权利要求3所述的RNA分子，其特征在于，5’UTR选自RNA结合蛋白家族或YTHDF家族；3’UTR选自人α-珠蛋白1、人α-珠蛋白2、小鼠α-珠蛋白、人β-珠蛋白、xrRNA或Apt-eIF4G。
  9. 一种权利要求1～8任一项所述体外制备的稳定的RNA分子的制备方法，其特征在于，包括如下步骤：
    S1、分别扩增RNA聚合酶启动子、保护性RNA二级结构序列、5’UTR序列、3’UTR 序列、IRES序列和目的蛋白的编码基因序列，连接到空载体上得到表达载体；
    S2、将S1步骤得到的表达载体经过酶切线性化，经过体外转录合成所述RNA分子。
  10. 根据权利要求9所述的方法，其特征在于，所述方法还包括采用递送系统封装所述RNA分子。
  11. 权利要求1～8任一项所述体外制备的稳定的RNA分子的应用，其特征在于，所述应用包括疫苗产品制备或体内原位CAR治疗产品制备中的应用。
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