技术领域Technical Field
本发明属于生物医药领域，具体涉及一种脂质纳米颗粒及其应用。The present invention belongs to the field of biomedicine, and in particular relates to lipid nanoparticles and applications thereof.
背景技术Background Art
众所周知，mRNA递送入胞过程中面临着两大技术障碍：(1)静电排斥致使的膜屏障；(2)递送途中的酶降解，因此需要特殊的修饰或包裹递送系统才能实现mRNA的胞内表达，改变mRNA胞内的生物分布、细胞靶向和摄取机制，促进mRNA的递送，发挥mRNA翻译后的效果。为弥补病毒载体的不足，非病毒递送系统因其低毒性、靶向递送的潜力、长期稳定性、DNA/mRNA装载量较高、化学结构可控、免疫原性较小、易于大量制备而受到越来越多的关注。LNP(Lipid Nanoparticle)被认为是最具潜力的用于外源mRNA递送的非病毒载体。As is well known, mRNA delivery into cells faces two major technical obstacles: (1) membrane barriers caused by electrostatic repulsion; (2) enzymatic degradation during delivery. Therefore, special modification or packaging delivery systems are required to achieve intracellular expression of mRNA, change the intracellular biodistribution, cell targeting and uptake mechanism of mRNA, promote mRNA delivery, and exert the post-translational effects of mRNA. To make up for the shortcomings of viral vectors, non-viral delivery systems have attracted increasing attention due to their low toxicity, potential for targeted delivery, long-term stability, high DNA/mRNA loading capacity, controllable chemical structure, low immunogenicity, and ease of large-scale preparation. LNP (Lipid Nanoparticle) is considered to be the most promising non-viral vector for exogenous mRNA delivery.
造血干细胞表面具有特定的受体和标志物，为了实现高效的转染，LNP需要能够特异性地识别并结合这些受体或标志物。然而，由于造血干细胞表面的受体和标志物表达水平较低且复杂多样，使得LNP的靶向性成为了一个难题。因此如何实现有效转染人造血干细胞，实现原代细胞内编辑效率的显著提高是本领域亟需解决的问题。Hematopoietic stem cells have specific receptors and markers on their surfaces. To achieve efficient transfection, LNPs need to be able to specifically recognize and bind to these receptors or markers. However, due to the low expression levels and complex and diverse expression of receptors and markers on the surface of hematopoietic stem cells, LNP targeting has become a challenge. Therefore, how to effectively transfect human hematopoietic stem cells and significantly improve the efficiency of editing in primary cells is an urgent problem in this field.
发明内容Summary of the Invention
为弥补现有技术的不足，本发明提供了一种脂质纳米颗粒及其应用。To overcome the deficiencies of the prior art, the present invention provides a lipid nanoparticle and its application.
为实现上述目的，本发明采用如下技术方案To achieve the above purpose, the present invention adopts the following technical solutions
本发明的第一方面提供了一种LNP-mRNA递送系统，所述LNP-mRNA递送系统包括负载一种或多种mRNA的LNP载体，所述LNP与mRNA的比例为(4-10)：1。The first aspect of the present invention provides an LNP-mRNA delivery system, which includes an LNP carrier loaded with one or more mRNAs, and the ratio of the LNP to the mRNA is (4-10):1.
进一步，所述LNP与mRNA的比例为(8-10)：1。Furthermore, the ratio of LNP to mRNA is (8-10):1.
进一步，所述LNP与mRNA的比例为8：1。Furthermore, the ratio of LNP to mRNA is 8:1.
进一步，所述LNP中油相的浓度为4-12mM。Furthermore, the concentration of the oil phase in the LNP is 4-12 mM.
进一步，所述LNP中油相的浓度为6mM。Furthermore, the concentration of the oil phase in the LNP is 6 mM.
进一步，所述mRNA为Cas9 mRNA。Furthermore, the mRNA is Cas9 mRNA.
进一步，所述LNP-mRNA递送系统还包括sgRNA。Furthermore, the LNP-mRNA delivery system also includes sgRNA.
进一步，mRNA与sgRNA的比例为(1-4)：(1-3)。Furthermore, the ratio of mRNA to sgRNA was (1-4):(1-3).
进一步，mRNA与sgRNA的比例为1：2。Furthermore, the ratio of mRNA to sgRNA was 1:2.
进一步，所述LNP包括可电离的氨基脂质、PEG脂质、磷脂、胆固醇。Furthermore, the LNP comprises ionizable amino lipids, PEG lipids, phospholipids, and cholesterol.
进一步，所述可电离的氨基脂质选自ALC0315。Furthermore, the ionizable amino lipid is selected from ALC0315.
进一步，所述PEG脂质选自ALC0159(DMG-PEG2000)。Furthermore, the PEG lipid is selected from ALC0159 (DMG-PEG2000).
进一步，所述磷脂选自DSPC。Furthermore, the phospholipid is selected from DSPC.
本发明的第二方面提供了GlutaMAX在制备提高细胞中蛋白翻译的产品中的应用。A second aspect of the present invention provides the use of GlutaMAX in preparing a product for increasing protein translation in cells.
进一步，所述细胞选自造血干细胞。Furthermore, the cells are selected from hematopoietic stem cells.
进一步，所述造血干细胞为人造血干细胞。Furthermore, the hematopoietic stem cells are human hematopoietic stem cells.
进一步，所述提高细胞中蛋白翻译为提高LNP转染后细胞内Cas9 mRNA的翻译。Furthermore, the increasing protein translation in the cells is to increase the translation of Cas9 mRNA in the cells after LNP transfection.
进一步，所述产品还包括细胞培养基。Furthermore, the product also includes cell culture medium.
进一步，所述细胞培养基选自IMDM培养基。Furthermore, the cell culture medium is selected from IMDM culture medium.
进一步，所述IMDM培养基还包括FBS、BSA、双抗、SCF、白细胞介素、β-巯基乙醇、丙酮酸钠。Furthermore, the IMDM culture medium further comprises FBS, BSA, double antibody, SCF, interleukin, β-mercaptoethanol, and sodium pyruvate.
进一步，所述白细胞介素选自IL-3。Furthermore, the interleukin is selected from IL-3.
本发明的第三方面提供了一种能够提高细胞中蛋白翻译的培养基，所述培养基包括GlutaMAX。A third aspect of the present invention provides a culture medium capable of increasing protein translation in cells, wherein the culture medium comprises GlutaMAX.
进一步，所述细胞选自造血干细胞。Furthermore, the cells are selected from hematopoietic stem cells.
进一步，所述造血干细胞为人造血干细胞。Furthermore, the hematopoietic stem cells are human hematopoietic stem cells.
进一步，所述提高细胞中蛋白翻译为提高LNP转染后细胞内Cas9 mRNA的翻译。Furthermore, the increasing protein translation in the cells is to increase the translation of Cas9 mRNA in the cells after LNP transfection.
进一步，所述培养基为细胞培养基。Furthermore, the culture medium is a cell culture medium.
进一步，所述细胞培养基选自IMDM培养基。Furthermore, the cell culture medium is selected from IMDM culture medium.
进一步，所述IMDM培养基还包括FBS、BSA、双抗、SCF、白细胞介素、β-巯基乙醇、丙酮酸钠。Furthermore, the IMDM culture medium further comprises FBS, BSA, double antibody, SCF, interleukin, β-mercaptoethanol, and sodium pyruvate.
进一步，所述白细胞介素选自IL-3。Furthermore, the interleukin is selected from IL-3.
本发明的第四方面提供了本发明第一方面所述的LNP-mRNA递送系统在转染和编辑人造血干细胞中的应用。The fourth aspect of the present invention provides the use of the LNP-mRNA delivery system described in the first aspect of the present invention in transfecting and editing human hematopoietic stem cells.
本发明的第五方面提供了本发明第一方面所述的LNP-mRNA递送系统在制备治疗疾病的药物组合物中的应用。The fifth aspect of the present invention provides use of the LNP-mRNA delivery system described in the first aspect of the present invention in preparing a pharmaceutical composition for treating a disease.
进一步，所述疾病选自镰刀型细胞贫血症。Furthermore, the disease is selected from sickle cell anemia.
进一步，所述药物组合物的动员方式为连续动员法。Furthermore, the mobilization method of the pharmaceutical composition is a continuous mobilization method.
进一步，所述连续动员法使用的动员剂为G-CSF和AMD3100，G-CSF选用Primegene品牌最佳。Furthermore, the mobilization agents used in the continuous mobilization method are G-CSF and AMD3100, and the Primegene brand is the best G-CSF.
进一步，所述药物组合物的注射方式为皮下注射。Furthermore, the pharmaceutical composition is injected subcutaneously.
进一步，所述皮下注射为脖颈部皮下注射。Furthermore, the subcutaneous injection is a subcutaneous injection in the neck.
进一步，所述药物组合物还包括药学上可接受的辅料。Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.
本发明的优点和有益效果：Advantages and beneficial effects of the present invention:
本申请对脂质材料配方的比例、最优细胞培养及编辑体系、编辑系统Cas9 mRNA：sgRNA比例、Cas9 mRNA设计优化进行一系列探索，摸索出适合于针对人造血干细胞转染及编辑的系统，本申请在节约成本的同时，可完全替代目前已有的市面转染试剂不能有效转染原代细胞的问题，实现原代细胞内编辑效率的显著提高，进而能在体内得到很好的治疗效果。This application conducts a series of explorations on the ratio of lipid material formulas, the optimal cell culture and editing system, the Cas9 mRNA:sgRNA ratio of the editing system, and the optimization of Cas9 mRNA design, and explores a system suitable for transfection and editing of human hematopoietic stem cells. While saving costs, this application can completely replace the problem that existing commercial transfection reagents cannot effectively transfect primary cells, achieve a significant improvement in the editing efficiency in primary cells, and thus achieve a good therapeutic effect in vivo.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1是常规合成LNP及转染试剂RNAiMax可高效转染K562细胞，但无法转染人造血干细胞CD34+细胞图；Figure 1 shows that conventional synthetic LNPs and the transfection reagent RNAiMax can efficiently transfect K562 cells, but cannot transfect human hematopoietic stem cell CD34+ cells;
图2是LNP配方对LNP-mRNA的粒径、PDI及表面电位的影响图；FIG2 is a graph showing the effect of LNP formulation on the particle size, PDI, and surface potential of LNP-mRNA;
图3是LNP-EGFP mRNA配方对CD34+细胞的转染效率及活性的影响图；FIG3 is a graph showing the effect of LNP-EGFP mRNA formulation on the transfection efficiency and activity of CD34+ cells;
图4是LNP包裹mRNA可稳定且有效转染人造血干细胞CD34+细胞图；FIG4 is a diagram showing that LNP-encapsulated mRNA can stably and effectively transfect human hematopoietic stem cell CD34+ cells;
图5是对人造血干细胞CD34+细胞的转染最优LNP油相浓度图；FIG5 is a graph showing the optimal LNP oil phase concentration for transfection of human hematopoietic stem cell CD34+ cells;
图6是sgRNAHBG靶向位点编辑策略图；Figure 6 is a diagram of the sgRNAHBG targeted site editing strategy;
图7是LNP包封Cas9 mRNA与sgRNA不同比例编辑细胞图(1×NLS Cas9mRNA)；Figure 7 shows cells edited with different ratios of LNP-encapsulated Cas9 mRNA and sgRNA (1×NLS Cas9 mRNA);
图8是GlutaMAXTM添加剂在人造血干细胞体外编辑中具有显著提高蛋白翻译的作用图(1×NLS Cas9 mRNA)；FIG8 shows that GlutaMAX™ additive significantly enhances protein translation in human hematopoietic stem cell editing in vitro (1×NLS Cas9 mRNA);
图9是LNP包封带有不同入核信号的Cas9 mRNA与sgRNA编辑细胞后编辑效率检测柱状图及原始峰图；Figure 9 is a bar graph and original peak graph of editing efficiency detection after LNP encapsulation of Cas9 mRNA and sgRNA with different nuclear entry signals edited cells;
图10是LNP包封带有不同入核信号的Cas9 mRNA与sgRNA编辑细胞后接种于固体培养基菌落CFU克隆形成成像及编辑效率图；Figure 10 is a graph showing the CFU colony formation imaging and editing efficiency of cells after LNP encapsulated Cas9 mRNA and sgRNA with different nuclear entry signals were inoculated on solid culture medium;
图11是LNP包封3×NLS Cas9 mRNA/sgRNA编辑细胞后分别检测HBG mRNA、HbF及CD235a+CD71+群体图；Figure 11 is a graph showing the detection of HBG mRNA, HbF, and CD235a+ CD71+ populations after LNP encapsulation of 3×NLS Cas9 mRNA/sgRNA edited cells;
图12是GROβ+AMD3100注射小鼠后，不同时间点取小鼠外周血，流式检测外周血中小鼠造血干细胞LSK的阳性率图；Figure 12 is a graph showing the positive rate of mouse hematopoietic stem cells LSK in peripheral blood of mice after GROβ+AMD3100 injection at different time points;
图13是G-CSF+AMD3100注射小鼠后，不同时间点取小鼠外周血，流式检测外周血中小鼠造血干细胞LSK的阳性率图；FIG13 is a graph showing the positive rate of mouse hematopoietic stem cells LSK in peripheral blood of mice after G-CSF+AMD3100 injection at different time points;
图14是不同动员剂皮下注射部位对小鼠造血干细胞递送效率的摸索图；FIG14 is a graph showing the delivery efficiency of different mobilization agents to mouse hematopoietic stem cells at subcutaneous injection sites;
图15是LNP-3×NLS Cas9 mRNA/sgRNA体内注射人源化镰刀型细胞贫血症小鼠1周后检测小鼠骨髓不同群体的编辑效率图；FIG15 is a graph showing the editing efficiency of different mouse bone marrow populations detected one week after LNP-3×NLS Cas9 mRNA/sgRNA was injected into humanized sickle cell anemia mice;
图16是LNP-3×NLS Cas9 mRNA/sgRNA体内注射人源化镰刀型细胞贫血症小鼠2周后使用高效液相色谱检测小鼠外周血中激活的珠蛋白含量图；FIG16 is a graph showing the activated globin content in the peripheral blood of humanized sickle cell anemia mice detected by high performance liquid chromatography 2 weeks after LNP-3×NLS Cas9 mRNA/sgRNA injection;
图17是LNP-3×NLS Cas9 mRNA/sgRNA体内注射人源化镰刀型细胞贫血症小鼠后不同时间点分别用流式、qPCR检测外周血珠蛋白表达细胞阳性率及mRNA含量图；FIG17 is a graph showing the positive rate of globin-expressing cells and mRNA content in peripheral blood detected by flow cytometry and qPCR at different time points after LNP-3×NLS Cas9 mRNA/sgRNA was injected into humanized sickle cell anemia mice in vivo;
图18是LNP-3×NLS Cas9 mRNA/sgRNA体内注射人源化镰刀型细胞贫血症小鼠后于治疗末期检测小鼠外周血血常规指标红细胞计数(RBC)、红细胞压积(HCT)、血红蛋白含量(HGB)图；FIG18 is a graph showing the peripheral blood routine indicators of red blood cell count (RBC), hematocrit (HCT), and hemoglobin content (HGB) detected in humanized sickle cell anemia mice after LNP-3×NLS Cas9 mRNA/sgRNA injection in vivo at the end of treatment;
图19是LNP-3×NLS Cas9 mRNA/sgRNA体内注射人源化镰刀型细胞贫血症小鼠后治疗末期检测小鼠外周血细胞形态、网织红细胞及镰刀型细胞图；FIG19 is a diagram showing the morphology of peripheral blood cells, reticulocytes, and sickle cells detected in mice at the end of treatment after LNP-3×NLS Cas9 mRNA/sgRNA was injected into humanized sickle cell anemia mice in vivo;
图20是LNP-3×NLS Cas9 mRNA/sgRNA体内注射人源化镰刀型细胞贫血症后治疗末期检测小鼠外周血肝炎相关因子谷丙转氨酶(ALT)、谷草转氨酶(AST)、白蛋白(ALB)及生存分析图。Figure 20 is a graph showing the hepatitis-related factors alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB) and survival analysis in the peripheral blood of mice after in vivo injection of LNP-3×NLS Cas9 mRNA/sgRNA into humanized sickle cell anemia at the end of treatment.
具体实施方式DETAILED DESCRIPTION
下文提供了本说明书中使用的一些术语的定义。除非另有说明，本文中使用的所有技术和科学用语通常具有和本发明所属领域的普通技术人员通常理解的意思相同的意思。The following provides definitions of some terms used in this specification. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
本发明提供了一种LNP-mRNA递送系统，所述LNP-mRNA递送系统包括负载一种或多种mRNA的LNP载体，所述LNP与mRNA的比例为(4-10)：1。The present invention provides an LNP-mRNA delivery system, which includes an LNP carrier loaded with one or more mRNAs, and the ratio of the LNP to the mRNA is (4-10):1.
在一些实施方式中，LNP包括可电离的氨基脂质、PEG脂质、磷脂、胆固醇。In some embodiments, the LNP comprises ionizable amino lipids, PEG lipids, phospholipids, cholesterol.
在一些实施方式中，可电离的氨基脂质是指可电离的氨基脂质是指一类含有氨基(-NH2或-NR2，R为烷基)且可以在特定条件下电离的脂质分子，包括但不限于ALC0315、SM-102。In some embodiments, ionizable amino lipids refer to a class of lipid molecules containing an amino group (-NH2 or -NR2, R is an alkyl group) and can be ionized under specific conditions, including but not limited to ALC0315 and SM-102.
在具体的实施方式中，所述可电离的氨基脂质选自ALC0315。In a specific embodiment, the ionizable amino lipid is selected from ALC0315.
在一些实施方式中，PEG脂质(聚乙二醇化脂质)是一类经过聚乙二醇(PEG)修饰的脂质分子。所述PEG脂质选自ALC0159(DMG-PEG2000)。In some embodiments, PEG lipids (PEGylated lipids) are lipid molecules modified with polyethylene glycol (PEG). The PEG lipids are selected from ALC0159 (DMG-PEG2000).
在一些实施方式中，磷脂是一类含有磷酸的脂类，是生物膜的主要组成成分。所述磷脂包括甘油磷脂、鞘磷脂、其他磷脂。In some embodiments, phospholipids are a type of lipid containing phosphoric acid and are the main components of biological membranes. The phospholipids include glycerophospholipids, sphingomyelin, and other phospholipids.
其中，甘油磷脂包括卵磷脂(磷脂酰胆碱)、脑磷脂(磷脂酰乙醇胺)、丝氨酸磷脂(磷脂酰丝氨酸)、肌醇磷脂(磷脂酰肌醇)、磷脂酰甘油。Among them, glycerophospholipids include phosphatidylcholine, cephalin (phosphatidylethanolamine), serine phospholipids (phosphatidylserine), inositol phospholipids (phosphatidylinositol), and phosphatidylglycerol.
在优选的实施方式中，磷脂选自甘油磷脂，所述甘油磷脂选自卵磷脂(磷脂酰胆碱)，所述卵磷脂(磷脂酰胆碱)选自DSPC(二硬脂酰磷脂酰胆碱，Distearoylphosphatidylcholine)。In a preferred embodiment, the phospholipid is selected from glycerophospholipids, the glycerophospholipid is selected from phosphatidylcholine, and the phosphatidylcholine is selected from DSPC (distearoylphosphatidylcholine).
本发明提供了GlutaMAX在制备提高细胞中蛋白翻译的产品中的应用。The present invention provides the use of GlutaMAX in preparing a product for improving protein translation in cells.
在一些实施方式中，所述产品可以为培养基。所述培养基为任意适于造血干细胞使用的培养基，所述培养基包括但不限于IMDM培养基、RPMI-1640培养基、DMEM/F-12培养基、培养基、StemSpanTMSFEM培养基。In some embodiments, the product can be a culture medium. The culture medium is any culture medium suitable for hematopoietic stem cells, including but not limited to IMDM culture medium, RPMI-1640 culture medium, DMEM/F-12 culture medium, Culture medium, StemSpan™ SFEM medium.
在具体的实施方式中，所述培养基选自IMDM培养基。In a specific embodiment, the culture medium is selected from IMDM medium.
在一些实施方式中，所述培养基还包括其他因子，所述其他因子包括但不限于FBS、BSA、双抗、SCF、白细胞介素、β-巯基乙醇、丙酮酸钠、促红细胞生成素(EPO)、粒细胞刺激因子(G-CSF)、巨核细胞刺激因子(M-CSF)。In some embodiments, the culture medium further comprises other factors, including but not limited to FBS, BSA, double antibody, SCF, interleukin, β-mercaptoethanol, sodium pyruvate, erythropoietin (EPO), granulocyte stimulating factor (G-CSF), and megakaryocyte stimulating factor (M-CSF).
本发明提供了上述LNP-mRNA递送系统或上述脂质纳米颗粒在制备治疗疾病的药物组合物中的应用。The present invention provides use of the LNP-mRNA delivery system or the lipid nanoparticles in preparing a pharmaceutical composition for treating a disease.
所述药物组合物还包括药学上可接受的辅料。The pharmaceutical composition further includes pharmaceutically acceptable excipients.
在一些实施方式中，药学上可接受的辅料用于指这样一种材料，该材料与接受对象，优选哺乳动物，更优选人相容，并且适于将活性剂递送到目标部位，同时不会终止该药剂的活性。与该药学上可接受的辅料相关的毒性或副作用(如果存在)优选与用于活性剂预定用途的合理风险/效益比相称。In some embodiments, a pharmaceutically acceptable excipient is used to refer to a material that is compatible with a recipient, preferably a mammal, more preferably a human, and suitable for delivering the active agent to the target site without disrupting the activity of the agent. The toxicity or side effects associated with the pharmaceutically acceptable excipient (if any) are preferably commensurate with a reasonable risk/benefit ratio for the intended use of the active agent.
药学上可接受的辅料包括但不限于稀释剂、粘合剂、表面活性剂、致湿剂、吸附载体、润滑剂、填充剂、崩解剂。这些辅料根据需要用于帮助配方的稳定性或有助于提高活性或它的生物有效性或在口服的情况下产生可接受的口感或气味，在这种药物中可以使用的制剂可以是其原始化合物本身的形式或任选地使用其药物学可接受的盐的形式，如此配制的药物组合物根据需要可选择本领域技术人员已知的任何适当的方式给药。Pharmaceutically acceptable excipients include, but are not limited to, diluents, binders, surfactants, humectants, adsorbents, lubricants, fillers, and disintegrants. These excipients are used, as needed, to enhance the stability of the formulation, improve its activity or bioavailability, or produce an acceptable taste or odor when taken orally. The formulations used in such drugs may be in the form of the original compound itself or, optionally, in the form of a pharmaceutically acceptable salt. The pharmaceutical composition thus formulated may be administered in any appropriate manner known to those skilled in the art.
其中，稀释剂包括但不限于乳糖、氯化钠、葡萄糖、尿素、淀粉、水。Among them, diluents include but are not limited to lactose, sodium chloride, glucose, urea, starch, and water.
粘合剂包括但不限于淀粉、预胶化淀粉、糊精、麦芽糖糊精、蔗糖、阿拉伯胶、明胶、甲基纤维素、羧甲基纤维素、乙基纤维素、聚乙烯醇、聚乙二醇、聚乙烯比咯烷酮、海藻酸及海藻酸盐、黄原胶、羟丙基纤维素和羟丙基甲基纤维素。Binders include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, and hydroxypropyl methylcellulose.
表面活性剂包括但不限于聚氧化乙烯山梨聚糖脂肪酸酯、十二烷基硫酸钠、硬脂酸单甘油酯、十六烷醇。Surfactants include, but are not limited to, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, and cetyl alcohol.
致湿剂包括但不限于甘油。Humectants include, but are not limited to, glycerin.
吸附载体包括但不限于斑脱土、硅胶、高岭土和皂粘土。Adsorptive supports include, but are not limited to, bentonite, silica gel, kaolin, and bentonite.
润滑剂包括但不限于硬脂酸锌、单硬脂酸甘油酯、聚乙二醇、滑石粉、硬脂酸钙和镁、聚乙二醇、硼酸粉末、氢化植物油、硬脂富马酸钠、聚氧乙烯单硬脂酸酯、单月桂蔗糖酸酯、月桂醇硫酸钠、月桂醇硫酸镁、十二烷基硫酸镁。Lubricants include, but are not limited to, zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium lauryl sulfate.
填充剂包括但不限于甘露醇(粒状或粉状)、木糖醇、山梨醇、麦芽糖、赤藓糖、微晶纤维素、聚合糖、偶合糖、葡萄糖、乳糖、蔗糖、糊精、淀粉、海藻酸钠、海带多糖粉末、琼脂粉末、碳酸钙和碳酸氢钠。Fillers include, but are not limited to, mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugars, coupling sugars, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, and sodium bicarbonate.
崩解剂包括但不限于交联乙烯吡咯烷酮、羧甲基淀粉钠、低取代羟丙基甲基、交联羧甲基纤维素钠、大豆多糖。Disintegrants include, but are not limited to, cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, and soybean polysaccharides.
下面结合具体实施例进一步阐述此发明。应理解的是，在此描述的特定实施方式通过举例的方式来表示，并不作为对本发明的限制。在不偏离本发明范围的情况下，本发明的主要特征可以用于各种实施方式。The present invention will be further described below with reference to specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the present invention. The main features of the present invention may be applied to various embodiments without departing from the scope of the present invention.
实施例1LNP载体的配方及制备工艺研究Example 1 Study on the formulation and preparation process of LNP carrier
1.实验材料及方法1. Materials and Methods
1)实验材料1) Experimental Materials
脂质材料配方：ALC0315(medkoo，556006)、ALC0159(medkoo，556014)、DSPC(Avanti，850365)、Cholesterol(Echelon Biosciences，L-6012)；Quant-iTTMRNA试剂(Thermo Fisher，R11490)、TritonTMX-100溶液(Sigma-Aldrich，93443)；柠檬酸(C6H8O7·H2O)、柠檬酸钠(C6H5Na3O7·2H2O)、DEPC水；无菌无酶超纯水、无水乙醇、PBS缓冲液；带螺旋口的注射器(新华)、1.5ml、15ml、50ml无菌无酶管、超滤管(Merck，UFC903024)、高速离心机、酶标仪、马尔文动态光散射仪。Lipid material formulation: ALC0315 (medkoo, 556006), ALC0159 (medkoo, 556014), DSPC (Avanti, 850365), Cholesterol (Echelon Biosciences, L-6012); Quant-iT™ RNA reagent (Thermo Fisher, R11490), Triton™ X-100 solution (Sigma-Aldrich, 93443); citric acid (C6 H8 O7 ·H2 O), sodium citrate (C6 H5 Na3 O7 ·2H2 O), DEPC water; sterile enzyme-free ultrapure water, anhydrous ethanol, PBS buffer; screw-cap syringe (Xinhua), 1.5 ml, 15 ml, and 50 ml sterile enzyme-free tubes, ultrafiltration tubes (Merck, UFC903024), high-speed centrifuge, microplate reader, and Malvern dynamic light scattering instrument.
2)实验方法2) Experimental methods
按照ALC0315：DSPC：Chol：ALC0159(DMG-PEG2000)摩尔比依次为50：10：38.5：1.5的比例制备脂质乙醇溶液，将mRNA测定浓度后根据脂质浓度使用pH＝4柠檬酸钠液稀释至所需浓度。利用注射泵、T-junction及连接管路搭建LNP制备装置，将脂质分子的乙醇溶液及mRNA水溶液按一定比例进行混合获得LNP-mRNA，对收集到的溶液用50倍的PBS稀释，超滤纯化后用动态光散射仪测定LNP-mRNA的粒径、均一度及ζ电位。使用Quant-iTTMRNA试剂检测用TritonTMX-100溶液破乳后及破乳前的核酸量，通过核酸量的差异计算得到载药量及包封率。A lipid ethanol solution was prepared with a molar ratio of ALC0315:DSPC:Chol:ALC0159(DMG-PEG2000) of 50:10:38.5:1.5. The mRNA concentration was measured and diluted to the desired concentration using pH = 4 sodium citrate solution based on the lipid concentration. A LNP preparation device was constructed using a syringe pump, T-junction, and connecting tubing. The ethanol solution of lipid molecules and the mRNA aqueous solution were mixed in a certain ratio to obtain LNP-mRNA. The collected solution was diluted 50 times with PBS and purified by ultrafiltration. The particle size, uniformity, and zeta potential of the LNP-mRNA were measured using a dynamic light scattering instrument. Quant-iT™ was used to analyze the LNP-mRNA. RNA reagent detection uses Triton™ X-100 solution to measure the amount of nucleic acid before and after demulsification. The drug loading capacity and encapsulation efficiency are calculated based on the difference in nucleic acid amount.
图1：包封EGFP-mRNA的LNP对人类髓性白血病细胞K562细胞、人造血干细胞CD34+细胞进行转染分组：K562细胞未处理阴性对照组(Control)、LNP-EGFP mRNA转染组(LNP)、RNA转染试剂RNAiMAX阳性对照组(RNAiMAX)；CD34+细胞未处理阴性对照组(Control)、LNP-EGFP mRNA转染组(LNP)、RNA转染试剂RNAiMAX阳性对照组(RNAiMAX)。Figure 1: Human myeloid leukemia K562 cells and human hematopoietic stem cell CD34+ cells were transfected with EGFP-mRNA-encapsulated LNPs and divided into the following groups: K562 cells were treated with an untreated negative control group (Control), a group transfected with LNP-EGFP mRNA (LNP), and a positive control group (RNAiMAX) using the RNA transfection reagent; CD34+ cells were treated with an untreated negative control group (Control), a group transfected with LNP-EGFP mRNA (LNP), and a positive control group (RNAiMAX) using the RNA transfection reagent.
轻柔吹打细胞，分别用含1％双抗、10％ FBS的1640培养基(K562细胞)；含30％FBS、10％ BSA、1％双抗、SCF、IL-3、β-巯基乙醇、丙酮酸钠的IMDM培养基收集细胞悬液(CD34+细胞)。室温800rpm离心5min(K562)，室温800g离心10min(CD34+细胞)，分别计数后取0.5×106个细胞/24孔板。用RNA转染试剂RNAiMAX作为对照转染实验。LNP转染组则直接加入包封mRNA完成的LNP，转染量达到1μg即可。通过流式细胞仪检测细胞EGFP阳性率、细胞活率。Gently pipette cells and collect the cell suspension (CD34+ cells) in 1640 medium supplemented with 1% double-antibody and 10% FBS (K562 cells); or in IMDM medium supplemented with 30% FBS, 10% BSA, 1% double-antibody, SCF, IL-3, β-mercaptoethanol, and sodium pyruvate. The cells were centrifuged at 800 rpm for 5 minutes at room temperature (K562 cells) and 800 g for 10 minutes at room temperature (CD34+ cells). Count and count 0.5×106 cells/24-well plate. RNAiMAX, an RNA transfection reagent, was used as a control transfection experiment. For the LNP transfection group, mRNA-encapsulated LNPs were directly added, with a transfection dose of 1 μg. Flow cytometry was used to determine EGFP positivity and cell viability.
不同阳离子脂质与mRNA的相对比例LNP合成LNP synthesis with different relative ratios of cationic lipids and mRNA
图2：合成过程见1)和2)，按照阳离子脂质与mRNA的相对质量比例分别为4：1-10：1合成4种LNP，使用马尔文粒径仪对颗粒电位、粒径、PDI进行物理表征检测。Figure 2: The synthesis process is shown in 1) and 2). Four types of LNPs were synthesized according to the relative mass ratio of cationic lipid to mRNA of 4:1-10:1, and the particle potential, particle size, and PDI were physically characterized and tested using a Malvern particle size analyzer.
图3：CD34+细胞未处理阴性对照组、LNP-EGFP mRNA转染组(按照阳离子脂质与mRNA的相对质量比例分别为4：1-10：1合成4种LNP转染CD34+细胞后，转染操作同图1的方法，转染剂量为1μg，于48h通过流式细胞仪检测细胞EGFP阳性率、细胞活率。Figure 3: CD34+ cells in the untreated negative control group and the LNP-EGFP mRNA transfection group (Four LNPs were synthesized at a relative mass ratio of cationic lipid to mRNA of 4:1-10:1 and transfected into CD34+ cells. The transfection procedure was the same as in Figure 1, with a transfection dose of 1 μg. The EGFP positivity rate and cell viability of the cells were detected by flow cytometry at 48 hours.
图4：CD34+细胞未处理阴性对照组、LNP-EGFP mRNA转染组(不同剂量组1、2、4、8μg)转染CD34+细胞后，转染操作同图1的方法，于不同时间点(48、72、96h)收样，通过流式细胞仪检测细胞EGFP阳性率、细胞活率。Figure 4: CD34+ cells were transfected into the untreated negative control group and the LNP-EGFP mRNA transfection group (different dose groups: 1, 2,4 , and 8 μg). The transfection procedure was the same as that in Figure 1. Samples were collected at different time points (48, 72, and 96 hours) and the EGFP positivity and cell viability of the cells were detected by flow cytometry.
图5：阳离子脂质与mRNA的比例确定为8：1后，以乙醇相中总脂质浓度分别配置为12mM、8mM、6mM、4mM分别与水相混合配置成不同LNP(不同剂量组2、4μg)转染CD34+细胞后，转染操作同图1的方法，于48h荧光显微镜下拍照观察细胞绿色荧光强度。Figure 5: After the ratio of cationic lipid to mRNA was determined to be 8:1, the total lipid concentrations in the ethanol phase were adjusted to 12mM, 8mM, 6mM, and 4mM, respectively, and mixed with the aqueous phase to prepare different LNPs (2 and 4μg in different dose groups). After transfection into CD34+ cells, the transfection operation was the same as that in Figure 1. The green fluorescence intensity of the cells was observed under a fluorescence microscope after 48 hours.
图7：不同Cas9 mRNA：sgRNA比例合成LNP，合成过程见图7上，按照1×NLS Cas9mRNA(云舟生物有限公司合成)：sgRNA(Sythgo公司合成，sgRNA序列：CUUGUCAAGGCUAUUGGUCA(SEQ ID NO：1))Cas9 mRNA、sgRNA比例分别为4：1、3：1、2：1、1：2、1：3合成4种LNP，使用马尔文粒径仪对颗粒电位、粒径、PDI进行物理表征检测。以4μg剂量转染CD34+细胞、K562细胞后，转染操作同图1的方法，室温800g离心10min后弃上清，使用DNA提取试剂盒(全式金，EE181-01)提取DNA，PCR扩增后进行琼脂糖电泳，切下条带送测进行Sanger一代测序检测基因编辑。Figure 7: LNPs synthesized at different Cas9 mRNA:sgRNA ratios. The synthesis process is shown in Figure 7 (top). Four types of LNPs were synthesized using 1×NLS Cas9 mRNA (synthesized by Yunzhou Biotechnology Co., Ltd.): sgRNA (synthesized by Sythgo, sgRNA sequence: CUUGUCAAGGCUAUUGGUCA (SEQ ID NO: 1)) at Cas9 mRNA:sgRNA ratios of 4:1, 3:1, 2:1, 1:2, and 1:3, respectively. Particle potential, size, and PDI were physically characterized using a Malvern particle size analyzer. CD34+ cells and K562 cells were transfected with a 4 μg dose, following the same transfection procedure as in Figure 1. The cells were centrifuged at 800 g for 10 minutes at room temperature, and the supernatant was discarded. DNA was extracted using a DNA extraction kit (Quanshijin, EE181-01). PCR amplification was performed by agarose gel electrophoresis, and the bands were excised and sent for Sanger sequencing to confirm gene editing.
编辑系统Cas9 mRNA：sgRNA比例、Cas9 mRNA入核信号设计、最优编辑体系的探索Cas9 mRNA editing system: sgRNA ratio, Cas9 mRNA nuclear entry signal design, and exploration of the optimal editing system
图8：GlutaMAXTM未添加组(0mM)、GlutaMAXTM添加低剂量组(1mM)、GlutaMAXTM添加中剂量组(2mM)、GlutaMAXTM添加高剂量组(4mM)。Figure 8: GlutaMAX™ -free group (0 mM), GlutaMAX™ -low-dose group (1 mM), GlutaMAX™ -medium-dose group (2 mM), and GlutaMAX™ -high-dose group (4 mM).
LNP-1×NLS Cas9 mRNA/sgRNA转染细胞以4μg剂量转染CD34+细胞，培养基体系见图1中所使用的培养体系，额外添加不同浓度剂量GlutaMAXTM，于转染后72h收集细胞裂解液处理后提取蛋白，进行Western blotting实验，Cas9蛋白试剂(Cell SignalingTechnology，14697)、GAPDH抗体(Proteintech，20536-1-AP)。CD34+ cells were transfected with LNP-1×NLS Cas9 mRNA/sgRNA at a dose of 4 μg. The culture medium system was shown in Figure 1 . GlutaMAX™ at various concentrations was added. Cell lysates were collected 72 hours after transfection and processed for protein extraction for Western blotting. Cas9 protein reagent (Cell Signaling Technology, 14697) and GAPDH antibody (Proteintech, 20536-1-AP) were used.
图9：CD34+细胞未处理阴性对照组、LNP-1×NLS Cas9 mRNA/sgRNA转染组、LNP-3×NLS Cas9 mRNA/sgRNA转染组在具有1个入核信号(NLS)的Cas9 mRNA的基础上，设计出带有3个NLS的Cas9 mRNA(云舟生物进行合成)。按照Cas9 mRNA：sgRNA比例为1：2合成LNP，以4μg剂量转染CD34+细胞，在转染72h(3dpt)后进行红系诱导分化，于不同时间节点(ED d4-EDd8)取细胞室温800g离心10min后弃上清，使用DNA提取试剂盒(全式金，EE181-01)提取DNA，PCR扩增后进行琼脂糖电泳，切下条带送测进行Sanger一代测序检测基因编辑。Figure 9: CD34+ cells in an untreated negative control group, a LNP-1×NLS Cas9 mRNA/sgRNA transfection group, and a LNP-3×NLS Cas9 mRNA/sgRNA transfection group. Based on the Cas9 mRNA with a single nuclear import signal (NLS), a Cas9 mRNA with three NLSs was designed (synthesized by Yunzhou Biotechnology). LNPs were synthesized at a 1:2 ratio of Cas9 mRNA:sgRNA and transfected into CD34+ cells at a dose of 4 μg. Erythroid differentiation was induced 72 hours after transfection (3 days post-transfection). At various time points (ED days 4-8), cells were harvested and centrifuged at 800 g for 10 minutes at room temperature. The supernatant was discarded, and DNA was extracted using a DNA extraction kit (Quanshijin, EE181-01). PCR amplification was performed on agarose gel electrophoresis, and the bands were excised and sent for Sanger sequencing to confirm gene editing.
图10：CD34+细胞未处理阴性对照组、LNP-1×NLS Cas9 mRNA/sgRNA转染组、LNP-3×NLS Cas9 mRNA/sgRNA转染组取转染72h后的CD34+细胞接种于固体培养基(stemcell，H4434)15天后镜下拍照并收集CFU菌落提取DNA后检测基因编辑。使用DNA提取试剂盒(全式金，EE181-01)提取DNA，PCR扩增后进行琼脂糖电泳，切下条带送测进行Sanger一代测序检测基因编辑。Figure 10: CD34+ cells from the untreated negative control group, the LNP-1×NLS Cas9 mRNA/sgRNA transfection group, and the LNP-3×NLS Cas9 mRNA/sgRNA transfection group. 72 hours after transfection, CD34+ cells were plated on solid culture medium (stem cell, H4434). 15 days later, images were taken under a microscope, and colony-forming units (CFUs) were collected for DNA extraction and detection of gene editing. DNA was extracted using a DNA extraction kit (Quanshijin, EE181-01), amplified by PCR, and then subjected to agarose gel electrophoresis. Bands were excised and sent for Sanger sequencing to detect gene editing.
图11：CD34+细胞未处理阴性对照组、LNP-3×NLS Cas9 mRNA/sgRNA转染组于转染后细胞红系诱导分化后第4天收集细胞并在PBS中洗涤，并使用根据制造商的方案分离总RNA含量(全式金，ER601-01-V2)。使用M-MLV逆转录酶(Promega，麦迪逊，威斯康星州，美国)进行逆转录。使用实时定量PCR分析GAPDH、HBB和HBG的表达水平。所有实时PCR反应均使用Biorad的实时PCR检测系统进行。通过熔解曲线分析确认产品的质量。使用以下表达引物：用于GAPDH的正向(F)引物CATTGCCCTCAACGACCACT(SEQ ID NO：2)和反向(R)引物GGTGGTCCAGGGGTCTTACT(SEQ ID NO：3)，用于HBB的F引物GCCCTGGCCCACAAGTATC(SEQ IDNO：4)和R引物GCCCTTCATAATATCCCCCAGTT(SEQ ID NO：5)，用于HBG的F引物GGTGACCGTTTTTGGCAATCC(SEQ ID NO：6)和R引物GTATCTGGAGGACAGGGCAC(SEQ ID NO：7)。HBG mRNA百分比是指相对于γ-珠蛋白和β-珠蛋白转录物丰度总和表达的HBG mRNA丰度([HBG/(HBB+HBG]*100))。Figure 11: CD34+ cells in the untreated negative control group and the LNP-3×NLS Cas9 mRNA/sgRNA transfection group were collected and washed in PBS on day 4 after erythroid differentiation induction after transfection, and the total RNA content was isolated using a total RNA isolation kit (Full-Form Gold, ER601-01-V2) according to the manufacturer's protocol. Reverse transcription was performed using M-MLV reverse transcriptase (Promega, Madison, WI, USA). Real-time quantitative PCR was used to analyze the expression levels of GAPDH, HBB, and HBG. All real-time PCR reactions were performed using a Biorad real-time PCR detection system. The quality of the product was confirmed by melting curve analysis. The following expression primers were used: forward (F) primer CATTGCCCTCAACGACCACT (SEQ ID NO: 2) and reverse (R) primer GGTGGTCCAGGGGTCTTACT (SEQ ID NO: 3) for GAPDH, F primer GCCCTGGCCCACAAGTATC (SEQ ID NO: 4) and R primer GCCCTTCATAATATCCCCCAGTT (SEQ ID NO: 5) for HBB, and F primer GGTGACCGTTTTTGGCAATCC (SEQ ID NO: 6) and R primer GTATCTGGAGGACAGGGCAC (SEQ ID NO: 7) for HBG. The HBG mRNA percentage refers to the abundance of HBG mRNA expressed relative to the sum of the abundance of γ-globin and β-globin transcripts ([HBG/(HBB+HBG]*100)).
为了确定细胞中HbF的表达，根据制造商的说明，使用细胞内标记试剂盒(InsideStain Kit，Miltenyi Biotec)和抗HbF-Fitc抗体(Miltenyi Biotec)对细胞进行固定、透化和染色后，流式上机检测。To determine the expression of HbF in cells, the cells were fixed, permeabilized, and stained using an intracellular labeling kit (InsideStain Kit, Miltenyi Biotec) and anti-HbF-Fitc antibody (Miltenyi Biotec) according to the manufacturer's instructions, and then detected by flow cytometry.
为了检测细胞红系分化能力，使用Brilliant Violet 421TManti-human CD71Antibody(Biolegend，334121)、APC anti-human CD235ab Antibody(Biolegend，306608)孵育细胞后，PBS洗涤后上机检测。To detect the erythroid differentiation ability of cells, the cells were incubated with Brilliant Violet 421™ anti-human CD71 Antibody (Biolegend, 334121) and APC anti-human CD235ab Antibody (Biolegend, 306608), washed with PBS and then tested on an immunoprecipitation machine.
2.实验结果2. Experimental Results
1)脂质材料配方的比例1) Ratio of lipid material formula
首先，按照文献常规描述进行LNP合成时，得到的颗粒与市面上常见转染试剂RNAiMAX(阴性对照)一样，可高效转染K562细胞，但无法在体外有效转染人造血干细胞(图1)。First, when LNP synthesis was performed according to the general description in the literature, the resulting particles, like the common transfection reagent RNAiMAX (negative control) on the market, could efficiently transfect K562 cells, but could not effectively transfect human hematopoietic stem cells in vitro (Figure 1).
进一步考察阳离子脂质与mRNA的相对比例(4：1-10：1)，LNP-mRNA理化性质的影响规律。首先，所制备的纳米颗粒粒径小、均一性好，粒径大小均保持在100nm左右，PDI良好(≤0.3为宜)。此外，随着阳离子脂质的占比增加，电位逐渐增加(图2)。在人造血干细胞CD34+进行研究显示，阳离子脂质占比太低或太高均会对转染阳性率有显著影响，二者比例为8：1、10：1时，转染效率最好且并不影响细胞活率(图3)。The influence of the relative ratio of cationic lipids to mRNA (4:1-10:1) and the physical and chemical properties of LNP-mRNA were further investigated. First, the prepared nanoparticles had a small particle size and good uniformity. The particle size was maintained at around 100 nm, and the PDI was good (≤0.3 is appropriate). In addition, as the proportion of cationic lipids increased, the potential gradually increased (Figure 2). Studies on human hematopoietic stem cells CD34+ showed that a too low or too high proportion of cationic lipids would have a significant effect on the transfection positive rate. When the ratio of the two was 8:1 and 10:1, the transfection efficiency was the best and did not affect the cell viability (Figure 3).
以上结果表明，阳离子脂质对LNP的转染人造血干细胞有较大影响，是脂质配方中的关键成分。在优化脂质配方时，需要综合考虑阳离子脂质的种类、比例及绝对浓度。阳离子脂质：EGFP mRNA比例为8：1时，粒径稳定(100nm左右)，PDI最小即粒径均一，转染效率良好，因此，后续将选择该比例进行后续实验。These results demonstrate that cationic lipids significantly impact LNP transfection into human hematopoietic stem cells and are a key component of the lipid formulation. When optimizing the lipid formulation, the type, ratio, and absolute concentration of the cationic lipid must be comprehensively considered. A cationic lipid:EGFP mRNA ratio of 8:1 resulted in a stable particle size (around 100 nm), minimal PDI (i.e., uniform particle size), and excellent transfection efficiency. Therefore, this ratio will be selected for subsequent experiments.
连续检测至96h的结果显示，随着时间延长，转染效率逐渐增高且最终维持在98％的高转染效率(图4)，细胞活性检测显示，该脂质体不会影响细胞正常生长。The results of continuous testing up to 96 hours showed that the transfection efficiency gradually increased with time and finally maintained at a high transfection efficiency of 98% (Figure 4). Cell activity detection showed that the liposomes did not affect the normal growth of cells.
2)转染造血干细胞的油相最优浓度2) Optimal concentration of oil phase for transfection of hematopoietic stem cells
合成LNP时，分为乙醇相(溶解脂质材料，简称油相)、水相(溶解mRNA，简称水相)，本申请研究发现，在可转染人造血干细胞的最优比例基础LNP配方上，仅通过调整油相浓度，可进一步增强脂质转染人造血干细胞并强表达EGFP荧光蛋白。对于人造血干细胞而言，当LNP合成时，保持油相浓度为6mM效果最好(图5)。During LNP synthesis, there is an ethanol phase (dissolving the lipid material, referred to as the oil phase) and an aqueous phase (dissolving the mRNA, referred to as the aqueous phase). This study found that, based on the optimal ratio of the basic LNP formulation for transfection of human hematopoietic stem cells, simply adjusting the oil phase concentration can further enhance lipid transfection of human hematopoietic stem cells and strongly express the EGFP fluorescent protein. For human hematopoietic stem cells, maintaining an oil phase concentration of 6 mM during LNP synthesis achieved the best results (Figure 5).
3)编辑系统Cas9 mRNA：sgRNA比例、Cas9 mRNA入核信号设计、最优编辑体系的探索目前，基因调控元件作为靶点再激活γ-珠蛋白(HBG)表达治疗地贫、镰贫以不受突变位点影响、使用范围广成为近两年的趋势；BCL11A蛋白对HbF蛋白的表达起负性调控作用；本申请针对HBG基因启动子区的BCL11A结合位点进行基因编辑以达到在激活γ-珠蛋白表达。在1×NLS Cas9 mRNA3) Editing system Cas9 mRNA: sgRNA ratio, Cas9 mRNA nuclear entry signal design, and exploration of the optimal editing system. At present, gene regulatory elements are used as targets to reactivate γ-globin (HBG) expression to treat thalassemia and sickle cell anemia. This trend has become widespread in the past two years because it is not affected by mutation sites and has a wide range of uses. BCL11A protein negatively regulates the expression of HbF protein. This application targets the BCL11A binding site in the HBG gene promoter region for gene editing to activate γ-globin expression. In 1×NLS Cas9 mRNA
(Cas9-Nucleoplasmin NLS)的基础上、额外添加2个入核信号即3×NLS Cas9mRNA(Myc NLS-Cas9-SV40 NLS-Nucleoplasmin NLS)，编辑策略sgRNA HBG靶向位点见图6。Based on the Cas9-Nucleoplasmin NLS, two additional nuclear import signals, namely 3×NLS Cas9mRNA (Myc NLS-Cas9-SV40 NLS-Nucleoplasmin NLS), were added. The editing strategy sgRNA HBG targeting site is shown in Figure 6.
用上述LNP基础配方包封编辑系统，鉴于原代细胞难以转染和编辑的特性，本申请合成了LNP-Cas9 mRNA：sgRNA-HBG＝4：1、3：1、2：1、1：2、1：3，体外转染细胞后，于48h检测编辑效率。结果显示，针对人造血干细胞而言，当比例为1：2时编辑效率最高，提示对于人造血干细胞，2种RNA的含量存在最优比，过多或过少均会显著影响原代细胞的编辑效率。此外，当比例为1：2时，对K562细胞进行高效编辑达80％，对CD34+细胞编辑达8％，进一步说明原代细胞转染及编辑难度远大于细胞系的转染与编辑(图7)。The editing system was encapsulated using the above-mentioned LNP basic formula. In view of the fact that primary cells are difficult to transfect and edit, this application synthesized LNP-Cas9 mRNA:sgRNA-HBG=4:1, 3:1, 2:1, 1:2, 1:3. After in vitro transfection of cells, the editing efficiency was detected at 48h. The results showed that for human hematopoietic stem cells, the editing efficiency was highest when the ratio was 1:2, suggesting that for human hematopoietic stem cells, there is an optimal ratio for the content of the two RNAs, and too much or too little will significantly affect the editing efficiency of primary cells. In addition, when the ratio was 1:2, K562 cells were efficiently edited by 80%, and CD34+ cells were edited by 8%, further illustrating that the difficulty of transfection and editing of primary cells is much greater than that of transfection and editing of cell lines (Figure 7).
为进一步提高编辑效率，本申请研究发现，在培养基中额外添加GlutaMAXTM添加剂可显著提高LNP转染人造血干细胞后细胞内Cas9 mRNA的翻译。后续实验使用该培养基体系进行下一步实验(图8)。To further enhance editing efficiency, this study found that adding GlutaMAX™ to the culture medium significantly increased Cas9 mRNA translation in human hematopoietic stem cells after LNP transfection. This culture medium system was used in subsequent experiments (Figure 8).
已知Cas9 mRNA翻译后入核进行编辑，本申请在具有1个入核信号(NLS)的Cas9mRNA的基础上，设计出带有3个NLS的入核信号，用LNP包封后4μg转染细胞进行人造血干细胞的编辑，结果显示3×NLS Cas9 mRNA达到最高的编辑效果(图9)，并且本申请的体外转染体系仅低剂量4μg便可实现高达70％的编辑，与现有研究相比，使用剂量更低。对于人造血干细胞这种原代细胞而言，入核信号的设计(种类选择、数量)至关重要。It is known that Cas9 mRNA is translated and imported into the nucleus for editing. Based on the Cas9 mRNA with one nuclear import signal (NLS), this application designed a nuclear import signal with three NLSs. After encapsulation with LNP, 4 μg of cells were transfected for editing of human hematopoietic stem cells. The results showed that 3×NLS Cas9 mRNA achieved the highest editing effect (Figure 9), and the in vitro transfection system of this application achieved up to 70% editing with only a low dose of 4 μg, which is a lower dose than existing studies. For primary cells such as human hematopoietic stem cells, the design of the nuclear import signal (type selection and quantity) is crucial.
进一步对LNP-1×NLS Cas9 mRNA/sgRNA、LNP-3×NLS Cas9 mRNA/sgRNA转染72h后的细胞接种固体培养基后，于15天后显微镜下拍照并检测编辑效率，结果显示，与未转染的细胞相比，LNP-1×NLS Cas9 mRNA/sgRNA、LNP-3×NLS Cas9 mRNA/sgRNA转染对祖细胞集落形成没有显著影响，同时，LNP-3×NLS Cas9 mRNA/sgRNA转染细胞后集落编辑效率更高(图10)。由于本申请的编辑策略是激活γ-珠蛋白的表达，紧接着，对LNP-3×NLS Cas9mRNA/sgRNA转染后的CD34+细胞进行红系诱导分化后表型检测，结果显示，与未处理的CD34+细胞相比，转染后的细胞于诱导后Day4，HBG mRNA表达显著升高，于Day4、Day8、Day11流式检测珠蛋白HbF表达，均显著高于未处理组，CD235a+CD71+群体阳性率2组未有显著影响，提示本申请优化后的编辑系统在高效编辑原代细胞CD34+细胞的同时，不会影响细胞正常的红系分化能力(图11)。After 72 hours of transfection with LNP-1×NLS Cas9 mRNA/sgRNA and LNP-3×NLS Cas9 mRNA/sgRNA, cells were inoculated into solid culture medium and photographed under a microscope 15 days later to detect the editing efficiency. The results showed that compared with untransfected cells, LNP-1×NLS Cas9 mRNA/sgRNA and LNP-3×NLS Cas9 mRNA/sgRNA transfection had no significant effect on progenitor cell colony formation. At the same time, the colony editing efficiency of cells transfected with LNP-3×NLS Cas9 mRNA/sgRNA was higher (Figure 10). Since the editing strategy of this application is to activate the expression of γ-globin, the CD34+ cells transfected with LNP-3×NLS Cas9mRNA/sgRNA were then subjected to phenotypic detection after erythroid differentiation induction. The results showed that compared with untreated CD34+ cells, the transfected cells had significantly increased HBG mRNA expression on Day 4 after induction, and the expression of globin HbF was detected by flow cytometry on Day 4, Day 8, and Day 11, which were significantly higher than those of the untreated group. The positive rate of the CD235a+ CD71+ population was not significantly affected in the two groups, indicating that the optimized editing system of this application can efficiently edit primary CD34+ cells without affecting the normal erythroid differentiation ability of the cells (Figure 11).
综上所述，本申请摸索出一套成熟的体外转染和编辑人造血干细胞的体系，从每个参数工艺的调整上，已成功实现不用连接任何抗体便可以使用基础LNP以低剂量实现高效转染、编辑人造血干细胞，并显著提高珠蛋白表达。与其他研究相比，节约成本，合成更加简便。In summary, this application has developed a mature in vitro system for transfecting and editing human hematopoietic stem cells. By adjusting each parameter and process, we have successfully achieved efficient transfection and editing of human hematopoietic stem cells at low doses using basic LNPs without conjugating them to any antibodies, and significantly increased globin expression. Compared to other studies, this system is cost-effective and simpler to synthesize.
实施例2体内递送影响因素研究Example 2 Study on factors affecting in vivo delivery
1.实验方法1. Experimental Methods
图12：按照试剂说明书溶解及注射小鼠(皮下)后，于不同时间点尾静脉取血50μL，孵育小鼠抗体PerCP Cy5 anti-Mouse Lin-、BV510 anti-Mouse Ckit、PE-Cy7 anti-MouseSca1，检测小鼠造血干细胞从骨髓中动员到外周血的阳性率。Figure 12: After dissolution and subcutaneous injection into mice according to the reagent instructions, 50 μL of blood was collected from the tail vein at different time points and incubated with mouse antibodies PerCP Cy5 anti-Mouse Lin- , BV510 anti-Mouse Ckit, and PE-Cy7 anti-MouseSca1 to detect the positive rate of mouse hematopoietic stem cell mobilization from the bone marrow to the peripheral blood.
图13：按照试剂说明书溶解及注射小鼠(皮下)后，于不同时间点尾静脉取血50μL，孵育小鼠抗体PerCP Cy5 anti-mouse Lin-、BV510 anti-mouse Ckit、PE-Cy7 anti-mouseSca1，检测小鼠造血干细胞从骨髓中动员到外周血的阳性率(抗体均来自于Biolegend)。Figure 13: After dissolution and subcutaneous injection into mice according to the reagent instructions, 50 μL of blood was collected from the tail vein at different time points and incubated with mouse antibodies PerCP Cy5 anti-mouse Lin- , BV510 anti-mouse Ckit, and PE-Cy7 anti-mouse Sca1 to detect the positive rate of mouse hematopoietic stem cell mobilization from the bone marrow to the peripheral blood (all antibodies are from Biolegend).
图14：PBS注射对照组、LNP-EGFP mRNA注射组(动员注射不同部位)。Figure 14: PBS injection control group and LNP-EGFP mRNA injection group (mobilization injection at different sites).
按照上述最优配方合成LNP-EGFP mRNA后通过尾静脉注射小鼠，于24h取小鼠骨髓细胞，孵育小鼠抗体BV605 anti-mouse Lin-、BV421 anti-mouse Ckit、PE-Cy7 anti-mouse Sca1、PE anti-mouse CD135、APC anti-mouse CD34，流式检测小鼠造血干细胞不同群体LSK、LT-LSK、ST-LSK中EGFP mRNA表达阳性率(抗体均来自于Biolegend)。LNP-EGFP mRNA was synthesized according to the optimal recipe and injected into mice via the tail vein. Bone marrow cells were harvested 24 hours later and incubated with mouse antibodies BV605 anti-mouseLin- , BV421 anti-mouse Ckit, PE-Cy7 anti-mouse Sca1, PE anti-mouse CD135, and APC anti-mouse CD34. The positive rates of EGFP mRNA expression in different mouse hematopoietic stem cell populations (LSK, LT-LSK, and ST-LSK) were detected by flow cytometry (all antibodies were from Biolegend).
图15：疾病小鼠未治疗组、疾病小鼠LNP治疗组Figure 15: Untreated disease mice group, LNP-treated disease mice group
LNP-Cas9 mRNA/sgRNA注射人源化镰刀型细胞贫血症小鼠后，尾静脉注射小鼠后于1周后取小鼠骨髓细胞，孵育小鼠抗体BV605 anti-mouse Lin-、BV421anti-mouse Ckit、PE-Cy7 anti-mouse Sca1(抗体均来自于Biolegend)。，流式分选出小鼠造血干细胞不同群体后，使用DNA提取试剂盒(全式金，EE181-01)提取DNA，PCR扩增后进行琼脂糖电泳，切下条带送测进行Sanger一代测序检测基因编辑。Humanized sickle cell anemia mice were injected with LNP-Cas9 mRNA/sgRNA. One week after tail vein injection, bone marrow cells were harvested and incubated with mouse antibodies BV605 anti-mouse Lin, BV421 anti-mouse Ckit, and PE-Cy7 anti-mouse Sca1 (all from Biolegend). Different populations of mouse hematopoietic stem cells were flow-sorted, and DNA was extracted using a DNA extraction kit (Quanshijin, EE181-01). PCR amplification was followed by agarose gel electrophoresis, and the excised bands were sent for Sanger sequencing to verify gene editing.
图16-图17：健康对照组、疾病小鼠未治疗组、疾病小鼠LNP治疗组Figure 16-17: Healthy control group, disease mouse untreated group, disease mouse LNP treated group
LNP-Cas9 mRNA/sgRNA注射人源化镰刀型细胞贫血症小鼠后，尾静脉注射小鼠后于2W、10W、12W(周)后取小鼠外周血，加入超纯水反复冻融后裂解取得蛋白上清后进行高效液相色谱(HPLC)检测γ珠蛋白链是否激活；小鼠外周血提取mRNA后，进行qPCR，详细操作同图11的方法。After LNP-Cas9 mRNA/sgRNA was injected into humanized sickle cell anemia mice, peripheral blood was collected from the mice at 2 weeks, 10 weeks, and 12 weeks after tail vein injection. The blood was added to ultrapure water and repeatedly frozen and thawed, and the protein supernatant was obtained and then subjected to high-performance liquid chromatography (HPLC) to detect whether the γ-globin chain was activated. After mRNA was extracted from the mouse peripheral blood, qPCR was performed. The detailed operation was the same as the method in Figure 11.
图18-图20：健康对照组、疾病小鼠未治疗组、疾病小鼠LNP治疗组LNP-Cas9 mRNA/sgRNA注射人源化镰刀型细胞贫血症小鼠后，尾静脉注射小鼠后于16W周后取小鼠外周血，分别于血常规检测仪器检测RBC、HGT、HGB指标；生化仪器检测ALT、AST、ALB；血涂片分别进行吉姆萨、新亚甲蓝染色、镰刀型实验后于显微镜下观察拍照；最后根据小鼠死亡情况做生存曲线。Figures 18-20: After LNP-Cas9 mRNA/sgRNA was injected into humanized sickle cell anemia mice in the healthy control group, the disease mouse untreated group, and the disease mouse LNP-treated group, peripheral blood was collected from the mice 16 weeks after tail vein injection. RBC, HGT, and HGB indicators were tested using a routine blood test instrument; ALT, AST, and ALB were tested using a biochemical instrument; blood smears were stained with Giemsa, new methylene blue, and sickle cell test, and then observed and photographed under a microscope; finally, survival curves were drawn based on the death of mice.
2.实验结果2. Experimental Results
1)不同动员药物组合、小鼠注射药物方式的探索1) Exploration of different mobilization drug combinations and drug injection methods in mice
按照上述最优配方合成LNP后。首先希望通过使用动员剂将骨髓里的造血干细胞动员到外周血后，更利于与尾静脉注射的LNP结合，从而提升递送效率。After synthesizing LNP according to the above optimal formula, we first hope to mobilize hematopoietic stem cells from the bone marrow into the peripheral blood by using a mobilizing agent, which will be more conducive to binding with the LNP injected through the tail vein, thereby improving delivery efficiency.
①不同动员剂组合对动员小鼠骨髓造血干进入外周血的探索：分为快速动员法(GROβ+AMD3100联合)、连续动员法(G-CSF+AMD3100联合，其中G-CSF含2种不同品牌)，结果显示，连续动员法动员小鼠造血干细胞LSK进入外周血的效果显著高于快速动员法，此外，连续动员法中，不同品牌的G-CSF效果也大有不同，后续将采用连续动员法(G-CSF-Primegene+AMD3100)组合进行下一步实验(图12-13)。① Exploration of different mobilization agent combinations to mobilize mouse bone marrow hematopoietic stem cells into peripheral blood: divided into rapid mobilization method (GROβ+AMD3100 combination) and continuous mobilization method (G-CSF+AMD3100 combination, in which G-CSF contains 2 different brands). The results showed that the continuous mobilization method was significantly more effective than the rapid mobilization method in mobilizing mouse hematopoietic stem cells LSK into peripheral blood. In addition, in the continuous mobilization method, the effects of different brands of G-CSF were also very different. The continuous mobilization method (G-CSF-Primegene+AMD3100) combination will be used in the next experiment (Figures 12-13).
②动员剂不同注射部位对小鼠造血干细胞递送效率的摸索：根据上述探索后确认了最佳动员剂组合，在此基础上，动员剂注射部位(脖颈部皮下、腹部皮下)不同，药物递送效率也大有不同：脖颈部皮下注射的小鼠造血干细胞(长期造血干细胞、短期造血干细胞)中EGFP mRNA表达显著高于腹部皮下注射(图14)。② Exploration of the delivery efficiency of mouse hematopoietic stem cells at different injection sites of mobilizers: Based on the above exploration, the optimal mobilizer combination was confirmed. On this basis, the drug delivery efficiency was greatly different depending on the mobilizer injection site (subcutaneous injection of the neck, subcutaneous injection of the abdomen): The EGFP mRNA expression in mouse hematopoietic stem cells (long-term hematopoietic stem cells and short-term hematopoietic stem cells) injected subcutaneously at the neck was significantly higher than that injected subcutaneously at the abdomen (Figure 14).
综上所述，本申请首次通过探索不同造血干细胞动员方式、不同给药方式，研究出一套可对体内造血干细胞递送LNP效率的最佳组合方法学。紧接着，使用这套方案，采用LNP包裹编辑系统，对人源化疾病小鼠进行体内治疗并取得了显著的成效。In summary, this application, for the first time, explored different hematopoietic stem cell mobilization methods and different administration routes to develop a set of optimal combined methodologies for LNP delivery efficiency to hematopoietic stem cells in vivo. Subsequently, this approach was used to treat humanized diseased mice in vivo using an LNP encapsulation editing system, achieving significant results.
2)人源化镰刀型细胞贫血症中实现在体内治疗2) Achieving in vivo treatment of humanized sickle cell anemia
低剂量、一次注射体内转染小鼠HSPC后1周，骨髓LSK编辑效率高达20％，珠蛋白表达显著升高、外周血珠蛋白阳性细胞比率增加，镰刀型等异常形态红细胞比例下降、异常增多网织红细胞显著降低，血常规等表型成功校正。生化指标ALT、AST显示，治疗组未有异常增高，即小鼠体内未引起明显的体内毒性。此外，显著延长疾病小鼠生存时间，治疗期间，治疗组疾病小鼠对比未治疗组，未出现小鼠死亡情况(图15-20)。One week after a low-dose, single-injection transfection of mouse HSPCs, bone marrow LSK editing efficiency reached 20%. Globin expression was significantly elevated, the proportion of globin-positive cells in peripheral blood increased, the proportion of abnormal erythrocytes, such as sickle-shaped ones, decreased, and the abnormally increased number of reticulocytes was significantly reduced. Blood routine tests successfully corrected these phenotypes. Biochemical markers such as ALT and AST showed no abnormal increases in the treated group, indicating no significant in vivo toxicity in the mice. Furthermore, the survival of the diseased mice was significantly prolonged. During treatment, no mice in the treated group died compared to the untreated group (Figures 15-20).
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.
| Application Number | Priority Date | Filing Date | Title |
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| CN202510079972.2ACN119876277B (en) | 2025-01-18 | 2025-01-18 | Lipid nanoparticle and application thereof |
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| CN202510079972.2ACN119876277B (en) | 2025-01-18 | 2025-01-18 | Lipid nanoparticle and application thereof |
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| CN119876277A CN119876277A (en) | 2025-04-25 |
| CN119876277Btrue CN119876277B (en) | 2025-08-15 |
| Application Number | Title | Priority Date | Filing Date |
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| CN202510079972.2AActiveCN119876277B (en) | 2025-01-18 | 2025-01-18 | Lipid nanoparticle and application thereof |
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| CN (1) | CN119876277B (en) |
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| CN115386599B (en)* | 2022-07-18 | 2024-01-12 | 江苏拓弘康恒医药有限公司 | mRNA-LNP delivery system, preparation process and application thereof in human mesenchymal stem cells |
| CN118356514A (en)* | 2023-01-17 | 2024-07-19 | 上海赛丽莱生物科技有限公司 | MRNA-LNP delivery system, preparation method and application thereof |
| CN116602936A (en)* | 2023-05-23 | 2023-08-18 | 温州医科大学附属眼视光医院 | A lipid nanoparticle and composition system and method for improving drug delivery performance and stability |
| CN119015257A (en)* | 2023-07-13 | 2024-11-26 | 深圳虹信生物科技有限公司 | Lipid nanoparticles and nucleic acid drugs and their applications |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116479041A (en)* | 2022-01-14 | 2023-07-25 | 北京干细胞与再生医学研究院 | A kind of gene construct and the method for producing multi-lineage hematopoietic stem progenitor cells |
| CN116271105A (en)* | 2023-05-18 | 2023-06-23 | 上海贝斯昂科生物科技有限公司 | Lipid nanoparticle suitable for RPE cell transfection and application thereof |
| Title |
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| In vivo hematopoietic stem cell modification by mRNA delivery;Laura Breda 等;Science;20230727;摘要，第6页第3段-第7页第1段* |
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| CN119876277A (en) | 2025-04-25 |
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