WO2023142168A1 - 阳离子脂质类似物在基因编辑核糖核蛋白复合物胞内递送中的应用 - Google Patents

阳离子脂质类似物在基因编辑核糖核蛋白复合物胞内递送中的应用 Download PDF

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WO2023142168A1
WO2023142168A1 PCT/CN2022/075915 CN2022075915W WO2023142168A1 WO 2023142168 A1 WO2023142168 A1 WO 2023142168A1 CN 2022075915 W CN2022075915 W CN 2022075915W WO 2023142168 A1 WO2023142168 A1 WO 2023142168A1
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cationic lipid
gene
a1i2r2c18
ribonucleoprotein complex
lipid analog
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French (fr)
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刘志佳
平渊
陈永明
乐志成
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中山大学
杭州睿导基因科技有限公司
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Definitions

  • the invention relates to the field of biotechnology, in particular to a nanocomplex containing cationic lipid analogs and the application of cationic lipid analogs in the intracellular delivery of gene editing ribonucleoprotein complexes.
  • the clustered regularly interspaced short palindromic repeats sequences (CRISPR) system is derived from the adaptive immune system of bacteria and archaea, which is mainly used to resist exogenous viruses such as phages and plasmids. Nucleic acid invasion.
  • the CRISPR/Cas system has been developed as a new type of gene editing technology, which can target the target sequence of the genome or target multiple target sequences of the genome simultaneously for genome editing.
  • the system has been widely used in the treatment of gene-related diseases, in vivo imaging of directional detection of genomic regions, identification of new disease-related targets, identification of gene functions, and establishment of animal disease models.
  • CRISPR/Cas9 as the most representative gene editing system, mainly consists of two core components: CRISPR/Cas9 endonuclease and single guided RNA (sgRNA).
  • CRISPR/Cas9-based gene editing technology was successfully applied to mammalian cell genome editing. This technology has been applied to a series of refractory diseases, including malignant tumors, sickle cell anemia, I-H mucopolysaccharidosis, al Alzheimer's disease, glycogen storage disease, hemophilia, cystic fibrosis, Duchenne muscular dystrophy, and others.
  • the purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a nanocomplex containing cationic lipid analogues, and the application of cationic lipid analogues in the intracellular delivery of gene editing ribonucleoprotein complexes.
  • Cationic lipid analogs deliver ribonucleoprotein complexes with high gene editing efficiency.
  • nanocomplex comprising a cationic lipid analog and a ribonucleoprotein complex
  • the cationic lipid analog has a structure as shown in formula (I):
  • m is independently selected from hydrogen (H), straight-chain alkyl, branched-chain alkyl;
  • n is an alkyl group, R2 is an alkyl group, R3 is an alkyl group, or R1 and R2 are connected to form a ring group;
  • n3 is independently selected from straight-chain alkyl, straight-chain alkenyl
  • m4 is independently selected from straight-chain alkyl groups and straight-chain alkyl groups containing ether linkages.
  • m2 contains a tertiary amine group, which ensures that the material has pH sensitivity and positive charge adjustable;
  • m3 contains a straight-chain alkyl group and a straight-chain alkenyl group, which can be adjusted by adjusting its chain length. The hydrophobicity of the material.
  • the cationic lipid analog designed in the present invention combines with the ribonucleoprotein complex (Cas9RNP protein) to form a nanocomplex, and the cationic lipid analog delivers the ribonucleoprotein complex to the cell with higher gene editing efficiency and delivery
  • the nanocomplex delivered into the cell still has biological activity, and at the same time, the nanocomplex itself and the delivery process have little toxicity to the cell.
  • the mass ratio of the cationic lipid analogue to the ribonucleoprotein complex is (1-4):1.
  • the mass ratio of the cationic lipid analogue to the ribonucleoprotein complex is 2:1.
  • the delivery efficiency and gene editing efficiency can be better improved, which is beneficial to the treatment of corresponding diseases.
  • the m is hydrogen (H)
  • m 2 is m 3 is m 4 is
  • the obtained nanocomplex delivers the ribonucleoprotein complex to the cells with high delivery efficiency.
  • the cationic lipid analog has any of the following 36 structures:
  • the inventors have found through experiments that the 36 small-molecule cationic lipid analogs obtained from the above screening can be co-assembled with ribonucleoprotein complexes to form small and stable nanocomplexes to achieve intracellular delivery of various positively and negatively charged proteins , and the protein delivered into cells can maintain biological activity and have therapeutic effect.
  • the cationic lipid analogs are A1I2R2C16, A1I2R2C18, A1I2R2C19, A1I2R2C20, A1I2R3C16, A1I2R3C18, A1I2R3C19, A1I2R3C20, A1I2R11C16, A1I2R11C18, A 1I2R11C19, A1I2R11C20, A1I2-1R2C18, A1I2 - at least one of 1R2C19, A1I2-1R2C20, A1I2-3R2C18, A1I2-3R2C19, A1I2-3R2C20, more preferably, the cationic lipid analog is A1I2R2C18, A1I2-1R2C18 or A1I2-3R2C18.
  • the gene-editing ribonucleoprotein complexes delivered by A1I2R2C18, A1I2-1R2C18 or A1I2-3R2C18 all have gene editing effects, and A1I2R2C18 has the highest
  • the mass ratio of A1I2R2C18 and ribonucleoprotein complexes is 2:1
  • the gene editing efficiency of nanocomplexes delivered to 293T cells is the best.
  • A12R2C18, A1I2-1R2C18 or A1I2-3R2C18 lies in the number of ethoxy repeating units at the linker position.
  • the gene editing efficiency of the vector is average for 1 and 3 repeating units, and the gene editing efficiency for 2 repeating units Vectors have the best gene editing efficiency.
  • the ribonucleoprotein complex is a CRISPR-Cas9 ribonucleoprotein complex.
  • the present invention also provides the application of the nanocomposite in gene editing and gene therapy.
  • the sites targeted by gene editing include AAVS1 sites, HBB sites, EGFP sites or KRAS sites.
  • the present invention also provides a modified nano-composite, including hyaluronic acid and the above-mentioned nano-composite.
  • hyaluronic acid is 0.1-2 mg/mL. More preferably, the concentration of hyaluronic acid is 0.2 mg/mL.
  • the inventors of the present invention have found through extensive research and experiments that the surface of the nanocomposite is modified with a layer of hyaluronic acid, so that the nanocomposite interacts with the nanocomposite through electrostatic interaction.
  • Hyaluronic acid (HA) a negatively charged natural polysaccharide with excellent biocompatibility, is used to shield the positive charge of nanocomplexes to exhibit long in vivo circulation and stealth properties.
  • Nanocomposites are surface-modified with an optimal concentration of hyaluronic acid, which can change the zeta potential of the nanocomposites from positive to negative.
  • the modified nanocomposite has antiserum ability, which can effectively prolong the blood circulation time in vivo.
  • the present invention also provides the application of the nanocomposite or the modified nanocomposite in the preparation of antitumor drugs.
  • the present invention provides a nanocomposite, which has a higher gene editing effect on the targeted mutation gene site in tumor tissue, and further demonstrates that cationic lipid analogue delivery ribonucleoprotein complex has a certain inhibitory effect on tumor growth .
  • the present invention also provides a method for intracellular delivery of the gene-edited ribonucleoprotein complex, which utilizes cationic lipid analogs to achieve intracellular delivery of the gene-edited ribonucleoprotein complex.
  • the present invention also provides the application of the cationic lipid analogue in the nanocomplex for intracellular delivery of the gene editing ribonucleoprotein complex.
  • delivery refers to the entry of drugs from the outside of the cell to the inside of the cell, making it localized in the cytosol or in the organelles of the cell.
  • cell or “intracellular” includes at least one of 293T cells, 293T-EGFP cells and SW-480 cells.
  • the present invention has the following beneficial effects:
  • the present invention provides a nanocomplex containing cationic lipid analogs with high gene editing effect on different gene loci (AAVS1, HBB, EGFP and KRAS) in tumor tissue, further illustrating the delivery of cationic lipid analogs
  • the ribonucleoprotein complex has a certain inhibitory effect on tumor growth; the gene-edited ribonucleoprotein complex delivery vector can achieve high delivery efficiency during intracellular delivery, is safe and effective, and does not require redundant chemical modifications, saving production costs .
  • Fig. 1 is the mass spectrum (a) and the proton nuclear magnetic resonance spectrum (b) of cationic lipid analog A1I2-1R2C18;
  • Fig. 2 is the mass spectrum (a) and the proton nuclear magnetic resonance spectrum (b) of cationic lipid analog A1I2R2C18;
  • Fig. 3 is the mass spectrum (a) and the proton nuclear magnetic resonance spectrum (b) of cationic lipid analog A1I2-3R2C18;
  • Figure 4 is a graph of the gene editing efficiency results of cationic lipid analogs A1I2-1R2C18 (a), A1I2R2C18 (b) and A1I2-3R2C18 (c) delivering gene editing ribonucleoprotein complex (RNP) targeting AAVS1 site;
  • Fig. 5 is the particle size distribution figure of the nanocomposite that embodiment 3 forms;
  • FIG. 6 is the T7E1 enzyme digestion experiment results and 4 gene loci of cationic lipid analogue A1I2R2C18 delivering Cas9RNP protein on 293T cells (a and b), 293T-EGFP cells (c) and SW-480 cells (d) The Sanger sequence result map of the T-A clone;
  • Figure 7 is a graph of the gene editing efficiency results of the four gene loci (AAVS1, HBB, EGFP and KRAS) of the cationic lipid analog A1I2R2C18 after the T7E1 enzyme digestion experiment and ImageJ software quantitative statistics, and compared with the commercial delivery reagent CMAX;
  • Figure 8 is the fluorescence image (a, the scale bar is 50 ⁇ m) and flow cytometry (FCM) detection of RNP/A1I2R2C18 nanocomplexes treated 293T-EGFP cells for 48 hours after RNP/A1I2R2C18 nanocomplexes Relative mean fluorescence intensity (MFI) (b);
  • Figure 9 shows the particle size (a) and zeta potential (b) of a series of modified nanocomposites (particles) obtained by modifying RNP/A1I2R2C18 nanocomposites with different concentrations of hyaluronic acid (HA);
  • Figure 10 is a diagram showing the stability results of modified nanocomplexes (particles) in serum-free medium and serum medium containing 10% FBS by mixing hyaluronic acid and RNP/A1I2R2C18 nanocomplexes;
  • Figure 11 is a diagram of the in vivo anti-tumor effect mediated by the RNP/A1I2R2C18 nanocomplex targeting the mutant KRAS site;
  • Fig. 12 is a graph showing the results of T7E1 enzyme digestion experiment of targeting mutant KRAS sites in tumors mediated by peritumoral injection of RNP/A1I2R2C18 nanocomplexes.
  • Embodiment 1 synthesis and characterization of cationic lipid analogs
  • the synthetic route of cationic lipid analog of the present invention is:
  • the amine compound m 2 -NH 2 is Carboxylic acid compound for Aldehydes for isocyanide for
  • the specific preparation method of the cationic lipid analog in this example is: respectively add 1 mmol of isobutyraldehyde and 1 mmol of amine compound into 0.5 mL of methanol solution, and then add 1 mmol of carboxylic acid compound and 0.5 mmol of isobutyraldehyde after reacting for 60 minutes. Cyanide compound, and reacted at 40° C. for 12 hours. After the reaction, the product was separated and purified by chromatographic column, wherein the mobile phase was a mixture of methanol and dichloromethane.
  • Cationic lipid analogs A1I2-1R2C18, A1I2R2C18 and A1I2-3R2C18 were selected as representative materials, and their structures were characterized.
  • the mass spectrum of A1I2-1R2C18 and the spectrogram of proton nuclear magnetic resonance spectrum are shown in Figure 1;
  • the mass spectrum of A1I2R2C18 and the spectrogram of proton nuclear magnetic resonance spectrum are shown in Figure 2;
  • the picture is shown in Figure 3.
  • the results of 1H NMR and mass spectrometry were consistent with the expected structure of the cationic lipid analogue.
  • Example 2 Intracellular effect of cationic lipid analogs A1I2-1R2C18, A1I2R2C18 and A1I2-3R2C18 delivering gene-edited ribonucleoprotein complex (RNP).
  • RNP gene-edited ribonucleoprotein complex
  • sgRNA (sgAAVS1) targeting AAVS1 was prepared by in vitro transcription IVT method (the target of AAVS1 is GGCTCCCTCCCAGGATCCTCTC).
  • the 293T cells were seeded into 24-well plates overnight, and the Cas9RNP protein delivery experiment was started when the 293T cell density reached more than 75%.
  • CRISPR-Cas9 protein and 0.5 ⁇ g sgAAVS1 were incubated at 37°C for 10 minutes to form a CRISPR-Cas9/sgAAVS1 complex (Cas9RNP), and then the CRISPR-Cas9/sgAAVS1 complex was mixed with A1I2-1R2C18, A1I2R2C18 and A1I2 -3R2C18
  • the three cationic lipid analogs were mixed, mixed quickly for 10-30 seconds, and immediately diluted with 450 ⁇ L serum-free DMEM medium to finally obtain a 500 ⁇ L system solution of Cas9RNP/cationic lipid analogs.
  • the dose of CRISPR-Cas9 was 1 ⁇ g per well, the dose of sgAAVS1 was 0.5 ⁇ g per well, and the doses of the three cationic lipid analogs A1I2-1R2C18, A1I2R2C18 and A1I2-3R2C18 were 1, 2, 3 and 4 ⁇ g per well, respectively .
  • AAVS1-FP CTATGTCCACTTCAGGACAGCATGT
  • AAVS1-RP CCTCTTGGGAAGTGTAAGGAAGCTG
  • heat denaturation annealing and renaturation treatment and finally add 0.3 ⁇ L of T7E1 nucleic acid Dicerase, after reacting at 37°C for 30 minutes, run 2% agarose gel electrophoresis to detect and analyze the enzyme digestion results.
  • A1I2R2C18 has better gene editing efficiency.
  • the difference between these three molecules is the number of ethoxy repeating units at the linker position.
  • the gene editing efficiency of carrier materials with 1 and 3 repeat units is average, and the effect of 2 repeat units is the best, indicating that optimizing the linker structure is of great significance for improving delivery and editing efficiency.
  • the cationic lipid analog A1I2R2C18 and the gene-edited ribonucleoprotein complex (RNP) were used to form the RNP/A1I2R2C18 nanocomplex of the present invention, and dynamic light scattering (DLS) was used to characterize the size and surface potential of the nanocomplex.
  • RNP gene-edited ribonucleoprotein complex
  • DLS dynamic light scattering
  • the specific operation method is as follows: quickly mix the ribonucleoprotein complex with the A1I2R2C18 solution, incubate at room temperature for 10-30 seconds, add 1 mL of deionized water to dilute, and use a laser nanometer to detect the particle size distribution and surface area of the nanoparticles in the solution. electric potential.
  • Example 4 Gene editing experiment of cationic lipid analog A1I2R2C18 delivering ribonucleoprotein complex (RNP) to different cells targeting different sites
  • Cationic lipid analog A1I2R2C18 delivered Cas9RNP protein targeting AAVS1 and HBB sites into 293T cells; delivered Cas9RNP protein targeting EGFP site into 293T-EGFP cells; delivered targeting KRAS site into SW-480 cells Spot the Cas9RNP protein.
  • the Sanger sequences of AAVS1 and HBB site T-A clones were obtained from 293T cells; the Sanger sequences of EGFP site T-A clones were obtained from 293T-EGFP cells; the Sanger sequences of KRAS site T-A clones were obtained from SW-480 cells.
  • the specific operation method is as follows: Firstly, the sgRNA targeting AAVS1 and HBB (sgAAVS1 and sgHBB) were prepared by IVT in vitro (the target of AAVS1 was GGCTCCCTCCCAGGATCCTCTC; the target of HBB was GGGTAACGGCAGACTTCTCTCTC); the sgRNA targeting EGFP (sgEGFP ) (the target of EGFP is GTGAACCGCATCGAGCTGAA); sgRNA (sgKRAS) targeting the mutant KRAS gene (the target of KRAS is GTTGGAGCTGATGGCGT). 293T cells, 293T-EGFP cells and SW-480 cells were seeded into 24-well plates overnight, and the Cas9RNP protein delivery experiment was started when the cell density reached above 75%.
  • CRISPR-Cas9 protein was incubated with sgAAVS1, sgHBB, sgEGFP, and sgKRAS at 37°C for 10 minutes to form a CRISPR-Cas9/sgRNA complex, and then the CRISPR-Cas9/sgRNA complex was fully mixed with the cationic lipid analog A1I2R2C18, and quickly After mixing for 10-30 seconds, immediately dilute with 450 ⁇ L serum-free DMEM medium to finally obtain 500 ⁇ L system solution of RNP/A1I2R2C18 nanocomplex.
  • the dose of CRISPR-Cas9 was 1 ⁇ g per well, the dose of sgRNA was 0.5 ⁇ g per well, and the dose of cationic lipid analog A1I2R2C18 was 2 ⁇ g.
  • the culture medium was removed, and 500 ⁇ L of DMEM medium containing 10% serum was added, and culture was continued for 48 hours.
  • the total genomic DNA was extracted using a commercial kit, and the target fragment with the mutation site was amplified by PCR.
  • the primers are shown in Table 2.
  • Example 5 Quantitative statistics of the gene editing efficiency of 4 gene loci (AAVS1, HBB, EGFP and KRAS) were carried out by T7E1 enzyme digestion experiment and ImageJ software.
  • T7E1 enzyme digestion experiment use a commercial kit to extract total genomic DNA, PCR amplify the target fragment with the mutation site, heat denaturation annealing and refolding treatment, finally add 0.3 ⁇ L of T7E1 endonuclease, and react at 37°C After 30 minutes, run 2% agarose gel electrophoresis to detect and analyze the digestion results.
  • ImageJ software performs quantitative statistics on the efficiency of gene editing cutting bands at 4 gene loci (AAVS1, HBB, EGFP and KRAS) as follows: First, open the gel image to be analyzed through File-Open for grayscale analysis of the bands, Next, convert the picture into an 8-bit grayscale image, then select a band with the rectangular tool box, number the bands through Analyze/gels/select first lane, and click on each peak with the Wandtool The calculation of the peak area can be completed one by one in the middle area of the area, and the calculation result is displayed in the Result window, which can be exported as an xls file through File. Finally, use the Indel percentage calculation formula: [1-(1-cut strip strength value)1/2] ⁇ 100%, the cutting efficiency value can be obtained, and then can be statistically quantified.
  • the specific operation method is as follows: 293T-EGFP cells were seeded into a 24-well plate overnight, and the Cas9RNP protein delivery experiment was started when the cell density reached above 75%. First, incubate the CRISPR-Cas9 protein and sgEGFP at 37°C for 10 minutes to form a CRISPR-Cas9/sgRNA complex, then mix the CRISPR-Cas9/sgRNA complex with the cationic lipid analog A1I2R2C18, and mix quickly for 10-30 seconds Immediately thereafter, it was diluted with 450 ⁇ L serum-free DMEM medium to finally obtain a 500 ⁇ L system solution (RNP/A1I2R2C18 nanocomposite) of Cas9 RNP/A1I2R2C18.
  • the dose of CRISPR-Cas9 is 1 ⁇ g per well, the dose of sgRNA is 0.5 ⁇ g per well, and the optimal dose of cationic lipid analog A1I2R2C18 is 2 ⁇ g.
  • the culture medium was removed, and 500 ⁇ L of DMEM medium containing 10% serum was added, and culture was continued for 48 hours. Then observe the fluorescence intensity and distribution in the cells under a fluorescence microscope.
  • the cells in the well plate were then collected for flow cytometry analysis and their relative average fluorescence intensity was detected.
  • a modified nanocomplex comprising hyaluronic acid and RNP/A1I2R2C18 nanocomplex.
  • concentration of hyaluronic acid is 0.1-2 mg/mL.
  • the preparation method of the above-mentioned modified nanocomposite adding different concentrations of hyaluronic acid (0.1-2 mg/mL) dropwise into the RNP/A1I2R2C18 nanocomposite solution, and obtaining the HA/RNP/A1I2R2C18 nanocomposite by electrostatic coating (modified nanocomposites). Then, the solution mixture was stirred gently at room temperature for 30 minutes. The optimal concentration of hyaluronic acid in the preparation of HA/RNP/A1I2R2C18 nanocomplexes was determined by DLS.
  • Example 8 verifying the stability and serum resistance of the HA/RNP/A1I2R2C18 nanocomplex (modified nanocomposite)
  • the specific operation method is as follows: the preparation of HA/RNP/A1I2R2C18 nanocomposites containing 0.2 mg/mL HA concentration was added to serum-free DMEM medium and DMEM medium containing 10% fetal bovine serum (FBS) to incubate for a period of time, After 0.5h, 24h, 36h and 48h respectively, the particle size change of the HA/RNP/A1I2R2C18 nanocomposite was detected by a dynamic light scattering instrument (DLS), so as to obtain the stability result.
  • DLS dynamic light scattering instrument
  • Example 9 In vivo anti-tumor effect mediated by RNP/A1I2R2C18 nanocomplexes targeting mutant KRAS sites
  • the way of administration is peritumoral injection of RNP/A1I2R2C18 nanocomposite.
  • the mean tumor growth volume changes in the different treatment groups were monitored and recorded three weeks after treatment.
  • SW-480 xenograft primary tumor model SW-480 cells (1 ⁇ 10 6 ) were subcutaneously injected into the right side of BALB/c nude mice. After 7 days, when tumors reached a size of approximately 50–80 mm, mice were randomly divided into 4 groups and treated with control, RNP only, RNP/A1I2R2C18 and Mock RNP/A1I2R2C18 groups. The nanocomplex was injected subcutaneously around the tumor once a week for three weeks, and tumor volume was measured in nude mice.
  • the specific operation method is as follows: collect the tumor tissues of the control group, the RNP-only group, the RNP/A1I2R2C18 group and the Mock RNP/A1I2R2C18 group, use a commercial kit to extract the total genomic DNA, and PCR amplify the target fragment with the mutation site (KRAS -FP: TGCAGTCAACTGGAATTTTCAT; KRAS-RP: GTTGGATCATATTCGTCCACAA), heat denaturation annealing and renaturation treatment, finally add 0.3 ⁇ L of T7E1 endonuclease, react at 37°C for 30 minutes, run 2% agarose gel electrophoresis to detect and analyze the enzyme digestion result.

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Abstract

一种含有阳离子脂质类似物的纳米复合物，及阳离子脂质类似物在基因编辑核糖核蛋白复合物胞内递送中的应用。纳米复合物包括阳离子脂质类似物和核糖核蛋白复合物；所述阳离子脂质类似物具有如式(I)所示结构。纳米复合物对肿瘤组织中的不同基因位点(AAVS1、HBB、EGFP和KRAS)有较高的基因编辑效果，进一步说明阳离子脂质类似物递送核糖核蛋白复合物对肿瘤生长有一定的抑制作用；基因编辑核糖核蛋白复合物递送载体在细胞内递送过程中可以达到高递送效率，安全有效。

Description

阳离子脂质类似物在基因编辑核糖核蛋白复合物胞内递送中的应用 技术领域
本发明涉及生物技术领域，尤其是涉及一种含有阳离子脂质类似物的纳米复合物，及阳离子脂质类似物在基因编辑核糖核蛋白复合物胞内递送中的应用。
背景技术
成簇的规律间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats sequences，CRISPR)系统源于细菌和古细菌的自适应性免疫系统，其主要用来抵御来自于噬菌体、质粒等外源性核酸的入侵。而利用这种天然存在的免疫系统，CRISPR/Cas系统已经被开发成为一种新型的基因编辑技术，它能够定点靶向基因组的目标序列或者同时靶向基因组多个目标序列用于基因组编辑。该系统目前已经被广泛应用于基因相关疾病的治疗，定向检测基因组区域的活体成像，疾病相关新靶点的鉴定，基因功能的识别以及动物疾病模型建立等多方面的研究。
在众多CRISPR/Cas系统中，CRISPR/Cas9作为最具有代表性的基因编辑系统，主要包含两个核心组分：CRISPR/Cas9核酸内切酶及单联导向RNA(single guided RNA，sgRNA)，自2013年以CRISPR/Cas9为基础的基因编辑技术被成功应用于哺乳细胞基因组编辑，该技术已经应用一系列难治愈的疾病上，包括恶性肿瘤、镰刀形细胞贫血症、I-H型粘多糖病、阿尔兹海默症、肝糖原贮积症、血友病、囊性纤维化、杜式肌肉营养不良症及其他疾病。
在基于CRISPR/Cas9系统的基因组编辑技术被应用于临床之前，除了需要提高基因编辑的特异性，降低脱靶和基因组突变率等复杂的技术挑战外。另一方面，如何安全有效地将CRISPR/Cas9基因编辑系统导入到特定的细胞、组织或者器官中以获得期望的治疗效果是另一个需要解决的关键性问题。但是由于目前能够高效安全递送CRISPR/Cas9的载体系统的匮乏，导致了CRISPR/Cas9基因编辑技术在临床应用上的潜力受到极大的限制。因此开发高效低毒的CRISPR/Cas9的递送载体系统，对推动CRISPR/Cas9基因编辑技术向临床应用 转化，降低毒副作用，提高治疗的安全性，具有极其重要的科学价值和研究意义。
发明内容
本发明的目的在于克服上述现有技术的不足之处而提供一种含有阳离子脂质类似物的纳米复合物，及阳离子脂质类似物在基因编辑核糖核蛋白复合物胞内递送中的应用。阳离子脂质类似物递送核糖核蛋白复合物具有较高的基因编辑效率。
为实现上述目的，本发明采取的技术方案为：
一种纳米复合物，包括阳离子脂质类似物和核糖核蛋白复合物；
所述阳离子脂质类似物具有如式(I)所示结构：
Figure PCTCN2022075915-appb-000001
式(I)中，m 1独立地选自氢(H)、直链烷基、支链烷基；
m 2为
Figure PCTCN2022075915-appb-000002
R1为烷基，R2为烷基，R3为烷基，或R1与R2连接为环基；
m 3独立地选自直链烷基、直链烯基；
m 4独立地选自直链烷基、含醚键的直链烷基。
本发明的阳离子脂质类似物中m 2含有叔胺基团，保证了材料具有pH敏感性和正电荷可调性；m3含有直链烷基、直链烯基，通过调控其链长，可以调控材料的疏水性。本发明设计得到的阳离子脂质类似物和核糖核蛋白复合物(Cas9RNP蛋白)结合形成纳米复合物，该阳离子脂质类似物递送核糖核蛋白复合物至胞内具有较高的基因编辑效率，递送至细胞内的纳米复合物仍具有生物活性，同时纳米复合物自身及递送过程对细胞产生的毒性小。
作为本发明所述纳米复合物的优选实施方式，所述阳离子脂质类似物和核糖核蛋白复合物的质量比为(1～4):1。优选地，阳离子脂质类似物和核糖核蛋白复合物的质量比为2:1。
当阳离子脂质类似物和核糖核蛋白复合物以上述特定质量比进行配比，可以更好地提高递送效率和基因编辑效率，有利于治疗相应的疾病。
作为本发明所述纳米复合物的优选实施方式，所述m 1为氢(H)、
Figure PCTCN2022075915-appb-000003
Figure PCTCN2022075915-appb-000004
m 2为
Figure PCTCN2022075915-appb-000005
Figure PCTCN2022075915-appb-000006
m 3为
Figure PCTCN2022075915-appb-000007
Figure PCTCN2022075915-appb-000008
Figure PCTCN2022075915-appb-000009
m 4为
Figure PCTCN2022075915-appb-000010
Figure PCTCN2022075915-appb-000011
所获得的纳米复合物递送核糖核蛋白复合物至细胞内的递送效率较高。
作为本发明所述纳米复合物的优选实施方式，所述阳离子脂质类似物具有如下36种结构中的任一种：
Figure PCTCN2022075915-appb-000012
Figure PCTCN2022075915-appb-000013
发明人经过试验发现，上述筛选得到的36种小分子阳离子脂质类似物可与核糖核蛋白复合物共组装形成尺寸小、稳定的纳米复合物，实现多种正、负电性蛋白质的胞内递送，且递送至细胞内的蛋白质能够保持着生物活性，具有治疗效果。
作为本发明所述纳米复合物的优选实施方式，所述阳离子脂质类似物为A1I2R2C16、A1I2R2C18、A1I2R2C19、A1I2R2C20、A1I2R3C16、A1I2R3C18、A1I2R3C19、A1I2R3C20、A1I2R11C16、A1I2R11C18、A1I2R11C19、A1I2R11C20、 A1I2-1R2C18、A1I2-1R2C19、A1I2-1R2C20、A1I2-3R2C18、A1I2-3R2C19、A1I2-3R2C20中的至少一种，更优选地，所述阳离子脂质类似物为A1I2R2C18、A1I2-1R2C18或A1I2-3R2C18。
经过将阳离子脂质类似物递送基因编辑核糖核蛋白复合物至细胞内的实验结果可知，A1I2R2C18、A1I2-1R2C18或A1I2-3R2C18递送基因编辑核糖核蛋白复合物均具有基因编辑效果，其中A1I2R2C18具有最高的基因编辑效率，同时当A1I2R2C18和核糖核蛋白复合物的质量比为2:1时形成的纳米复合物递送至293T细胞中的基因编辑效率最佳。A12R2C18、A1I2-1R2C18或A1I2-3R2C18不同之处在于连接基团(linker)位置乙氧基重复单元数量的不同，其中1个和3个重复单元时载体的基因编辑效率一般，2个重复单元时载体的基因编辑效率最好。
优选地，所述核糖核蛋白复合物为CRISPR-Cas9核糖核蛋白复合物。
本发明还提供了上述纳米复合物在基因编辑和基因治疗中的应用。
作为本发明所述应用的优选实施方式，基因编辑靶向的位点包括AAVS1位点、HBB位点、EGFP位点或KRAS位点。
本发明还提供了一种修饰后的纳米复合物，包括透明质酸和上述的纳米复合物。优选地，透明质酸的浓度为0.1～2mg/mL。更优选地，透明质酸的浓度为0.2mg/mL。
由于纳米复合物带正电荷会被网状内皮系统迅速清除，因此本发明发明人经过大量研究及试验发现，用一层透明质酸修饰纳米复合物的表面，使得纳米复合物通过静电相互作用与透明质酸的阴离子壳包覆以生成修饰后的纳米复合物。透明质酸(HA)是一种具有优异生物相容性的带负电荷的天然多糖，用于屏蔽纳米复合物的正电荷以呈现长体内循环和隐身特性。
纳米复合物采用优选浓度的透明质酸进行表面修饰，可以使得纳米复合物的zeta电位从正变成负电荷。此外，修饰后的纳米复合物具有抗血清能力，可以有效延长体内血流循环时间。
本发明还提供了上述纳米复合物或修饰后的纳米复合物在制备抗肿瘤药物中的应用。
本发明提供了一种纳米复合物，对肿瘤组织中的靶向突变基因位点有较高的基因编辑效果，进一步说明阳离子脂质类似物递送核糖核蛋白复合物对肿瘤 生长有一定的抑制作用。
本发明还提供了一种基因编辑核糖核蛋白复合物细胞内递送方法，所述方法利用阳离子脂质类似物实现在基因编辑核糖核蛋白复合物胞内递送。
本发明还提供了上述纳米复合物中的阳离子脂质类似物在基因编辑核糖核蛋白复合物胞内递送的应用。
分别在293T细胞、293T-EGFP细胞、SW-480细胞等细胞系中递送基因编辑核糖核蛋白复合物编辑不同的基因，实验结果表明，本发明的阳离子脂质类似物在细胞内递送过程中可以达到较高的递送效率；本发明的阳离子脂质类似物可以高效地递送核糖核蛋白复合物至对应细胞内编辑4个基因位点(AAVS1、HBB、EGFP和KRAS)，效率优于商业化递送试剂Lipofectamine CRISPRMAX(以下简称CMAX)。
需要说明的是，本文所用术语“递送”或者“胞内递送”指的是使药物从细胞的外部进入到细胞的内部，使其局限在细胞溶质中或在细胞的细胞器内。术语“细胞”或“胞内”包括293T细胞、293T-EGFP细胞和SW-480细胞中的至少一种。
与现有技术相比，本发明具有以下的有益效果：
本发明提供了一种包含阳离子脂质类似物的纳米复合物对肿瘤组织中的不同基因位点(AAVS1、HBB、EGFP和KRAS)有较高的基因编辑效果，进一步说明阳离子脂质类似物递送核糖核蛋白复合物对肿瘤生长有一定的抑制作用；基因编辑核糖核蛋白复合物递送载体在细胞内递送过程中可以达到高递送效率，安全有效，且不需要多余的化学修饰，节省了生产成本。
附图说明
图1为阳离子脂质类似物A1I2-1R2C18的质谱(a)和核磁共振氢谱(b)；
图2为阳离子脂质类似物A1I2R2C18的质谱(a)和核磁共振氢谱(b)；
图3为阳离子脂质类似物A1I2-3R2C18的质谱(a)和核磁共振氢谱(b)；
图4为阳离子脂质类似物A1I2-1R2C18(a)、A1I2R2C18(b)和A1I2-3R2C18(c)递送基因编辑核糖核蛋白复合物(RNP)靶向AAVS1位点的基因编辑效率结果图；
图5为实施例3形成的纳米复合物的粒径分布图；
图6为阳离子脂质类似物A1I2R2C18在293T细胞(a和b)，293T-EGFP 细胞(c)和SW-480细胞(d)上递送Cas9RNP蛋白的T7E1酶切实验结果图以及4个基因位点的T-A克隆的Sanger序列结果图；
图7为经过T7E1酶切实验和ImageJ软件定量统计阳离子脂质类似物A1I2R2C18对4个基因位点(AAVS1、HBB、EGFP和KRAS)的基因编辑效率结果图，并与商业递送试剂CMAX进行对比；
图8为RNP/A1I2R2C18纳米复合物处理293T-EGFP细胞48小时后的荧光图像(a，比例尺为50μm)和流式细胞术(FCM)检测RNP/A1I2R2C18纳米复合物处理293T-EGFP细胞48小时的相对平均荧光强度(MFI)(b)；
图9为利用不同浓度的透明质酸(HA)修饰RNP/A1I2R2C18纳米复合物得到的一系列修饰后的纳米复合物(颗粒)的粒径大小(a)和zeta电位(b)；
图10为利用透明质酸与RNP/A1I2R2C18纳米复合物混合得到修饰后的纳米复合物(颗粒)在无血清培养基和含10％FBS的血清培养基的稳定性结果图；
图11为通过靶向突变KRAS位点的RNP/A1I2R2C18纳米复合物介导的体内抗肿瘤效果图；
图12为通过瘤旁注射RNP/A1I2R2C18纳米复合物介导的肿瘤中的靶向突变KRAS位点的T7E1酶切实验结果图。
具体实施方式
为更好的说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。
在以下实施例中，所使用的实验方法如无特殊说明，均为常规方法，所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、阳离子脂质类似物的合成与表征
本发明的阳离子脂质类似物的合成路线为：
Figure PCTCN2022075915-appb-000014
其中，胺类化合物m 2-NH 2为
Figure PCTCN2022075915-appb-000015
羧酸类化合物
Figure PCTCN2022075915-appb-000016
为
Figure PCTCN2022075915-appb-000017
醛类化合物
Figure PCTCN2022075915-appb-000018
为
Figure PCTCN2022075915-appb-000019
异氰化合物
Figure PCTCN2022075915-appb-000020
为
Figure PCTCN2022075915-appb-000021
本实施例的阳离子脂质类似物的具体制备方法为：分别将1mmol的异丁醛和1mmol的胺类化合物加入到0.5mL甲醇溶液中，反应60min后依次加入1mmol羧酸类化合物和0.5mmol异氰化合物，并于40℃反应12h，反应结束后经层析色谱柱分离提纯产物，其中，流动相采用甲醇和二氯甲烷的混合液。
本实施例采用的原料及合成的阳离子脂质类似物结构如表1所示。
表1
Figure PCTCN2022075915-appb-000022
选取阳离子脂质类似物A1I2-1R2C18、A1I2R2C18和A1I2-3R2C18作为代表材料，并对其结构进行表征。其中，A1I2-1R2C18的质谱和核磁共振氢谱的谱图如图1所示；A1I2R2C18的质谱和核磁共振氢谱的谱图如图2所示；A1I2-3R2C18的质谱和核磁共振氢谱的谱图如图3所示。核磁共振氢谱和质谱的结果与预期阳离子脂质类似物的结构一致。
实施例2、阳离子脂质类似物A1I2-1R2C18、A1I2R2C18和A1I2-3R2C18递送基因编辑核糖核蛋白复合物(RNP)的胞内效果。
具体操作方法如下：首先通过体外转录IVT的方法制备靶向AAVS1的sgRNA(sgAAVS1)(AAVS1的靶点为GGCTCCCTCCCAGGATCCTCTC)。将293T细胞接种到24孔板中过夜，待293T细胞密度达到75％以上时，开始Cas9RNP蛋白递送实验。
首先将分别为1μg CRISPR-Cas9蛋白与0.5μg sgAAVS1在37℃孵育10分钟后形成CRISPR-Cas9/sgAAVS1复合物(Cas9RNP)，再将CRISPR-Cas9/sgAAVS1复合物分别与A1I2-1R2C18、A1I2R2C18和A1I2-3R2C18 这三个阳离子脂质类似物混合，快速混匀10～30秒后立即用450μL无血清DMEM培养基进行稀释，最终得到Cas9RNP/阳离子脂质类似物的500μL体系溶液。其中CRISPR-Cas9的剂量为每孔1μg，sgAAVS1的剂量为每孔0.5μg，A1I2-1R2C18、A1I2R2C18和A1I2-3R2C18这三个阳离子脂质类似物的剂量为每孔分别1、2、3和4μg。移除细胞培养基，使用PBS清洗两次后，加入Cas9RNP/阳离子脂质类似物的500μL体系溶液，37℃培养箱孵育4小时。移除培养基，加入500μL含有10％血清的DMEM培养基，继续培养48小时。使用商业化试剂盒提取基因组总DNA，PCR扩增带有突变位点的目的片段(AAVS1-FP:CTATGTCCACTTCAGGACAGCATGT，AAVS1-RP:CCTCTTGGGAAGTGTAAGGAAGCTG)，加热变性退火复性处理，最后加入0.3μL的T7E1核酸内切酶，37℃反应30分钟后，跑2％的琼脂糖凝胶电泳检测分析酶切结果。
实验结果：从图4的结果表明A1I2-1R2C18、A1I2R2C18和A1I2-3R2C18这三个阳离子脂质类似物递送Cas9RNP蛋白具有基因编辑效率。其中，A1I2R2C18具有最高的基因编辑递送效率。同时，结果表明A1I2R2C18的剂量为2μg时与Cas9RNP蛋白(CRISPR-Cas9蛋白与sgAAVS1的剂量分别为1μg和0.5μg)形成的纳米复合物递送至293T细胞中的基因编辑效率最佳。对比结构相似分子A1I2-1R2C18和A1I2-3R2C18，发明人发现A1I2R2C18具有更好的基因编辑效率，这三个分子的不同之处在于连接基团(linker)位置乙氧基重复单元数量的不同，其中1个和3个重复单元时载体材料的基因编辑效率一般，2个重复单元效果最好，表明优化linker结构对提升递送和编辑效率具有重要意义。
实施例3、纳米复合物的粒径分布
将阳离子脂质类似物A1I2R2C18与基因编辑核糖核蛋白复合物(RNP)形成本发明的RNP/A1I2R2C18纳米复合物，并利用动态光散射(DLS)表征纳米复合物的尺寸及表面电势。
具体操作方法如下：将核糖核蛋白复合物与A1I2R2C18溶液快速混合均匀，室温孵育10～30秒后，加入1mL去离子水稀释后，使用激光纳米粒度仪检测溶液中纳米颗粒的粒径分布和表面电势。
实验结果：从图5的结果表明，本发明制备的A1I2R2C18与核糖核蛋白复合物形成的纳米复合物DLS表征的颗粒直径约为460nm。
实施例4、阳离子脂质类似物A1I2R2C18递送核糖核蛋白复合物(RNP)至不同细胞内靶向不同位点的基因编辑实验
阳离子脂质类似物A1I2R2C18分别向293T细胞内递送靶向AAVS1和HBB位点的Cas9RNP蛋白；向293T-EGFP细胞内递送靶向EGFP位点的Cas9RNP蛋白；向SW-480细胞内递送靶向KRAS位点的Cas9RNP蛋白。此外，从293T细胞中获得AAVS1和HBB位点T-A克隆的Sanger序列；从293T-EGFP细胞获得EGFP位点T-A克隆的Sanger序列；从SW-480细胞获得KRAS位点T-A克隆的Sanger序列。
具体操作方法如下：首先通过体外转录IVT的方法分别制备：靶向AAVS1和HBB的sgRNA(sgAAVS1和sgHBB)(AAVS1的靶点为GGCTCCCTCCCAGGATCCTCTC；HBB的靶点为GGGTAACGGCAGACTTCTCCTC)；靶向EGFP的sgRNA(sgEGFP)(EGFP的靶点为GTGAACCGCATCGAGCTGAA)；靶向突变KRAS基因的sgRNA(sgKRAS)(KRAS的靶点为GTTGGAGCTGATGGCGT)。将293T细胞、293T-EGFP细胞和SW-480细胞接种到24孔板中过夜，待其细胞密度达到75％以上时，开始Cas9RNP蛋白递送实验。
首先将CRISPR-Cas9蛋白与sgAAVS1、sgHBB、sgEGFP和sgKRAS在37℃孵育10分钟后形成CRISPR-Cas9/sgRNA复合物，再将CRISPR-Cas9/sgRNA复合物与阳离子脂质类似物A1I2R2C18充分混合，快速混匀10～30秒后立即用450μL无血清DMEM培养基进行稀释，最终得到RNP/A1I2R2C18纳米复合物的500μL体系溶液。其中CRISPR-Cas9的剂量为每孔1μg，sgRNA的剂量为每孔0.5μg，阳离子脂质类似物A1I2R2C18的剂量为2μg。移除细胞培养基，使用PBS清洗两次后，加入RNP/A1I2R2C18纳米复合物的500μL体系溶液，37℃培养箱孵育4小时。移除培养基，加入500μL含有10％血清的DMEM培养基，继续培养48小时。使用商业化试剂盒提取基因组总DNA，PCR扩增带有突变位点的目的片段，引物如表2所示。
表2
Figure PCTCN2022075915-appb-000023
加热变性退火复性处理，最后加入0.3μL的T7E1核酸内切酶，37℃反应30分钟后，跑2％的琼脂糖凝胶电泳检测分析酶切结果。
T-A克隆的Sanger序列检测实验步骤：基因克隆选用Taq DNA聚合酶，进行PCR扩增再跑胶后纯化；连接反应：连接T载体并室温孵育5分钟；接下来转化：取感受态100μL加入上述10μL连接产物，轻轻吹打搅拌混匀后冰上静置5分钟，42℃热激45秒后迅速转移至冰浴中静置2分钟，之后涂板培养过夜并测序。
实验结果：从图6的结果表明本发明制备的阳离子脂质类似物A1I2R2C18在293T细胞、293T-EGFP细胞和SW-480细胞上递送Cas9RNP蛋白的T7E1酶切实验检测基因编辑效果以及4个基因位点的T-A克隆的Sanger序列结果。结果表明本发明制备的阳离子脂质类似物A1I2R2C18可安全高效地递送Cas9 RNP蛋白至对应细胞内编辑相关基因的位点，并且优于商业化试剂Lipofectamine CRISPRMAX(CMAX)。
实施例5、通过T7E1酶切实验和ImageJ软件对4个基因位点(AAVS1、HBB、EGFP和KRAS)的基因编辑效率进行定量统计。
具体操作方法如下：
T7E1酶切实验简述:使用商业化试剂盒提取基因组总DNA，PCR扩增带有突变位点的目的片段，加热变性退火复性处理，最后加入0.3μL的T7E1核酸内切酶，37℃反应30分钟后，跑2％的琼脂糖凝胶电泳检测分析酶切结果。ImageJ软件对4个基因位点(AAVS1、HBB、EGFP和KRAS)的基因编辑切割条带的效率 进行定量统计步骤如下：首先通过File—Open打开要分析的胶图进行条带的灰度分析，接下来将图片转为为8位的灰度图，然后用矩形工具框任选一个条带，通过Analyze/gels/select first lane为条带编号，再用魔棒工具(Wandtool)点击每个峰的中间区域即可逐一完成对峰面积的计算，计算的结果在Result窗口显示，可通过File导出为xls文件。最后用Indel百分率计算公式:[1-(1-切割条带强度值)1/2]×100％，可得到切割效率数值，进而可统计定量。
实验结果：从图7的结果表明本发明制备的阳离子脂质类似物A1I2R2C18在293T细胞、293T-EGFP细胞和SW-480细胞上递送Cas9RNP蛋白的T7E1酶切实验检测基因编辑效果。
结果表明本发明制备的阳离子脂质类似物A1I2R2C18可安全高效地递送Cas9RNP蛋白至对应细胞内编辑4个基因位点(AAVS1、HBB、EGFP和KRAS)，并且都优于商业化试剂Lipofectamine CRISPRMAX(CMAX)。
实施例6、RNP/A1I2R2C18纳米复合物对293T-EGFP细胞的影响
RNP/A1I2R2C18纳米复合物处理293T-EGFP细胞48小时后的使用荧光显微镜观察细胞内的荧光强度和分布。再通过流式细胞术分析RNP/A1I2R2C18纳米复合物处理48小时的293T-EGFP细胞并检测其相对平均荧光强度。
具体操作方法如下：将293T-EGFP细胞接种到24孔板中过夜，待其细胞密度达到75％以上时，开始Cas9RNP蛋白递送实验。首先将CRISPR-Cas9蛋白与sgEGFP在37℃孵育10分钟后形成CRISPR-Cas9/sgRNA复合物，再将CRISPR-Cas9/sgRNA复合物与阳离子脂质类似物A1I2R2C18充分混合，快速混匀10～30秒后立即用450μL无血清DMEM培养基进行稀释，最终得到Cas9 RNP/A1I2R2C18的500μL体系溶液(RNP/A1I2R2C18纳米复合物)。其中CRISPR-Cas9的剂量为每孔1μg，sgRNA的剂量为每孔0.5μg，阳离子脂质类似物A1I2R2C18的最佳剂量为2μg。移除细胞培养基，使用PBS清洗两次后，加入RNP/A1I2R2C18纳米复合物的500μL体系溶液，37℃培养箱孵育4小时。移除培养基，加入500μL含有10％血清的DMEM培养基，继续培养48小时。之后再荧光显微镜下观察细胞内的荧光强度和分布。再收集孔板内细胞进行流式细胞术分析并检测其相对平均荧光强度。
实验结果：从图8的结果表明通过荧光显微镜观察细胞内的荧光强度、荧 光分布和流式细胞术分析，相较于商用试剂CMAX阳性对照组，RNP/A1I2R2C18纳米复合物处理组有更低的荧光荧光强度，从而说明阳离子脂质类似物A1I2R2C18可安全高效地递送Cas9RNP蛋白至293T-EGFP细胞中对EGFP位点进行高效的基因编辑。
实施例7、制备修饰后的RNP/A1I2R2C18纳米复合物
一种修饰后的纳米复合物，包括透明质酸和RNP/A1I2R2C18纳米复合物。透明质酸的浓度为0.1～2mg/mL。
上述修饰后的纳米复合物的制备方法：将不同浓度的透明质酸(0.1～2mg/mL)逐滴加入RNP/A1I2R2C18纳米复合物溶液中，通过静电涂覆获得HA/RNP/A1I2R2C18纳米复合物(修饰后的纳米复合物)。然后，将溶液混合物在室温下轻轻搅拌30分钟。HA/RNP/A1I2R2C18纳米复合物制备中的最佳透明质酸的浓度由DLS确定。
实验结果：从图9的结果表明，DLS测定显示，涂层包裹的最佳透明质酸是浓度为0.2mg/mL。透明质酸修饰后，RNP/A1I2R2C18纳米复合物的zeta电位从正(约+19mV)变为负电荷(约-31mV)。
实施例8、验证HA/RNP/A1I2R2C18纳米复合物(修饰后的纳米复合物)的稳定性和血清抗性
在含血清的培养基中进行血清抗性研究，无血清的培养基作为阴性对照组。
具体操作方法如下：将制备含HA浓度为0.2mg/mL的HA/RNP/A1I2R2C18纳米复合物分别加入无血清的DMEM培养基和含有10％胎牛血清(FBS)的DMEM培养基孵育一段时间，分别再0.5h、24h、36h和48h通过光动态散射仪(DLS)检测HA/RNP/A1I2R2C18纳米复合物的粒径变化，从而得出稳定性结果。
实验结果：从图10的结果表明，DLS分析表明HA/RNP/A1I2R2C18纳米复合物在含10％FBS的培养基中孵育0.5h时，相同孵育时间的粒径大小仅略大于无血清培养基中的粒径大小，并且进一步孵育不会导致粒径明显增加。在含血清培养基中延长的孵育时间不会进一步导致粒度方面的显著变化。因此，HA/RNP/A1I2R2C18纳米复合物的抗血清能力将有效延长体内血流循环时间。
实施例9、通过靶向突变KRAS位点的RNP/A1I2R2C18纳米复合物介导的体内抗肿瘤效果
给药方式为瘤旁注射RNP/A1I2R2C18纳米复合物。监测并记录不同治疗组在治疗后三周的平均肿瘤生长体积变化。
具体操作方法如下：在SW-480异种移植原发肿瘤模型中，将SW-480细胞(1×10 6)皮下注射到BALB/c裸鼠右侧。7天后，当肿瘤达到大约50–80mm 3的大小时，将小鼠随机分成4组并用对照组、仅RNP组、RNP/A1I2R2C18组和Mock RNP/A1I2R2C18组处理。纳米复合物每周一次通过肿瘤周围皮下注射，持续三周，测量裸鼠的肿瘤体积。
实验结果：从图11的结果表明，监测并记录不同治疗组在治疗后三周的平均肿瘤生长体积变化。相较于对照组，给予RNP/A1I2R2C18纳米复合物瘤旁皮下注射治疗后，肿瘤生长体积有明显的抑制作用。
实施例10、RNP/A1I2R2C18纳米复合物介导肿瘤中的靶向突变KRAS位点的T7E1酶切实验
具体操作方法如下：收集对照组、仅RNP组、RNP/A1I2R2C18组和Mock RNP/A1I2R2C18组的肿瘤组织，使用商业化试剂盒提取基因组总DNA，PCR扩增带有突变位点的目的片段(KRAS-FP:TGCAGTCAACTGGAATTTTCAT；KRAS-RP:GTTGGATCATATTCGTCCACAA)，加热变性退火复性处理，最后加入0.3μL的T7E1核酸内切酶，37℃反应30分钟后，跑2％的琼脂糖凝胶电泳检测分析酶切结果。
实验结果：从图11的结果表明RNP/A1I2R2C18纳米复合物通过肿瘤周围皮下注射治疗，对肿瘤组织中的靶向突变KRAS位点有较高的基因编辑效果，从而说明阳离子脂质类似物分子A1I2R2C18递送Cas RNP蛋白对肿瘤生长有一定的抑制作用。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

Claims (11)

  1. 一种纳米复合物，其特征在于，包括阳离子脂质类似物和核糖核蛋白复合物；
    所述阳离子脂质类似物具有如式(I)所示结构：
    Figure PCTCN2022075915-appb-100001
    式(I)中，m 1独立地选自氢、直链烷基、支链烷基；
    m 2为
    Figure PCTCN2022075915-appb-100002
    R1为烷基，R2为烷基，R3为烷基，或R1与R2连接为环基；
    m 3独立地选自直链烷基、直链烯基；
    m 4独立地选自直链烷基、含醚键的直链烷基。
  2. 如权利要求1所述的纳米复合物，其特征在于，所述阳离子脂质类似物和核糖核蛋白复合物的质量比为(1～4):1。
  3. 如权利要求1所述的纳米复合物，其特征在于，所述m 1为氢、
    Figure PCTCN2022075915-appb-100003
    Figure PCTCN2022075915-appb-100004
    m 2为
    Figure PCTCN2022075915-appb-100005
    Figure PCTCN2022075915-appb-100006
    m 3为
    Figure PCTCN2022075915-appb-100007
    Figure PCTCN2022075915-appb-100008
    Figure PCTCN2022075915-appb-100009
    m 4为
    Figure PCTCN2022075915-appb-100010
    Figure PCTCN2022075915-appb-100011
  4. 如权利要求1所述的纳米复合物，其特征在于，所述阳离子脂质类似物具有如下36种结构中的任一种：
    Figure PCTCN2022075915-appb-100012
    Figure PCTCN2022075915-appb-100013
  5. 如权利要求4所述的纳米复合物，其特征在于，所述阳离子脂质类似物为A1I2R2C16、A1I2R2C18、A1I2R2C19、A1I2R2C20、A1I2R3C16、A1I2R3C18、A1I2R3C19、A1I2R3C20、A1I2R11C16、A1I2R11C18、A1I2R11C19、A1I2R11C20、A1I2-1R2C18、A1I2-1R2C19、A1I2-1R2C20、A1I2-3R2C18、A1I2-3R2C19、A1I2-3R2C20中的至少一种，更优选地，所述阳离子脂质类似物为A1I2R2C18、A1I2-1R2C18或A1I2-3R2C18。
  6. 如权利要求1～5任一项所述的纳米复合物在基因编辑和基因治疗中的应用。
  7. 如权利要求6所述的应用，其特征在于，基因编辑靶向的位点包括AAVS1位点、HBB位点、EGFP位点或KRAS位点。
  8. 一种修饰后的纳米复合物，其特征在于，包括透明质酸和如权利要求1～5任一项所述的纳米复合物。
  9. 如权利要求1～5任一项所述的纳米复合物或如权利要求8所述的修饰后的纳米复合物在制备抗肿瘤药物中的应用。
  10. 一种基因编辑核糖核蛋白复合物细胞内递送方法，其特征在于，所述方法利用权利要求1中的阳离子脂质类似物实现在基因编辑核糖核蛋白复合物胞内递送。
  11. 如权利要求1～5任一项所述的纳米复合物中的阳离子脂质类似物在基因编辑核糖核蛋白复合物胞内递送的应用。
PCT/CN2022/075915 2022-01-27 2022-02-10 阳离子脂质类似物在基因编辑核糖核蛋白复合物胞内递送中的应用 WO2023142168A1 (zh)

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