阅读说明：本技术 含苯硼酸修饰的高分子材料及其在基因编辑核糖核蛋白复合物胞内递送中的应用 (Phenylboronic acid modified high polymer material and application thereof in intracellular delivery of gene editing ribonucleoprotein complex ) 是由 程义云 平渊 万涛 刘崇懿 吕佳 于 2019-04-10 设计创作，主要内容包括：本发明提供一种含苯硼酸修饰的高分子材料在基因编辑核糖核蛋白复合物胞内递送中的应用,所述含苯硼酸修饰的高分子材料由阳离子聚合物和含苯硼酸的功能基团组成,所述含苯硼酸功能基团共价连接在阳离子聚合物上；所述阳离子高分子包括聚酰胺-胺树形高分子、聚丙烯亚胺、聚赖氨酸等；所述含苯硼酸修饰的高分子材料可以作为基因编辑核糖核蛋白复合物的胞内递送载体,即将基因编辑核糖核蛋白复合物由细胞外递送到细胞内的载体。本发明提供的含苯硼酸修饰的高分子材料作为基因编辑核糖核蛋白复合物胞内递送载体的方法可以达到高效率,在同一细胞不同基因位点以及不同种类细胞均有较好的基因编辑效果,并且可以保持核糖核蛋白复合物的生物活性,同时对细胞产生的毒性小,具有良好的生物相容性。(The invention provides an application of a high molecular material modified by phenylboronic acid in intracellular delivery of a gene-editing ribonucleoprotein complex, wherein the high molecular material modified by the phenylboronic acid consists of a cationic polymer and a functional group containing the phenylboronic acid, and the functional group containing the phenylboronic acid is covalently connected to the cationic polymer; the cationic polymer comprises polyamide-amine dendrimer, polypropylene imine, polylysine and the like; the high molecular material modified by phenylboronic acid can be used as an intracellular delivery carrier of a gene-editing ribonucleoprotein complex, namely a carrier for delivering the gene-editing ribonucleoprotein complex from the outside to the inside of a cell. The method for using the phenylboronic acid modified high polymer material as the intracellular delivery carrier of the gene editing ribonucleoprotein complex can achieve high efficiency, has good gene editing effect on different gene sites and different types of cells of the same cell, can keep the bioactivity of the ribonucleoprotein complex, has low toxicity on the cell, and has good biocompatibility.)

1. The polymer material modified by the phenylboronic acid is characterized by comprising a cationic polymer and a functional group containing the phenylboronic acid, wherein the functional group containing the phenylboronic acid is connected to the cationic polymer through a covalent bond; the structure of the phenylboronic acid-modified polymer material is shown in the following formulas (1) to (3):

in the formulae (1) to (3),

R₁is a cationic polymer;

R₂is a connecting bond;

R₃、R₄、R₅、R₆are respectively a chemical functional group and are respectively and independently selected from H, halogen, C1-C5 alkyl, C1-C5 methoxyl and nitryl;

x is the connecting number of the phenylboronic acid-containing functional group and is an integer between 1 and 256.

2. The phenylboronic acid-modified polymeric material of claim 1, wherein the R linkage is₂Is selected from-NH-CH₂-, -NH-C (═ O) -O-, -NH-C (═ O) -NH-, or-NH-C (═ S) -NH-.

3. The phenylboronic acid-modified polymeric material of claim 1, wherein the cationic polymer is selected from the group consisting of: a polyamide-amine dendrimer represented by the formula (4), a branched polyethyleneimine represented by the formula (5), a linear polyethyleneimine represented by the formula (6), an alpha-polylysine represented by the formula (7), or a polylysine represented by the formula (8):

in the formula (4), n is an integer between 1 and 10; m is an integer between 2 and 4; m is the core of a polyamide-amine dendrimer; when the core is ethylenediamine, butanediamine, hexanediamine, octanediamine, decanediamine and 1, 12-dodecanediamine, m is 4; when the core is ammonia, m is 3; when the core is methylamine, ethylamine, n-propylamine, n-butylamine, n-pentylamine, n-hexylamine, n-octylamine, n-decylamine and n-dodecylamine, m is 2;

in the formula (5), n is an integer between 2 and 100;

in the formula (6), n is an integer between 20 and 1500;

in the formula (7), n is an integer between 20 and 1000;

in the formula (8), n is an integer of 20 to 1000.

4. The phenylboronic acid-modified polymer material according to claim 1, wherein the structure of the phenylboronic acid-modified polymer material is represented by formula (1) to formula (3); the structural formula of the cationic polymer is shown in a formula (4), wherein n is an integer between 1 and 10, M is an integer between 2 and 4, and M is 4 when M is ethylenediamine, butanediamine, hexanediamine, octanediamine, decanediamine and 1, 12-dodecanediamine; when the core is ammonia, m is 3; when the core is methylamine, ethylamine, n-propylamine, n-butylamine, n-pentylamine, n-hexylamine, n-octylamine, n-decylamine and n-dodecylamine, m is 2; connecting bond R₂is-NH-CH₂-；R₃、R₄、R₅、R₆Each independently selected from H, F, Cl, Br, methyl, methoxy, nitro; x is an integer between 1 and 256.

5. The phenylboronic acid-modified polymeric material of claim 1, wherein the structure of the phenylboronic acid-modified polymeric material comprises the following:

in the formula (9), R₁The polyamide-amine dendrimer is a 5 th generation polyamide-amine dendrimer, wherein M is ethylenediamine, n is 5, M is 4, the theoretical molecular weight is 28826, 128 primary amine groups are arranged on the surface of the polymer, and X is respectively: 44,72, 84, 58 and 59;

in the formula (10), R₁The polymer is a 5 th generation polyamide-amine dendrimer, M is ethylenediamine, n is 5, M is 4, the theoretical molecular weight is 28826, 128 primary amine groups are arranged on the surface of the polymer, and the average connecting number X of phenylboronic acid is 61;

in the formula (11), R₁The polymer is a 5 th generation polyamide-amine dendrimer, M is ethylenediamine, n is 5, M is 4, the theoretical molecular weight is 28826, 128 primary amine groups are arranged on the surface of the polymer, and the average connecting number X of phenylboronic acid is 59;

in the formula (12), R₁The polyamide-amine dendrimer is a 5 th generation polyamide-amine dendrimer, wherein M is ethylenediamine, n is 5, M is 4, the theoretical molecular weight is 28826, 128 primary amine groups are arranged on the surface of the polymer, and the average connecting number of 2, 3-difluoro-4-formylphenylboronic acid is 53;

in the formula (13), R₁The polyamide-amine dendrimer is a 5 th generation polyamide-amine dendrimer, wherein M is ethylenediamine, n is 5, M is 4, the theoretical molecular weight is 28826, 128 primary amine groups are arranged on the surface of the polymer, and the average connecting number of 2-fluoro-4-formylphenylboronic acid is 61;

in the formula (14), R₁The polyamide-amine dendrimer is a 5 th generation polyamide-amine dendrimer, wherein M is ethylenediamine, n is 5, M is 4, the theoretical molecular weight is 28826, 128 primary amine groups are arranged on the surface of the polymer, and the average connecting number of 4-formyl-2-methoxyphenylboronic acid is 55.

6. The use of the phenylboronic acid-modified polymeric material of any one of claims 1-5 for intracellular delivery of a gene-editing ribonucleoprotein complex.

7. The use of claim 6, wherein the gene-editing ribonucleoprotein complex is a CRISPR-Cas9 ribonucleoprotein complex.

8. A gene-editing ribonucleoprotein complex intracellular delivery vector, which comprises the phenylboronic acid-modified polymeric material according to any one of claims 1 to 5.

9. A complex comprising a polymer material modified with phenylboronic acid according to any one of claims 1 to 5, and a gene-editing ribonucleoprotein complex; wherein the high molecular material carries the gene-editing ribonucleoprotein complex.

10. The phenylboronic acid-modified polymeric material of any one of claims 1-5, the gene-editing ribonucleoprotein complex intracellular delivery vector of claim 8, or the use of the complex of claim 9 in gene editing and gene therapy.

11. A method for intracellular delivery of a gene-editing ribonucleoprotein complex, wherein the intracellular delivery of the gene-editing ribonucleoprotein complex is achieved by using the phenylboronic acid-modified polymeric material according to any one of claims 1 to 5.

Technical Field

The invention relates to the fields of biotechnology, polymer chemistry, cell biology and the like, in particular to a novel polymer material modified by phenylboronic acid and application thereof in intracellular delivery of a gene-editing ribonucleoprotein complex.

Background

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems are derived from the adaptive immune system of bacteria and archaea, which are mainly used to protect against the invasion of exogenous nucleic acids from phages, plasmids and the like. With this naturally occurring immune system, the CRISPR/Cas system has been developed as a novel gene editing technology that can target a target sequence of a genome at a site or multiple target sequences of a genome simultaneously for genome editing. The system is widely applied to the treatment of gene-related diseases, the living body imaging of a directional detection genome region, the identification of new disease-related targets, the identification of gene functions, the establishment of animal disease models and other researches at present. Among the numerous CRISPR/Cas systems, CRISPR/Cas9, the most representative gene editing system, mainly comprises two core components: CRISPR/Cas9 endonuclease and single-stranded guide RNA (sgRNA), a CRISPR/Cas 9-based gene editing technique that has been successfully applied to mammalian cell genome editing since 2013, has been applied to a range of intractable diseases including malignancies, sickle cell anemia, mucopolysaccharidosis type I-H, alzheimer's disease, hepatic glycogen storage disease, hemophilia, cystic fibrosis, duchenne muscular dystrophy and others. Before genome editing technology based on the CRISPR/Cas9 system is applied to clinic, complex technical challenges such as improvement of specificity of gene editing, reduction of off-target and genome mutation rate and the like are required. On the other hand, how to safely and effectively introduce the CRISPR/Cas9 gene editing system into a specific cell, tissue or organ to obtain a desired therapeutic effect is another critical issue to be solved. However, due to the lack of the current vector system capable of efficiently and safely delivering CRISPR/Cas9, the potential of the CRISPR/Cas9 gene editing technology in clinical application is greatly limited. Therefore, the development of a high-efficiency and low-toxicity CRISPR/Cas9 delivery vector system has extremely important scientific value and research significance for promoting the transformation of the CRISPR/Cas9 gene editing technology to clinical application, reducing toxic and side effects and improving the safety of treatment. Currently there are three general ways to perform genome editing using the CRISPR/Cas9 system in vitro or in vivo. The first approach is to encode the Cas9 protein and sgRNA into the same pDNA vector for genome editing. The second approach is to deliver Cas9mRNA and sgRNA simultaneously for genome editing. The third approach is genome editing using a complex formed by Cas9 ribonucleoprotein complex and sgRNA. Each of these three delivery schemes has advantages and disadvantages, and each presents unique challenges. Of the three delivery formats of CRSPR-Cas9, the simplest and most efficient method is to deliver a complex formed by the Cas9 protein and the sgRNA. Delivery of the complex formed by Cas9 protein and sgRNA can avoid the insertion risk and the process of transcription and translation caused by the delivery of pDNA, compared to the mode of delivery based on plasmid pDNA. On the other hand, since pDNA exists in cells for a long time, Cas9 protein is expressed continuously, resulting in that Cas9 protein exists in cells for a long time for gene editing, which also results in strong immune response and higher off-target effect. In contrast to the mode of delivering mRNA, delivery of Cas9 protein can avoid the translation process of mRNA and also protect against intracellular RNA interference. However, due to the large molecular weight and the structural complexity of the Cas9 protein, the efficiency of lysosome escape is low after being taken up by cells, and the protein is easily degraded and inactivated during transportation and storage, so the development of nanocarriers capable of delivering Cas9 ribonucleoprotein complexes faces a huge challenge. Currently reported non-viral vectors for delivery of Cas9 protein include cationic polymers, cationic liposomes, lipid-like derivatives, liposome complexes, hydrogel nanoparticles, gold nanoparticles, DNA nanowire spheres, metal organic frameworks and ultrathin two-dimensional black phosphorus nanosheets. Compared with numerous delivery modes, the high-molecular carrier has the advantages of low synthesis cost, easiness in chemical modification, easiness in implementation of endosome escape, good biocompatibility and the like, and has a wide application prospect in intracellular delivery of the gene editing ribonucleoprotein complex. The existing delivery method for the ribonucleoprotein complex generally needs chemical modification or biological modification on endonuclease such as Cas9, has high synthesis cost, can destroy the original biological activity of the protein, and is inconvenient to popularize and apply.

Disclosure of Invention

In order to solve the problem that the polymer carrier in the prior art can not be well combined with the ribonucleoprotein complex, the invention innovatively provides a polymer material modified by phenylboronic acid as a carrier for intracellular delivery of the ribonucleoprotein complex. The carrier has high intracellular delivery efficiency, and the complex delivered into cells still has biological activity. Meanwhile, the toxicity of the material and the delivery operation process to cells is small.

The invention provides a method for realizing high-efficiency delivery of a ribonucleoprotein complex by using a phenylboronic acid-containing cationic polymer. In the invention, on one hand, the phenylboronic acid molecule can form a nitrogen-boron coordination bond with a cationic group on the surface of endonuclease, such as amino, imidazole and the like, under a neutral condition; on the other hand, the cationic group on the polymer can be bonded to the nucleic acid in the complex and the negatively charged region on the protein surface by electrostatic interaction, thereby forming a stable complex. The invention designs and synthesizes cationic macromolecules containing phenylboronic acid molecules by using the innovative concept, and further obtains a high-efficiency and low-toxicity ribonucleoprotein complex delivery carrier.

The invention provides a polymer material modified by phenylboronic acid, which comprises a cationic polymer and a functional group containing the phenylboronic acid, wherein the functional group containing the phenylboronic acid is connected to the cationic polymer through a covalent bond.

The structure of the phenylboronic acid modified polymer material is shown in formulas (1) to (3):

in the formulae (1) to (3),

R₁is a cationic polymer and is characterized in that,

R₂is a connecting bond comprising-NH-CH₂-, -NH-C (═ O) -O-, -NH-C (═ O) -NH-, -NH-C (═ S) -NH-, and the like;

R₃、R₄、R₅、R₆are chemical functional groups and are respectively and independently selected from H, halogen, C1-C5 alkyl, C1-C5 methoxyl, nitryl and the like; preferably, R₃、R₄、R₅、R₆Each independently selected from H, F, Cl, Br, methyl, methoxy, nitro; further preferably, R₃、R₄、R₅、R₆Each independently selected from H.

X is the connecting number of the functional group containing the phenylboronic acid, and is an integer between 1 and 256; preferably, X is an integer between 42 and 128; further preferably, X is an integer between 42 and 84.

Wherein R is₁Including but not limited to polyamide-amine dendrimer as shown in formula (4), branched polyethyleneimine as shown in formula (5), linear polyethyleneimine as shown in formula (6), alpha-polylysine as shown in formula (7), polylysine as shown in formula (8), etc.;

in the formula (4), n is an integer between 1 and 10; preferably, n is an integer between 4 and 6; further preferably, n ═ 5;

m is an integer between 2 and 4; preferably, m is 3 or 4; further preferably, m is 4;

m is the core of a polyamide-amine dendrimer; when the core is ethylenediamine, butanediamine, hexanediamine, octanediamine, decanediamine and 1, 12-dodecanediamine, m is 4; when the core is ammonia, m is 3; when the core is methylamine, ethylamine, n-propylamine, n-butylamine, n-pentylamine, n-hexylamine, n-octylamine, n-decylamine and n-dodecylamine, m is 2; preferably, the core is ammonia or ethylenediamine.

In the formula (5), n is an integer of 2 to 100.

In the formula (6), n is an integer of 20 to 1500.

In the formula (7), n is an integer of 20 to 1000.

In the formula (8), n is an integer of 20 to 1000.

Preferably, the structure of the phenylboronic acid modified polymer material is shown as formula (1) to formula (3); the structural formula of the cationic polymer is shown in a formula (4), wherein n is an integer between 1 and 10, M is an integer between 2 and 4, and M is 4 when M is ethylenediamine, butanediamine, hexanediamine, octanediamine, decanediamine and 1, 12-dodecanediamine; when the core is ammonia, m is 3; when the core is methylamine, ethylamine, n-propylamine, n-butylamine, n-pentylamine, n-hexylamine, n-octylamine, n-decylamine and n-dodecylamine, m is 2; connecting bond R₂is-NH-CH₂-；R₃、R₄、R₅、R₆Each independently selected from H, F, Cl, Br, methyl, methoxy, nitro; x is an integer between 1 and 256. In a specific embodiment, the structure of the phenylboronic acid-modified polymeric material comprises the following:

in the formula (9), R1 is a 5 th generation polyamidoamine dendrimer, M is ethylenediamine, n is 5, M is 4, the theoretical molecular weight is 28826, 128 primary amine groups are on the surface of the polymer, and X is: 44,72, 84, 58 and 59;

in the formula (10), R1 is a 5 th generation polyamide-amine dendrimer, M is ethylenediamine, n is 5, M is 4, the theoretical molecular weight is 28826, 128 primary amine groups are arranged on the surface of the polymer, and the average connecting number X of phenylboronic acid is 61;

in the formula (11), R1 is a 5 th generation polyamide-amine dendrimer, M is ethylenediamine, n is 5, M is 4, the theoretical molecular weight is 28826, the polymer surface has 128 primary amine groups, and the average connecting number X of phenylboronic acid is 59;

in the formula (12), R1 is a 5 th generation polyamidoamine dendrimer, M is ethylenediamine, n is 5, M is 4, the theoretical molecular weight is 28826, 128 primary amine groups are on the surface of the polymer, and the average number of the connecting strips of 2, 3-difluoro-4-formylphenylboronic acid is 53;

in the formula (13), R1 is a 5 th generation polyamidoamine dendrimer, M is ethylenediamine, n is 5, M is 4, the theoretical molecular weight is 28826, 128 primary amine groups are on the surface of the polymer, and the average number of the 2-fluoro-4-formylphenylboronic acid connecting strips is 61;

in the formula (14), R1 is a 5 th generation polyamidoamine dendrimer, M is ethylenediamine, n is 5, M is 4, the theoretical molecular weight is 28826, 128 primary amine groups are on the surface of the polymer, and the average number of the connecting strips of 4-formyl-2-methoxyphenylboronic acid is 55.

The invention also provides application of the phenylboronic acid modified high polymer material in intracellular delivery of a gene editing ribonucleoprotein complex. The phenylboronic acid-modified high polymer material is shown in a formula (1) -formula (3), and is used as a carrier for intracellular delivery of a ribonucleoprotein complex. Preferably, the gene-editing ribonucleoprotein complex is a CRISPR-Cas9 ribonucleoprotein complex.

The invention also provides a gene editing ribonucleoprotein complex intracellular delivery vector, which comprises the phenylboronic acid modified high molecular material.

The invention also provides a novel compound which comprises the phenylboronic acid modified high polymer material and the ribonucleoprotein compound shown in the formulas (1) to (3); wherein the gene-editing ribonucleoprotein complex is carried by the polymer material.

The invention also provides the phenylboronic acid modified high polymer material, the gene editing ribonucleoprotein complex intracellular delivery vector and application of the complex in gene editing and gene therapy.

The invention also provides a gene-editing ribonucleoprotein complex intracellular delivery method, which utilizes the phenylboronic acid-modified high polymer material shown in the formulas (1) to (3) to realize intracellular delivery of the gene-editing ribonucleoprotein complex.

The invention is used for delivering gene editing ribonucleoprotein complexes to edit different genes in 293T, 293T-EGFP, HCT-116, HT-29 and other cell lines. The experimental result shows that the invention has the following advantages: the invention provides a phenylboronic acid modified polymer material, application of the phenylboronic acid modified polymer material in intracellular delivery of a gene-editing ribonucleoprotein complex, and a geneThe intracellular delivery method of the edited ribonucleoprotein complex has higher intracellular delivery efficiency which is far higher than that of a commercial transfection reagent Lipofectamine CRISPRMAX^TM(ii) a The material prepared by the invention can efficiently deliver CRISPR-Cas9 protein to 293T-EGFP cell for editing EGFP gene, and is far superior to a commercial reagent Lipofectamine CRISPRMAX^TM(ii) a The polymer material modified by phenylboronic acid can efficiently deliver CRISPR-Cas9 protein to 293T cells to edit AAVS1 and HBB genes, and is far superior to a commercial reagent Lipofectamine CRISPRMAX^TM(ii) a The polymer material modified by phenylboronic acid can efficiently deliver CRISPR-Cas9 protein to HT-116 and HT-29 intracellular editing AAVS1 and HBB genes; the gene-editing ribonucleoprotein complex delivery vector provided by the invention can achieve high delivery efficiency in the intracellular delivery process, has low preparation cost and low material cytotoxicity, can effectively and safely deliver the gene-editing ribonucleoprotein complex to cytoplasm, and does not need to chemically modify protein and polypeptide molecules.

Drawings

FIG. 1 is a dynamic light scattering characterization of the complex formed by the phenylboronic acid-modified polymer material P4 and the gene-editing ribonucleoprotein complex in example 1.

FIG. 2 is a graph of the efficiency of delivering the gene-editing ribonucleoprotein complex-editing EGFP gene into 293T-EGFP cells by phenylboronic acid-modified polymeric material P4 in example 1, compared to the commercial reagent Lipofectamine CRISPRMAX.

FIG. 3 shows the efficiency of delivering the gene-editing ribonucleoprotein complex-editing AAVS1 and HBB gene into 293T cells by the phenylboronic acid-modified polymer material P4 in example 1, and the comparison with the commercial reagent Lipofectamine CRISPRMAX.

FIG. 4 shows the efficiency of delivering the gene-editing ribonucleoprotein complex editing AAVS1 and HBB gene into HCT-116 cells and HT-29 cells by the phenylboronic acid-modified polymer material P4 in example 1.

FIG. 5 is a diagram of the synthetic route and the structure of a carrier of polymer material P7-P11 modified with phenylboronic acid in example 7.

FIG. 6 is a synthetic route and a structure diagram of a carrier of polymer material P12 modified with phenylboronic acid in example 7.

FIG. 7 is a synthetic route and a structure diagram of a carrier of polymer material P13 modified with phenylboronic acid in example 7.

FIG. 8 is a synthetic route and a structure diagram of a carrier of the polymer material P14 modified with phenylboronic acid in example 7.

FIG. 9 shows the efficiency of delivering the gene-editing ribonucleoprotein complex-editing KRAS gene into SW-480 cells by the phenylboronic acid-modified polymeric material P4 in example 1 and the polymeric material P7 in example 7.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and drawings, and the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.