Supramolecular nano gene vector as well as preparation method and application thereof

Abstract

The invention relates to a supramolecular nano gene vector as well as a preparation method and application thereof. The supramolecular nano-carrier is prepared from beta-cyclodextrin grafted polyethyleneimine, 8-adamantane grafted polyamide-amine dendrimer, adamantane grafted polyethylene glycol and adamantane grafted polyethylene glycol modified by using a cell penetrating peptide (TAT). A CRISPR / Cas9 system is loaded through self-assembly of a nano material monomer, the prescription is screened, and the subsequent research on cells and retinal organs proves that the transfection efficiency and the RS1 protein expression quantity are good. The supramolecular nanoparticle loaded gene editing system has a good potential of treating the X-linked retina cleavage disease. Moreover, the supramolecular nanoparticle gene vector is high in biological safety, high in transfection efficiency, simple and easily available in preparation mode, and good in clinical application potential in the future.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and particularly relates to a supramolecular nano gene carrier, a preparation method thereof, and an application thereof. Background Art

[0002] Gene editing for treating diseases mainly intervenes the expression of specific genes in target cells by targeting the knockout or knock-in of exogenous genes, so as to achieve the purpose of treating diseases. Gene therapy is widely used, involving cancer, hemophilia, retinal diseases, etc. However, the key problems faced by gene drugs are that naked nucleic acids are easily degraded by nucleases, and exogenous genes are easily recognized and cleared by the immune system; there is a strong electrostatic repulsion between DNA or RNA and negatively charged phospholipid bilayers, making it quite challenging for cells to uptake genes. Therefore, developing safe and effective gene delivery vectors to improve the targeted delivery efficiency and transfection efficiency is not only the key to the success of gene therapy but also a key technical problem at present.

[0003] At present, two major categories of vectors, viral vectors and non-viral vectors, have been developed. Although viral vectors have high efficiency, problems such as potential biosafety risks and high production costs have greatly reduced their clinical applications. Non-viral vectors mainly include lipid nanoparticles (LNPs), which have unique advantages such as simple preparation, low immunogenicity, and not being easily integrated with host genes, and have become important candidates for nucleic acid drug delivery. However, the LNP system also has some deficiencies, such as toxicity, poor biodistribution, and low transfection efficiency. Moreover, LNP delivery is mainly limited to the liver and has insufficient targeting ability for non-liver organs. The rapid development of nanotechnology has provided new ideas for solving gene delivery. Rational utilization of the advantages of various nanoparticles to design the structure of nano carriers is beneficial to combining several excellent gene drug delivery platforms together, thereby improving the delivery efficiency of gene drugs, helping to optimize the therapeutic effect of gene drugs, and having great potential for future gene-based therapies. Therefore, designing a delivery system with high biosafety, low toxicity, high targeting, and high transfection efficiency has important application potential in the fields of gene therapy, gene editing, gene vaccines, etc.

[0004] The eye is a very important organ of the human body. As the most important sense of the human body, visual diseases will deeply affect the quality of human life. X-linked retinoschisis (XLRS) is a common clinical genetic disease. XLRS is caused by retinoschisin 1

[0005] (Retinoschisin 1, RS1) gene mutation causes the disease. The RS1 gene is responsible for encoding a protein of 224 amino acids, which is called RS1 protein. The RS1 protein is related to cell - cell interaction and cell adhesion, can maintain the inter - layer structure of retinal cells, ensure the normal connection of synapses and promote the transduction of visual signals. There is still no clear and effective treatment for this disease. At present, gene therapy methods have been widely concerned by researchers, but inflammatory reactions have occurred in clinical trials using viral vectors to carry genes. Therefore, developing a supramolecular nano - delivery system for the treatment of XLRS is of positive significance in terms of the effectiveness and safety of treatment. Summary of the Invention

[0006] Aiming at the clinical needs of treating X - linked retinoschisis, the purpose of the present invention is to provide a supramolecular nano - gene vector, its preparation method and application. The supramolecular nano - gene vector provided by the present invention consists of four parts, including β - cyclodextrin - grafted polyethyleneimine (β - CD - PEI), 8 - adamantane - grafted polyamidoamine dendrimer (8 - Ad - PAMAM), adamantane - grafted polyethylene glycol (Ad - PEG) and adamantane - grafted polyethylene glycol modified with cell - penetrating peptide (TAT) (Ad - PEG - TAT). The supramolecular nano - gene vector described in the present invention can load the target gene in nanoparticles to form a stable and highly efficient gene delivery vector. Through the evaluation of its loading rate, the highest loading rate is 92%. Through the transfection of HEK293T cells, the prepared supramolecular nano - gene vector has a high transfection efficiency. When RS1 gene delivery is carried out in the retinal organoids of retinoschisis patients, good fluorescence protein expression and the expression levels of RS1 protein and photoreceptor - related proteins are found, showing the potential to cure the disease at the genetic level. The transfection reagent provided by the present invention has the advantages of low toxicity, high transfection efficiency and high targeting, can effectively overcome the problems existing in the current gene delivery system, and has an important application prospect in the field of realizing precise and efficient gene knock - in.

[0007] The present invention is achieved through the following technical solutions:

[0008] The first object of the present invention is to provide a supramolecular nano - gene vector, including β - cyclodextrin - grafted polyethyleneimine, 8 - adamantane - grafted polyamidoamine dendrimer, adamantane - grafted polyethylene glycol and adamantane - grafted polyethylene glycol modified with cell - penetrating peptide.

[0009] In an embodiment of the present invention, the 8 - adamantane - grafted polyamidoamine dendrimer is prepared by the following method:

[0010] Dissolve PAMAM in an organic solvent to form a PAMAM solution. Add 1-adamantyl isocyanate and stir the reaction at room temperature. Then remove the solvent. Add ether to the reaction residue to produce a white precipitate. Collect, wash, and dry it by filtration to obtain 8-adamantyl-grafted polyamide-amine dendrimer (white solid, 8-Ad-PAMAM).

[0011] In one embodiment of the present invention, the adamantyl-grafted polyethylene glycol is prepared by the following method:

[0012] Dissolve 1-adamantylamine hydrochloride in an organic solvent, sequentially add triethylamine and mPEG-NHS, stir at room temperature, remove the solvent in vacuo, and add water to the reaction residue. Centrifuge to remove unreacted 1-adamantylamine, filter, and then dialyze overnight using a dialysis cassette and lyophilize to obtain adamantyl-grafted polyethylene glycol (white powder, Ad-PEG).

[0013] In one embodiment of the present invention, the adamantyl-grafted polyethylene glycol modified with a cell-penetrating peptide is prepared by the following method:

[0014] Add triethylamine and MAL-PEG-NHS to an organic solution of 1-adamantylamine hydrochloride and stir at room temperature. After the reaction is completed, remove the solvent in vacuo, and add a PBS buffer solution containing CGRKKRRQRRR (TAT peptide) to the reaction residue. Stir the resulting mixture at room temperature, centrifuge, dialyze, and lyophilize to obtain adamantyl-grafted polyethylene glycol modified with a cell-penetrating peptide (white powder, Ad-PEG-TAT).

[0015] In one embodiment of the present invention, the β-cyclodextrin-grafted polyethyleneimine is prepared by the following method:

[0016] Dissolve branched polyethyleneimine in an organic solvent, add 6-p-toluenesulfonyl-β-cyclodextrin (6-OTs-β-CD) and react. After dialysis, filter the resulting mixture to remove unreacted 6-OTs-β-CD (white precipitate). Freeze-dry the filtrate overnight to obtain β-cyclodextrin-grafted polyethyleneimine (white fluffy solid, β-CD-PEI).

[0017] In one embodiment of the present invention, the mass ratio of the 8-adamantyl-grafted polyamide-amine dendrimer to the β-cyclodextrin-grafted polyethyleneimine is 3.75:1 - 15:1; preferably 6:1.

[0018] In one embodiment of the present invention, the mass ratio of the β-cyclodextrin-grafted polyethyleneimine to the adamantyl-grafted polyethylene glycol is 1:2.5 - 1:10; preferably 1:4.

[0019] In one embodiment of the present invention, the mass ratio of the adamantane-grafted polyethylene glycol to the adamantane-grafted polyethylene glycol modified with a cell-penetrating peptide is 8:1 - 12:1; preferably 10:1.

[0020] In one embodiment of the present invention, the particle size of the supramolecular nano gene carrier is 30 nm - 140 nm.

[0021] The second object of the present invention is to provide a method for preparing the supramolecular nano gene carrier, comprising the following steps:

[0022] (1) Prepare β-cyclodextrin-grafted polyethyleneimine, 8-adamantane-grafted polyamidoamine dendrimer, adamantane-grafted polyethylene glycol, and adamantane-grafted polyethylene glycol modified with a cell-penetrating peptide respectively;

[0023] (2) Dissolve the 8-adamantane-grafted polyamidoamine dendrimer in an organic solvent to obtain a solution of the 8-adamantane-grafted polyamidoamine dendrimer, add a solvent, vortex and mix evenly, cool in an ice bath, add β-cyclodextrin-grafted polyethyleneimine and adamantane-grafted polyethylene glycol, vortex and mix evenly, cool in an ice bath; continue to add the adamantane-grafted polyethylene glycol modified with a cell-penetrating peptide, oscillate and mix evenly, cool in an ice bath to obtain the supramolecular nano gene carrier.

[0024] In one embodiment of the present invention, in step (2), the solvent is enzyme-free water and / or PBS. Preferably it is enzyme-free water.

[0025] The third object of the present invention is to provide a nano gene delivery preparation, comprising the supramolecular nano gene carrier and a plasmid loaded in the nanoparticles.

[0026] In one embodiment of the present invention, the plasmid includes one or more of Donor RS1 / GFP plasmid, Cas9 / sgRNA plasmid, and GFP plasmid.

[0027] In one embodiment of the present invention, the particle size of the nano gene delivery preparation is about 120 nm, the particle size distribution is uniform, and the dispersibility is good, which is suitable for gene drug delivery.

[0028] The fourth object of the present invention is to provide the application of the nano gene delivery preparation in the preparation of a drug for treating single-gene genetic diseases.

[0029] In one embodiment of the present invention, the genetic diseases include one or more of treating X-linked retinoschisis, sickle cell anemia, β-thalassemia, and Duchenne muscular dystrophy.

[0030] The supramolecular nanoparticles modified by Ad-PEG-TAT of the present invention can significantly improve the cell transfection efficiency.

[0031] The above technical solution of the present invention has the following advantages compared with the prior art:

[0032] (1) The raw materials for synthesizing the nanomaterial monomers are convenient to obtain;

[0033] (2) The self-assembly process of the supramolecular nanoparticles is simple, fast, and efficient;

[0034] (3) The nano gene delivery preparation provided by the present invention includes a supramolecular nano gene carrier and a plasmid loaded in the nanoparticles; considering that the supramolecular nanoparticles can effectively encapsulate the plasmid and can be taken up by cells through endocytosis, a supramolecular nano gene carrier with a particle size of 100 nm is preferably used to encapsulate the plasmid, and a nano gene delivery preparation with a particle size of about 120 nm is obtained, which has good dispersibility;

[0035] (4) The encapsulation rate of the gene is high;

[0036] (5) It has low cytotoxicity, high biological safety, and is easily metabolized and excreted from the body;

[0037] (6) It has high transfection efficiency, has a good expression level of RS1 protein on the retinal organoids of XLRS patients, and is a good gene delivery vector. Description of the Drawings

[0038] In order to make the content of the present invention easier to be clearly understood, the following further detailed description of the present invention is made according to the specific embodiments of the present invention in conjunction with the drawings, wherein,

[0039] Figure 1 is the transmission electron microscope image of the supramolecular nano carrier in the present invention;

[0040] Figure 2 is the in vitro stability evaluation of the supramolecular nano carrier in the present invention; wherein, A is the stability under temperature change; B is the stability over time; C is the stability before and after ultrasonic treatment;

[0041] Figure 3 is the cell transfection fluorescence image of the two kinds of SMNPs obtained in Example 4 and Comparative Example 1 of the present invention; wherein, (a) is the fluorescence image after transfection with the three-component SMNPs, and (b) is the fluorescence image after transfection with the four-component SMNPs;

[0042] Figure 4 is the cell transfection fluorescence of the supramolecular nano carrier in the present invention;

[0043] Figure 5 is the result of the inhibitory effect of different concentrations of SMNPs on the activity of HEK293T cells in the present invention;

[0044] Figure 6 It shows the fluorescence protein expression of XLRS patients on Day 30 and Day 39 after the third gene knock-in in the present invention. Detailed implementation manners

[0045] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited are not intended to limit the present invention.

[0046] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0047] The sources of the materials and reagents used in the present invention are as follows:

[0048] PAMAM was purchased from Shanghai Merck Chemical Technology Co., Ltd., with the product number 536709;

[0049] 1-adamantyl isocyanate was purchased from Shanghai Merck Chemical Technology Co., Ltd., with the product number M54534;

[0050] 1-adamantylamine hydrochloride was purchased from Shanghai Merck Chemical Technology Co., Ltd., with the product number A1260;

[0051] Triethylamine was purchased from Sinopharm Chemical Reagent Co., Ltd., with the product number 80134318;

[0052] mPEG-NHS was purchased from Shanghai Macklin Biochemical Co., Ltd., with the product number MKL-P968648;

[0053] The dialysis cassette was purchased from Thermo Fisher Scientific, USA, with the product numbers 2KD 66230; 3.5KD 66110; 20KD 66030;

[0054] MAL-PEG-NHS was purchased from ponsure biology, with the product number PS2-MH-5K;

[0055] The TAT peptide was purchased from GenScript Biotech Corporation, with the product number RP20256;

[0056] Branched polyethyleneimine was purchased from Shanghai Beyotime Biotechnology Co., Ltd., with the product number C0539 and the specification of 100 mL;

[0057] 6-p-toluenesulfonyl-β-cyclodextrin was purchased from Shanghai Macklin Biochemical Co., Ltd., M830137-5g;

[0058] The GFP plasmid was purchased from Shanghai Beyotime Biotechnology Co., Ltd., with the product number D2626;

[0059] The Donor-RS1 / GFP plasmid was custom-made by Shanghai Sangon Biotech Co., Ltd.;

[0060] The Cas9 / sgRNA plasmid was synthesized by Shanghai Sangon Biochemical Engineering Co., Ltd.;

[0061] HEK293T cells were purchased from Shanghai Beyotime Biotechnology Co., Ltd., with the product number C6008;

[0062] The RMM-2 medium was prepared from the following substances: 200 mL of high-glucose DMEM medium (purchased from Thermo Fisher Scientific, USA, product number 11965092); 200 mL of DMEM / F12 medium (Thermo Fisher Scientific, USA, product number 11320033); 4 mL of NEAA (Shanghai Beyotime Biotechnology Co., Ltd., China, product number C0332-100ml); 4 mL of double antibody (Shanghai Beyotime Biotechnology Co., Ltd., China, product number C0222); 8 mL of B27 additive (Thermo Fisher Scientific, USA, product number 17504044); 4 mL of GlutaMAX solution (Thermo Fisher Scientific, USA, product number A1286001).

[0063] Retinal organoids ROs were differentiated and cultured from human induced pluripotent stem cells of healthy and patients with X-linked retinoschisis;

[0064] Lipo3000 was purchased from Thermo Fisher Scientific, USA, with the product number L3000001;

[0065] High-glucose DMEM complete medium: Prepared according to the ratio of DMEM:FBS:double antibody as 100:10:1; FBS was purchased from Thermo Fisher Scientific, USA, with the product number 10099141C.

[0066] The synthesis methods of β-CD-PEI, Ad-PEG, 8-Ad-PAMAM, and Ad-PEG-TAT used in the examples of the present invention are as follows:

[0067] (1) Synthesis of 8-Ad-PAMAM

[0068] A methanol solution containing PAMAM (20 wt%, 100 mg, 0.07 mmol) was added to a round-bottom flask. Methanol was evaporated in vacuo and it was dissolved in 10 mL of dry DMF to form a PAMAM solution. 1-Adamantyl isocyanate (244.6 mg, 1.4 mmol) in 10 mL of dry DMF was added to the PAMAM solution. After the mixture was stirred at room temperature for 2 h, the solvent was removed in vacuo. Diethyl ether (100 mL) was added to the reaction residue to produce a white precipitate, which was collected by filtration. The white precipitate was washed with diethyl ether (100 mL × 3) and dried to obtain the white solid 8-Ad-PAMAM.

[0069] (2) Synthesis of Ad-PEG

[0070] To a solution of 1-adamantylamine hydrochloride (187.7 mg, 1.0 mmol, 5.0 equiv) dissolved in 10 mL of CH2Cl2, triethylamine (105.0 mg, 1.0 mmol, 5.1 equiv) and mPEG-NHS (1.0 g, 0.2 mmol, 1.0 equiv) were added successively. It was stirred at room temperature for 2 h, then the solvent was removed in vacuo and water was added to the reaction residue. The solution was transferred to a centrifuge tube and centrifuged at 10000 rpm for 10 min to remove unreacted 1-adamantylamine. It was filtered through a 0.45 μm filter, and then dialyzed overnight using a dialysis cassette (MWCO, 2 kD) and lyophilized to obtain a white powder.

[0071] (3) Synthesis of Ad-PEG-TAT

[0072] To a solution of 1-adamantylamine hydrochloride (0.94 mg, 5.0 μmol, 5.0 equiv) and CH2Cl2 (1.0 mL), triethylamine (0.6 mg, 5.0 μmol, 5.0 equiv) and MAL-PEG-NHS (5.0 mg, 1.0 mmol, 1.0 equiv) were added. The reaction mixture was stirred at room temperature for 2 h. After the reaction was completed, the solvent was removed in vacuo, and a PBS buffer solution (1 mL) containing CGRKKRRQRRR (TAT peptide, 7.5 mg, 5.0 μmol, 5.0 equiv) was added to the reaction residue. The mixture was stirred at room temperature for another 2 h. The solution was transferred to a centrifuge tube and centrifuged at 10000 rpm for 10 min to remove unreacted 1-adamantylamine. The solution was filtered through a 0.45 μm filter, and then dialyzed using a Slide-A-Lyzer dialysis cassette (MWCO, 3.5 kD) overnight and lyophilized to obtain the white powder Ad-PEG-TAT.

[0073] (4) Synthesis of β-CD-PEI

[0074] Branched polyethyleneimine (100 mg, 10.0 μmol) was dissolved in 100 mL of dimethyl sulfoxide, and 6-p-toluenesulfonyl-β-cyclodextrin (6-OTs-β-CD) (1.29 g, 1.0 mmol) was added. After the mixture was reacted at 70 °C for 3 days, it was transferred to a dialysis cassette (MWCO, 10 kDa) and dialyzed with deionized water for 6 days. After dialysis, the reaction mixture was filtered to remove unreacted 6-OTs-β-CD (white precipitate), and the filtrate was freeze-dried overnight to obtain the product β-CD-PEI as a white soft solid.

[0075] Example 1 Preparation of Supramolecular Nanogene Vector

[0076] This example provides a method for preparing a supramolecular nanogene vector, wherein the mixing mass ratio of 8-Ad-PAMAM, β-CD-PEI and Ad-PEG is 6:1:4, specifically as follows:

[0077] Precisely weigh 7.5 μg of 8-Ad-PAMAM into a centrifuge tube, add 10 μL of DMSO to dissolve 8-Ad-PAMAM to form an 8-Ad-PAMAM solution. Subsequently, add enzyme-free water (to construct a 50 μL system) to the centrifuge tube and vortex to mix evenly. Add 1.25 μg of β-CD-PEI and 5 μg of Ad-PEG and vortex to mix evenly, and place in an ice bath for 30 min; continue to add 0.5 μg of Ad-PEG-TAT and vortex to mix evenly, and place in an ice bath for 30 min. A supramolecular nanogene vector (SMNPs) with a particle size of 100 nm was obtained.

[0078] Example 2

[0079] This example provides a method for preparing a supramolecular nanogene vector, which is similar to Example 1, except that: the mixing mass ratio of 8-Ad-PAMAM, β-CD-PEI, and Ad-PEG is 15:4:10; the remaining operations are the same, and a supramolecular nanogene vector (SMNPs) with a particle size of 30 nm was obtained.

[0080] Example 3

[0081] This example provides a method for preparing a supramolecular nanogene vector, which is similar to Example 1, except that: the mixing mass ratio of 8-Ad-PAMAM, β-CD-PEI, and Ad-PEG is 15:1:10; the remaining operations are the same, and a supramolecular nanogene vector (SMNPs) with a particle size of 140 nm was obtained.

[0082] Test Example

[0083] (1) Stability with temperature change: To understand the thermal stability of SMNPs, the size changes of the obtained SMNPs with particle sizes of 30 nm, 100 nm, and 140 nm were monitored by real-time DLS measurement at different temperatures from 10 °C to 50 °C. The samples were equilibrated at the given temperature for 20 min.

[0084] (2) In vitro stability test conditions: Stability with time change. After mixing four molecular building blocks in their respective ratios, SMNPs with particle sizes of 30 nm, 100 nm, and 140 nm were prepared, and real-time DLS measurement was used to monitor the size changes of SMNPs at different times. The size of SMNPs was recorded every 4 minutes for 36 minutes. The results showed that the nanoparticles exhibited good stability during the change with time.

[0085] (3) Dynamic stability: Sonication was used to provide the energy barrier required to overcome the reorganization of SMNPs. SMNPs with particle sizes of 30 nm, 100 nm, and 140 nm were prepared, and the size of SMNPs in solution was measured. Before and after sonication (1 MHz, 50 W, 10 min) of each SMNP at room temperature for 10 min, it was monitored by DLS. No significant size change was observed under the experimental conditions, indicating that sonication alone could not decompose SMNP. (The results are as Figure 2 shown)

[0086] Example 4

[0087] Dissolve 8-Ad-PAMAM (7.5 μg) in 10 μL DMSO, add 1.5 μg of GFP plasmid, add enzyme-free water (to construct a 50 μL system), mix well by oscillation, and incubate on ice for 10 min; add Ad-PEG (5.0 μg) and β-CD-PEI (1.25 μg), mix well by oscillation and incubate on ice for 30 min. Then continue to add Ad-PEG-TAT (0.5 μg), mix well by oscillation and incubate on ice for 30 min, and then obtain GFP plasmid SMNPs.

[0088] Comparative Example 1

[0089] Dissolve 8-Ad-PAMAM (7.5 μg) in 10 μL DMSO, add 1.5 μg of GFP plasmid, add enzyme-free water (to construct a 50 μL system), mix well by oscillation, and incubate on ice for 10 min; add Ad-PEG (5.0 μg) and β-CD-PEI (1.25 μg), mix well by oscillation and incubate on ice for 30 min to obtain a three-component supramolecular nanoparticle loaded with GFP plasmid.

[0090] Seed the cells in a 24-well plate with a cell density of 5×10 4per well. After 18 h, discard the old culture medium, add the two kinds of SMNPs obtained in Example 4 and Comparative Example 1 into the well plate (3 groups for each kind of SMNPs, a total of 6 wells are used), and supplement with 1 mL / well of serum-containing culture medium. After 48 h, analyze with a confocal fluorescence microscope, and the results are as Figure 3 shown; the results show that the fluorescence efficiency of cells is higher after adding the Ad-PEG-TAT component, and the transfection efficiency is increased from 55% to 75%. Therefore, the Ad-PEG-TAT component is indispensable in the gene delivery process of SMNPs.

[0091] Example 5 Donor RS1 / GFP SMNPs and Cas 9 / sgRNA Co-delivery transfection of HEK293T cells with SMNPs:

[0092] Self-assemble the four synthesized nanomaterial monomers into supramolecular nanoparticles: by adjusting the following 3 parameters: the mass ratio of 8-Ad-PAMAM, β-CD-PEI, and Ad-PEG is 6:1:4. The specific synthesis scheme is as follows:

[0093] Weigh 8-Ad-PAMAM (7.5 μg) and dissolve it in 10 μL of DMSO, add 1.5 μg of Donor-RS1 / GFP plasmid, add enzyme-free water (to construct a 50 μL system), shake and mix well and ice-bath for 10 min; add Ad-PEG (5.0 μg), β-CD-PEI (1.25 μg), shake and mix well and ice-bath for 30 min; add Ad-PEG-TAT (0.5 μg), shake and mix well and ice-bath for 30 min. Subsequently, obtain DonorRS1 / GFP SMNPs. After the mixture is allowed to stand at 4 °C for 30 min, the particle size of Donor RS1 / GFP SMNPs is characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM).

[0094] Prepare Cas9 / sgRNA SMNPs by a similar method, with the only difference being that the Donor-RS1 / GFP plasmid is replaced with the Cas9 / sgRNA plasmid, and the rest of the operations are the same. After storing the mixture at 4 °C for 1 h, the size of the nanoparticles is characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM).

[0095] After characterization, Donor RS1 / GFP SMNPs and Cas9 / sgRNA The particle sizes of SMNPs are all around 120 nm, with a uniform particle size distribution and good morphological dispersion. After co-transfecting HEK293T cells, it shows a good transfection efficiency of 65% under a fluorescence microscope. The MTT assay shows low cytotoxicity.

[0096] Example 6: Donor RS1 / GFP&Cas 9 / sgRNA Transfection of HEK 293T cells with SMNPs:

[0097] The synthesized nanomaterial monomers are self-assembled to synthesize supramolecular nanoparticles. A similar self-assembly method is applied to prepare Donor RS1 / GFP&Cas 9 / sgRNA SMNPs. The synthesis formula is as follows:

[0098] Weigh 8-Ad-PAMAM and dissolve it in 10 μL of DMSO, add Cas9 / sgRNA plasmid (0.75 μg), donor RS1 / GFP plasmid (0.75 μg), and nuclease-free water (to construct a 50 μL system), vortex and mix well, and place on ice for 10 min; add Ad-PEG (5.0 μg) and β-CD-PEI (1.25 μg), oscillate to dissolve and place on ice for 30 min; add Ad-PEG-TAT (0.5 μg), oscillate and mix well, and place on ice for 30 min. Subsequently, Donor RS1 / GFP&Cas9 / sgRNA SMNPs are obtained. After storing the mixture at 4 °C for 1 h, the nanoparticle size is characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM). The particle size is around 120 nm, with a uniform particle size distribution and good morphological dispersion. When transfected into HEK293T cells, it shows a good transfection efficiency of 75% under a fluorescence microscope, as Figure 4 shown.

[0099] The MTT assay shows that this delivery system has low cytotoxicity:

[0100] The MTT method was used to investigate the inhibitory effect on the activity of HEK293T cells at different concentrations of SMNPs (25 μL / mL - 200 μL / mL) under the optimal formulation. It can be seen from Figure 5 this that the SMNP delivery systems at different concentrations show a concentration-dependent inhibitory effect. At 150 μL / mL, there is still an 80% cell survival rate, and at 200 μL / mL, the cell survival rate drops to 60%. However, in the study, when using 50 μL / mL SMNPs, a relatively high transfection efficiency can be achieved. Therefore, the SMNPs used in the present invention are basically non-toxic to cells, and the SMNP delivery system has good biosafety.

[0101] Comparative Example 2

[0102] Prepare DonorRS1 / GFP separately LNP, Cas9 / sgRNA LNP, Donor RS

[0103] 1 / GFP & Cas 9 / sgRNA Transfect with LNP by liposome transfection: In a 24-well plate seeded with cells, perform transfection during the logarithmic growth phase of the cells. Among them, DonorRS1 / GFP LNP, Cas9 / sgRNA LNP and Donor RS1 / GFP & Cas 9 / sgRNA The preparation method of LNP is similar to that of Example 5 and Example 6. The specific operation is as follows: Take a 1.5 mL centrifuge tube, add 2 μL of liposome reagent Lipo3000 and dilute it with 100 μL of serum-free DMEM medium, and vortex to mix evenly; Take another 1.5 mL centrifuge tube, add 1.5 μg of plasmid (1.5 μg of Cas9 / sgRNA plasmid, 1.5 μg of donor RS1 / GFP plasmid or 0.75 μg of Cas9 / sgRNA plasmid and 0.75 μg of donorRS1 / GFP plasmid) and dilute it with 100 μL of serum-free medium, and vortex to mix evenly. Mix the two tubes well, incubate in the dark for 20 min to obtain Donor RS1 / GFP LNP, Cas9 / sgRNA LNP, DonorRS1 / GFP & Cas 9 / sgRNA LNP.

[0104] Take out the seeded cells, discard the old medium and wash with PBS, add the incubated reagents respectively, gently shake to mix evenly, place in the incubator, discard the culture medium after 6 h, replace it with complete medium, and perform fluorescence quantification using a confocal fluorescence microscope after culturing for 48 h. The transfection efficiency ranges from 45% to 55%, which is lower than the efficiency of using SMNPs for delivery.

[0105] Example 7 Donor RS1 / GFP & Cas9 / sgRNA Transfect retinal organoids with SMNPs:

[0106] Take ROs and incubate them in 24-well plates treated with Matrigel at a density of 1000 ROs per well. Culture them using RMM-2 medium, and take the ROs of XLRS as the blank control (without delivery, replace the fresh RMM-2 medium every 2 days). Treatment group: Take the ROs of XLRS, and perform three deliveries of Donor RS1 / GFP&Cas9 / sgRNA every 14 days (Day0, Day14, Day28). Deliver SMNPs (prepared in Example 6). After the third gene knock-in, significant green fluorescent protein expression was observed on Day30 and Day39. The results showed that after the delivery of the RS1 / GFP gene by this supramolecular nanoparticle, significant fluorescent protein expression was observed under a fluorescence microscope, as Figure 6 shown. It shows that this supramolecular nanoparticle delivery system has good potential for precise gene knock-in therapy for X-linked retinoschisis.

[0107] Obviously, the above examples are only for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of this invention.

Claims

1. A supramolecular nano gene carrier, characterized in that, It includes β-cyclodextrin-grafted polyethyleneimine, 8-adamantane-grafted polyamidoamine dendrimer, adamantane-grafted polyethylene glycol, and adamantane-grafted polyethylene glycol modified with a cell-penetrating peptide.

2. The supramolecular nano gene carrier according to claim 1, characterized in that, The mass ratio of the 8-adamantane-grafted polyamidoamine dendrimer to the β-cyclodextrin-grafted polyethyleneimine is 3.75:1 - 15:

1.

3. The supramolecular nano gene carrier according to claim 1, wherein The mass ratio of the β-cyclodextrin-grafted polyethyleneimine to the adamantane-grafted polyethylene glycol is 1:2.5 - 1:

10.

4. The supramolecular nano gene carrier according to claim 1, wherein The mass ratio of the adamantane-grafted polyethylene glycol to the adamantane-grafted polyethylene glycol modified with a cell-penetrating peptide is 8:1 - 12:

1.

5. The supramolecular nano gene carrier according to claim 1, wherein The particle size of the supramolecular nano gene carrier is 30 nm - 140 nm.

6. The preparation method of the supramolecular nano gene carrier according to any one of claims 1-5, characterized in that, It includes the following steps: (1) Prepare β-cyclodextrin-grafted polyethyleneimine, 8-adamantane-grafted polyamidoamine dendrimer, adamantane-grafted polyethylene glycol, and adamantane-grafted polyethylene glycol modified with a cell-penetrating peptide respectively; (2) Dissolve the 8-adamantane-grafted polyamidoamine dendrimer in an organic solvent to obtain an 8-adamantane-grafted polyamidoamine dendrimer solution, add a solvent, vortex and mix evenly, cool in an ice bath, add β-cyclodextrin-grafted polyethyleneimine and adamantane-grafted polyethylene glycol, vortex and mix evenly, cool in an ice bath; continue to add adamantane-grafted polyethylene glycol modified with a cell-penetrating peptide, oscillate and mix evenly, cool in an ice bath to obtain the supramolecular nano gene carrier.

7. A nano gene delivery preparation, which includes the supramolecular nano gene carrier according to any one of claims 1 - 5 and a plasmid loaded in the nanoparticles.

8. The nano gene delivery preparation according to claim 7, wherein The plasmid includes one or more of Donor RS1 / GFP plasmid, Cas9 / sgRNA plasmid, and GFP plasmid.

9. Use of the nano gene delivery preparation according to any one of claims 7 - 8 in the preparation of a drug for treating monogenic hereditary diseases.

10. The application according to claim 9, characterized in that, The hereditary diseases include one or more of treating X-linked retinoschisis, sickle cell anemia, β-thalassemia, and Duchenne muscular dystrophy.

Images