CN116271105A - Lipid nanoparticle suitable for RPE cell transfection and application thereof - Google Patents

Abstract

The application discloses a lipid nanoparticle and a lipid nanoparticle-nucleic acid complex suitable for RPE cell transfection, which enable the transfection rate to be close to 100% after the lipid nanoparticle-nucleic acid complex is used for transfecting retinal pigment epithelial cells, enable the relevant gene editing efficiency to reach 78%, and enable the relevant base editing efficiency to reach 56%, thereby providing a new technical scheme for preparing therapeutic drugs for eye relevant diseases.

Description

Lipid nanoparticle suitable for RPE cell transfection and application thereof

Technical Field

The specification relates to the field of biological medicine, in particular to lipid nanoparticles suitable for RPE cell transfection and application thereof.

Background

Lipid nanoparticle (Lipid NanoParticles, LNP) delivery system is a delivery system that comprises cholesterol, a helper phospholipid, a cationic lipid, and a pegylated lipid as the main components, and encapsulates nucleic acid substances to form nanoparticles, thereby delivering genes into cells. Because LNP components are bionic from cell membranes, the safety and biocompatibility of the LNP are good. LNP is a clinically very excellent gene delivery vector at present, and is especially directed to nucleic acid substances such as mRNA with a large number of bases. LNP can efficiently and targeted deliver genes to different cells through the proportion change of the components and the selection of different substances of the components.

Retinal pigment epithelium (Retinal Pigment Epithelium, hereinafter RPE) is composed of a single layer of retinal pigment epithelial cells (Retinal Pigment Epithelial Cells, hereinafter RPE cells) that are located between the retina and the choroid. RPE plays an important role in normal physiological activities of the eye and eye health, which is not only one of the components of the blood-retinal barrier, controlling the ingress and egress of substances and immune-exemption. Furthermore, it secretes vascular endothelial growth factor (Vascular Endothelial Growth Factor, hereinafter referred to as VEGF), especially VEGFA (one of VEGF), which plays an important role in the growth of blood vessels in the choroid.

Age-related macular degeneration disease (Age-related Macular Degeneration, AMD) is an acquired retinal degeneration disease that is particularly well established in the elderly population. Serious central vision impairment is caused by the formation of non-neovascular (drusen and RPE abnormalities) and neovascular disorders. Wherein the non-neovascular form is atrophic or dry AMD, and the neovascular form is exudative or wet AMD (hereinafter referred to as wAMD). Choroidal Neovascularization (CNV) of the wtd infiltrates into the retina, severely affecting the patient's vision. Growth of CNV is regulated by VEGFA. By reducing the content of eye VEGFA, the atrophy of CNV is caused, so that the disease development and even the healing are controlled.

RPE cells secrete VEGFA in large amounts to the side near the choroid to maintain normal growth of the choroidal blood vessels. For wAMD patients, VEGFA secreted by RPE cells is counter-productive and promotes the development of CNV. Therefore, limiting VEGFA secretion by RPE cells is the most important means to treat wcmd. The current clinical treatment regimen is to inject neutralizing antibodies to VEGFA, such as ravigneaux, etc. However, antibodies are metabolized in the body and cannot be maintained for a long period of time, and thus require frequent injections (once every 2-3 months). Yet another therapy in clinical trials is to deliver mRNA of VEGFA monoclonal antibodies to RPE cells using AAV (adeno-associated virus) to produce constantly neutralizing antibodies to VEGFA. Thus, one injection can obviously treat the wAMD. However, mRNA delivered by AAV has a slow onset, often takes weeks or even tens of weeks to produce sufficient VEGFA neutralizing antibodies, and AAV enters the body to produce an immune response. Although the immune response is mild, in cases where a secondary injection of AAV is required, the secondary and subsequent injections of AAV may result in an inability to deliver the gene normally due to the immune response. AAV is also not delivered for genes above 5 Kbp due to size limitations.

Based on this, the development of more efficient, specific RPE cell delivery systems is a very important tool for ophthalmic gene therapy. Thus, the development of ophthalmic gene therapy, especially against diseases caused by RPE cells, has resulted in a very great promotion.

Disclosure of Invention

In order to solve the above problems, the present application provides a lipid nanoparticle-nucleic acid complex for delivering drugs for ophthalmic diseases into RPE cells.

The present application provides a lipid nanoparticle suitable for RPE cell transfection, the starting material of the lipid nanoparticle comprising a steroid lipid, a cationic lipid, a helper lipid and a pegylated lipid, the molar ratio of the steroid lipid, the helper lipid, the cationic lipid and the pegylated lipid being 37:10:51 to 51.5:1.5 to 2; the steroid lipid is selected from at least one of cholesterol or beta-sitosterol, the auxiliary lipid is selected from at least one of cardiolipin or DSPC, the cationic lipid is selected from at least two of SM102, DOTAP or ALC-0315, the polyethylene glycol lipid is selected from at least one of DSPE-PEG-Biotin, DMG-PEG or PEG-folic acid, the cationic lipid comprises ionizable cationic lipid and permanent cationic lipid, and the mole ratio of the ionizable cationic lipid to the permanent cationic lipid is 25-50: 10 to 25.

The application also provides the application of the lipid nanoparticle, which is used as a delivery carrier of nucleic acid.

The application also provides a lipid nanoparticle-nucleic acid complex comprising a nucleic acid and the lipid nanoparticle described above, the nucleic acid being loaded in the lipid nanoparticle.

The application also provides the use of the lipid nanoparticle-nucleic acid complexes described above for gene editing.

The application also provides application of the lipid nanoparticle-nucleic acid complex in preparing a medicament for treating eye related diseases.

The present application also provides a method for delivering a nucleic acid to a cell comprising contacting the lipid nanoparticle-nucleic acid complex described above with a cell.

The present application also provides a method of gene editing comprising contacting the lipid nanoparticle-nucleic acid complex described above with a cell containing a gene to be edited.

The application also provides the use of DSPE-PEG-Biotin in preparing the above lipid nanoparticle and the above lipid nanoparticle-nucleic acid complex.

Benefits provided by embodiments of the present description include, but are not limited to: (1) The lipid nanoparticle-nucleic acid complex can deliver nucleic acid to cells, resulting in a nucleic acid transfection efficiency approaching 100%; (2) By using lipid nanoparticle-nucleic acid complexes to deliver target nucleic acids to cells, the efficiency of gene editing can be 78%; (3) By using lipid nanoparticle-nucleic acid complexes to deliver target nucleic acids to cells, the relevant base editing efficiency can be 56%.

Drawings

The present application will be further illustrated by way of example embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein:

FIG. 1 is a diagram of the constituent components of schemes one through eight shown in some embodiments of the present application;

FIG. 2 is a diagram of the constituent components of schemes nine through sixteen according to some embodiments of the present application;

FIG. 3 is a diagram of the constituent components of schemes seventeen through twenty-two shown in some embodiments of the present application;

FIG. 4 is a histogram of lipid nanoparticle-nucleic acid complex transfection efficiency according to some embodiments of the present application;

FIG. 5 is a flow chart illustrating transfection effects of lipid nanoparticle-nucleic acid complexes according to some embodiments of the present application;

FIG. 6 is a bar graph of lipid nanoparticle-nucleic acid complex base editing efficiency according to some embodiments of the present application;

FIG. 7 is a histogram of lipid nanoparticle-nucleic acid complex gene editing efficiency according to some embodiments of the present application.

Detailed Description

In order to more clearly illustrate the technical solutions of the embodiments of the present specification, the drawings that are required to be used in the description of the embodiments will be briefly described below. It is apparent that the drawings in the following description are only some examples or embodiments of the present specification, and it is possible for those of ordinary skill in the art to apply the present specification to other similar situations according to the drawings without inventive effort. Unless otherwise apparent from the context of the language or otherwise specified, like reference numerals in the figures refer to like structures or operations.

As used in this specification and the claims, the terms "a," "an," "the," and/or "the" are not specific to a singular, but may include a plurality, unless the context clearly dictates otherwise. In general, the terms "comprises" and "comprising" merely indicate that the steps and elements are explicitly identified, and they do not constitute an exclusive list, as other steps or elements may be included in a method or apparatus.

A flowchart is used in this specification to describe the operations performed by the system according to embodiments of the present specification. It should be appreciated that the preceding or following operations are not necessarily performed in order precisely. Rather, the steps may be processed in reverse order or simultaneously. Also, other operations may be added to or removed from these processes.

Nucleic acid drugs, due to their nature, such as electronegativity, susceptibility to degradation by nucleases, etc., are not able to effectively penetrate cell membranes into cells and degrade away quickly in vivo, so that a good delivery system is needed to deliver nucleic acids stably to target sites and to act. A common difficulty in mRNA and siRNA development is how to deliver them efficiently into cells at the target site. During delivery, consideration is also given to how to avoid rapid clearance, avoid degradation by nucleases, improve post-endocytic escape, etc.

Nucleic acids bear a large number of phosphates and are thus negatively charged. In order to enable better encapsulation by lipid nanoparticles, a special class of lipids, cationic lipids, needs to be used. Cationic lipids tend to have a hydrophilic end with an ammonium group that can be positively charged in combination with hydrogen ions in an acidic environment. By electrostatic adsorption of the two, the nucleic acid can be encapsulated in the lipid nanoparticle. The outside of the wrapped structure is hydrophobic because the hydrophobic end of the cationic lipid is outwards, at the moment, a lipid of which one end is modified with polyethylene glycol (PEG) commonly used in the traditional liposome synthesis, namely PEG-lipid, can be added, so that the hydrophobic end of the PEG-lipid is combined with the hydrophobic end of the cationic lipid, and the hydrophilic end (connected with PEG) of the PEG-lipid outwards forms the shell of the nucleic acid lipid nanoparticle. In order to increase the stability of the nucleic acid lipid nanoparticle, a proper amount of cholesterol and other components can be added, so that the hydrophobic end of PEG-lipid and the hydrophobic end of cationic lipid are combined more tightly, and finally the finished product of the nucleic acid lipid nanoparticle is obtained. When the nucleic acid lipid nanoparticle is endocytosed by a cell, the ionizable lipid has pH sensitivity, the ionizable cationic lipid can be ionized in an acidic environment to damage an endosomal membrane, so that endosome escape of the Lipid Nanoparticle (LNP) is realized, and nucleic acid in vivo transfection is realized.

Although lipid carriers are the same, the formulation, method of preparation, and the release of different drug molecules in the target tissue or cells are different.

The present application provides a lipid nanoparticle suitable for RPE cell transfection, the starting material of the lipid nanoparticle comprising a steroid lipid, a cationic lipid, a helper lipid and a pegylated lipid, the molar ratio of the steroid lipid, the helper lipid, the cationic lipid and the pegylated lipid being 37:10:51 to 51.5:1.5 to 2; the steroid lipid is selected from at least one of cholesterol or beta-sitosterol, the auxiliary lipid is selected from at least one of cardiolipin or DSPC, the cationic lipid is selected from at least two of SM102, DOTAP or ALC-0315, the polyethylene glycol lipid is selected from at least one of DSPE-PEG-Biotin, DMG-PEG or PEG-folic acid, the cationic lipid comprises ionizable cationic lipid and permanent cationic lipid, and the mole ratio of the ionizable cationic lipid to the permanent cationic lipid is 25-50: 10 to 25.

The term "lipid nanoparticle" as used herein refers to a particle comprising a plurality of (e.g., more than one) lipid molecules physically bound to each other by intermolecular forces. LNP can be, for example, microspheres (including unilamellar and multilamellar vesicles, e.g., "liposomes" -lamellar phase lipid bilayers, which in some embodiments are substantially spherical, and in more specific embodiments can comprise an aqueous core, e.g., comprising a substantial portion of an RNA molecule), a dispersed phase in an emulsion, a micelle, or an internal phase in a suspension. Emulsions, micelles and suspensions may be suitable compositions for topical and/or external delivery.

Cationic lipids refer to lipid molecules capable of being positively charged. In some embodiments, the cationic lipid may include at least one of an ionizable cationic lipid and a permanent cationic lipid. In some embodiments, the cationic lipid may include at least one of an ionizable cationic lipid and a permanent cationic lipid. In some embodiments, the molar ratio of the ionizable cationic lipid to the permanent cationic lipid may be from 27.5 to 47.5:10 to 22.5. In some embodiments, the molar ratio of the ionizable cationic lipid to the permanent cationic lipid may be from 30 to 45:10 to 20. In some embodiments, the molar ratio of the ionizable cationic lipid to the permanent cationic lipid can be from 32.5 to 42.5:10 to 17.5. In some embodiments, the mole ratio of the ionizable cationic lipid to the permanent cationic lipid may be from 35 to 40:10 to 15. In some embodiments, the molar ratio of the ionizable cationic lipid to the permanent cationic lipid may be from 37.5 to 40:12.5 to 15.

In some embodiments, preferably, the molar ratio of the ionizable cationic lipid to the permanent cationic lipid may be from 30 to 41.5:10 to 25. In some embodiments, more preferably, the molar ratio of the ionizable cationic lipid to the permanent cationic lipid may be from 41 to 41.5:10.

the ionizable cationic lipid has pH sensitive ammonium head group, its pKa is between 6.0 and 7.0, in the environment of lower pH, the polar head of this lipid is positively charged, can combine with gene medicament of negative charge through the electrostatic action, the lipid keeps the electroneutrality or electronegativity under the condition of pH7.4, has guaranteed the stability in serum, can avoid the degradation of the gene medicament in the endosome or lysosome that pH presents weak acidity at the same time, realize endosome/lysosome escape; the ionizable cationic lipids also include two C18 long carbon chains with two unsaturated double bonds on each carbon chain, which significantly increases the hydrophobic region in such lipids, thereby facilitating membrane fusion and facilitating disruption of the bilayer membrane structure. In some embodiments, the ionizable cationic lipid may comprise any of 1-octyl nonyl 8- [ (2-hydroxyethyl) [6-O-6- (undecyloxy) hexyl ] amino ] -octanoate (SM 102) or ((4-hydroxybutyl) azadialkyl) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (ALC-0315).

A permanently cationic lipid is a lipid that itself has a positive charge and does not change electrical properties as the pH of the solution changes. In some embodiments, the permanent cationic lipid may be (2, 3-dioleoyl-propyl) -trimethylammonium chloride (DOTAP).

Pegylated lipids refer to molecules comprising a polyethylene glycol moiety and a lipid moiety. Polyethylene glycol (PEG) lipid is beneficial to controlling the particle size of lipid nano particles, forming a hydration layer outside the particles, stabilizing the charge on the surfaces of the particles, avoiding fusion between the particles, and playing an important role in the preparation process and intracellular delivery process. In some embodiments, the pegylated lipid may comprise any one or more of 1, 2-distearoyl-SN-glycero-3-phosphorylethanolamine-N-polyethylene glycol 2000-Biotin (DSPE-PEG 2000-Biotin), 1, 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000), 1, 2-distearoyl-SN-glycero-3-phosphorylethanolamine-N-polyethylene glycol 2000-folic acid (DSPE-PEG 2000-folic acid).

The application also provides the application of the lipid nanoparticle, which is used as a delivery carrier of nucleic acid.

In some embodiments, the nucleic acid may be a gene editing nucleic acid.

The application also provides a lipid nanoparticle-nucleic acid complex comprising a nucleic acid and the lipid nanoparticle described above, the nucleic acid being loaded in the lipid nanoparticle.

In some embodiments, the molar ratio of the lipid nanoparticle to the nucleic acid may be 4-8. In some embodiments, the molar ratio of the lipid nanoparticle to the nucleic acid may be 5-7. In some embodiments, the molar ratio of the lipid nanoparticle to the nucleic acid is 6.

Nucleic acid refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-or double-stranded form and includes DNA, RNA, and hybrids thereof. In some embodiments, the nucleic acid may comprise at least one of DNA or RNA.

In some embodiments, the RNA can include one or more of antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, sgRNA, tsRNA.

In some embodiments, the mRNA may include Cas9 nuclease mRNA or cytosine base editor mRNA. The sgRNA is TIGIT gene sgRNA.

The sgrnas can direct Cas nucleases to target sequences on a target nucleic acid molecule, wherein the sgrnas hybridize to the target sequences and the Cas nucleases cleave or modulate the target sequences.

A Base Editor (Base Editor) is an engineered gene editing tool derived from fusion of deaminase to a programmable DNA binding protein that can effect precise nucleotide changes at a genomic locus of interest. Unlike the gene editing tools such as CRISPR-Cas9 that cut, the base editor does not require double strand breaks and thus has unique safety and accuracy advantages. The cytosine base editor (Cytosine base editor, CBE) enables efficient substitution of cytosine to thymine (c→t) at the target site without creating a double strand break.

In some embodiments, the mRNA may also include mRNA for a reporter molecule. In some embodiments, the reporter may include, but is not limited to, green Fluorescent Protein (GFP), blue Fluorescent Protein (BFP), enhanced GFP (EGFP), red Fluorescent Protein (RFP).

In some embodiments, the mass ratio of mRNA to sgRNA can be 1-5:1-5. In some embodiments, the mass ratio of mRNA to sgRNA can be 1-4:1-4. In some embodiments, the mass ratio of mRNA to sgRNA can be 1-3:1-3. In some embodiments, the mass ratio of mRNA to sgRNA can be 1-2:1-2. In some embodiments, the mass ratio of mRNA to sgRNA can be 1:1.

In some embodiments, preferably, the mass ratio of mRNA to sgRNA can be 5:1.

The application also provides the use of the lipid nanoparticle-nucleic acid complexes described above for gene editing.

In some embodiments, the lipid nanoparticle-nucleic acid complex may have at least one of the following effects:

a, improving the transfection efficiency of nucleic acid; b, improving the gene editing efficiency; and c, improving the base editing efficiency.

The application also provides application of the lipid nanoparticle-nucleic acid complex in preparing a medicament for treating eye related diseases.

In some embodiments, the eye-related disease may be an acquired retinal degenerative disease. In some embodiments, the acquired retinal degenerative disease may be an age-related macular degeneration disease. In some embodiments, the age-related macular degeneration disease may include a dry age-related macular degeneration disease and a wet age-related macular degeneration disease. In some embodiments, the lipid nanoparticle-nucleic acid complexes may be used for retinal pigment epithelial cell transfection, gene editing, base editing.

The present application also provides a method for delivering a nucleic acid to a cell comprising contacting the lipid nanoparticle-nucleic acid complex described above with a cell. In some embodiments, the cell may be a retinal pigment epithelial cell. In some embodiments, the cell may be an ARPE-19 cell. In some embodiments, the lipid nanoparticle-nucleic acid complex is contacted with an ARPE-19 cell or 293T cell.

The present application also provides a method of gene editing comprising contacting the lipid nanoparticle-nucleic acid complex described above with a cell containing a gene to be edited. In some embodiments, the cell containing the gene to be edited may be a retinal pigment epithelial cell. In some embodiments, the cell containing the gene to be edited may be an ARPE-19 cell.

The application also provides the use of DSPE-PEG-Biotin in preparing the above lipid nanoparticle and the above lipid nanoparticle-nucleic acid complex.

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagent companies. The quantitative tests in the following examples were all set up in triplicate and the results averaged.

Earlier we have had some transfection capacity of ARPE-19 cell lines in vitro by testing LNP prepared from DLin-MC3-DMA cationic lipids. Thus, 22 different protocols were designed to test the ARPE19 cell line. Four schemes are preferably selected, namely scheme six, scheme ten, scheme twenty-one and scheme twenty-two (see fig. 1-3).

The four schemes, scheme six, scheme eleven, scheme twenty-one, scheme twenty-two, differ from the conventional LNP composition. The conventional LNP component is cholesterol, an ionizable cationic lipid, a helper phospholipid, and a pegylated lipid. Of the four schemes, the LNP component is cholesterol, an ionizable cationic lipid, a permanent cationic lipid, a helper phospholipid, a pegylated lipid (or a modified pegylated lipid).

The LNP-coated content is mRNA, sgRNA, siRNA, miRNA, ssDNA, plasmid or a mixture of these nucleic acid materials mixed in any ratio.

In the examples described below, both Cas9mRNA and sgRNA are of the prior art, wherein Cas9mRNA is purchased from Absin, cat No. abs 6074. The TIGIT sgRNA-3 of example 3 of patent WO2022CN93747 was used for sgrnas. CE-CEB mRNA was transcribed from CE-CEB of example 1 of patent 202010163058.3 using CE-CEB of example 1 of patent 202010163058.3.

In the following examples, the methods for preparing LNP and LNP-RNA complexes, and methods for detecting transfection efficiency and editing efficiency in 22 protocols are not shown in total, and the other examples are described in examples 1 to 13.

Example 1: LNP formulation one of green fluorescent protein EGFP mRNA

The nitrogen to phosphorus ratio is suitable for parameters in LNP for preparing nucleic acid materials. The ratio of nitrogen to phosphorus determines the ratio of nucleic acid to lipid, typically in molar ratio. Nitrogen refers to cationic lipids and phosphorus refers to the phosphate group in the nucleic acid. The nitrogen to phosphorus ratio is generally 4 to 8.

The optimum nitrogen-to-phosphorus ratio is 6, and all the schemes in the invention are not specifically described as nitrogen-to-phosphorus ratio 6.

Taking nucleic acid substance mRNA, scheme six as an example, 2 μg of green fluorescent protein EGFP mRNA. A nitrogen to phosphorus ratio of 6, 17.802 nmol/. Mu.g of cationic lipid was required. Cationic lipid is 51.5% of total LNP lipid, and the total lipid mass is required to be 34.567 nmol/. Mu.g.

According to the sixth ratio, cholesterol 9.890 μg, DSPC 5.463 μg, SM-102.375 μg, DOTAP 4.829 μg, DMG-PEG 2.602 μg.

Example 2: LNP formula II of green fluorescent protein EGFP mRNA

Taking nucleic acid substance mRNA, scheme ten as an example, 2 μg of green fluorescent protein EGFP mRNA. Cationic lipid 17.802 nmol/. Mu.g was required. Cationic lipid is 51.5% of total LNP lipid, and the total lipid mass is required to be 34.567 nmol/. Mu.g.

According to the composition ratios of scheme ten, 10.608. Mu.g of beta-sitosterol, 5.463. Mu.g of DSPC, SM-102.375. Mu.g, DOTAP 4.829. Mu.g, DMG-PEG 2.602. Mu.g.

Example 3: LNP formulation III of green fluorescent protein EGFP mRNA

Taking nucleic acid substance mRNA, protocol twenty-one as an example, 2 μg of mRNA of green fluorescent protein EGFP. Cationic lipid 17.802 nmol/. Mu.g was required. Cationic lipid is 51.0% of total LNP lipid, and the total lipid mass is required to be 34.906 nmol/. Mu.g.

According to the twenty-one ratio of the protocol, 9.987 μg of cholesterol, 5.516 μg of DSPC, 20.327 μg of SM-102, 4.877 μg of DOTAP, 3.840 μg of DSPE-PEG-Biotin.

Example 4: LNP formulation IV of green fluorescent protein EGFP mRNA

Taking nucleic acid substance mRNA, protocol twenty-two as an example, 2 μg of mRNA of green fluorescent protein EGFP. Cationic lipid 17.802 nmol/. Mu.g was required. Cationic lipid is 51.5% of total LNP lipid, and the total lipid mass is required to be 34.567 nmol/. Mu.g.

According to the twenty-two ratio of the protocol, cholesterol 9.890 μg, DSPC 5.463 μg, ALC-0315.20.375 μg, DOTAP 4.829 μg, DMG-PEG 2.602 μg.

Example 5: LNP formulation of CRISPR-Cas9 System

The CRSPR-Cas9 system requires mRNA and sgRNA of Cas9 protein in a ratio of from 5:1. 2: 1. 1:1. 1:2 to 1:5. the invention adopts the proportion of 5:1, unless otherwise specified.

Taking the nucleic acid material mRNA 2.5. Mu.g and sgRNA 0.5. Mu.g, scheme six as an example, 3. Mu.g of RNA of the CRSPR-Cas9 system. Cationic lipid 17.802 nmol/. Mu.g was required. Cationic lipid is 51.5% of total LNP lipid, and the total lipid mass is required to be 34.567 nmol/. Mu.g.

According to the sixth ratio, cholesterol 14.836 μg, DSPC 8.194 μg, SM-102.563 μg, DOTAP 7.244 μg, DMG-PEG 3.903 μg.

Example 6: LNP formulation II of CRISPR-Cas9 system

Taking the nucleic acid material mRNA 2.5. Mu.g and sgRNA 0.5. Mu.g, scheme ten as an example, 3. Mu.g of RNA of the CRSPR-Cas9 system. Cationic lipid 17.802 nmol/. Mu.g was required. Cationic lipid is 51.5% of total LNP lipid, and the total lipid mass is required to be 34.567 nmol/. Mu.g.

According to the ten-scheme ratio, cholesterol 14.836 μg, DSPC 8.194 μg, SM-102.563 μg, DOTAP 7.244 μg, DMG-PEG 3.903 μg.

Example 7: LNP formulation III of CRISPR-Cas9 system

Taking the nucleic acid material mRNA 2.5. Mu.g and sgRNA 0.5. Mu.g, scheme twenty-one as an example, 3. Mu.g of RNA of the CRSPR-Cas9 system. Cationic lipid 17.802 nmol/. Mu.g was required. Cationic lipid is 51.0% of total LNP lipid, and the total lipid mass is required to be 34.906 nmol/. Mu.g.

According to the twenty-one ratio of the protocol, 14.981 μg of cholesterol, 8.274 μg of DSPC, 30.491 μg of SM-102, 7.315 μg of DOTAP, 5.759 μg of DSPE-PEG-Biotin.

Example 8: LNP formulation IV of CRISPR-Cas9 system

Taking the nucleic acid material mRNA 2.5. Mu.g and sgRNA 0.5. Mu.g, protocol twenty-two as an example, 3. Mu.g of RNA of the CRSPR-Cas9 system. Cationic lipid 17.802 nmol/. Mu.g was required. Cationic lipid is 51.5% of total LNP lipid, and the total lipid mass is required to be 34.567 nmol/. Mu.g.

According to the twenty-two ratio of the protocol, cholesterol 14.836 μg, DSPC 8.194 μg, ALC-0315.32.977 μg, DOTAP 7.244 μg, DMG-PEG 3.903 μg.

The formulation is not limited to EGFP and CRISPR-Cas9 mRNAs, but also mRNAs to sgRNAs (w/w) in a 5:1 ratio.

Example 9: preparation method 1 of laboratory-grade trace LNP-RNA complex

1. 2.5. Mu.l, 20. Mu.l, 200. Mu.l, 1000. Mu.l pipette tips, 10, 200, 1000. Mu.l non-ribozyme tips, 0.6 ml centrifuge tubes, PCR octant tubes, 15ml centrifuge tubes, 50ml centrifuge tubes, 15ml100kDa ultrafiltration centrifuge tubes, DEPC water, 20XPBS, sodium citrate buffer (50 mmol, pH=4), ribozyme scavenger, 75% alcohol, absolute ethanol, PCR octant rack, 1.5 ml centrifuge rack, 15/50 ml centrifuge rack, 50ml syringe, 0.22 μm needle filters (all of the above mentioned liquids, tubes, etc. are non-ribozyme sterile).

2. The working solutions of cholesterol, DSPC, SM-102, DOTAP and DMG-PEG2000 are prepared by absolute ethyl alcohol in advance, the concentrations of the working solutions are all 2 mg/ml, and the working solutions are sealed and stored in a refrigerator to prevent the concentration from being influenced by the volatilization of the ethyl alcohol.

3. The operating environment was sterilized with 75% alcohol and then treated with a ribozyme scavenger to create a sterile, ribozyme-free environment (super clean bench).

4. 1xPBS was prepared with 20xPBS and DEPC water, then filtered with a syringe into a 50ml centrifuge tube for use, and then 6 ml PBS was loaded into a 15ml centrifuge tube.

5. Cholesterol, DSPC, SM-102, DOTAP, DMG-PEG were removed and mixed in one well of a PCR octant as described in example 1; then another 148. Mu.l of sodium citrate buffer was added to the other well and then 2. Mu.g EGFPmRNA was added and mixed well.

6. Absolute ethanol was added to the organic phase of the lipid to a volume of 50 μl and mixed well. 50. Mu.l of the organic phase and 150. Mu.l of the aqueous phase were aspirated (both hands combined) with two guns, respectively, and added rapidly at the same time to a 0.6 ml centrifuge tube, which was then placed in a vortex mixer (2000 rpm,10 min).

7. An equal volume (200. Mu.l total of organic and aqueous) of PBS solution was added (step 4 formulation) and placed in a vortex mixer (2000 rpm,10 min).

8. The mixed liquid from step 7 was added to the 15ml centrifuge tube from step 4.

9. The LNP-mRNA mixture was concentrated by ultrafiltration centrifuge (3000 rpm, 5 min), and after completion of the concentration, the PBS prepared in step 4 of 2 ml was added and the LNP was washed 3 times by centrifugation.

10. The concentrated LNP was transferred to a PCR octant, and the total amount was 50-100. Mu.l, and stored at 2-4 ℃.

Example 10: preparation method 2 of laboratory-grade trace LNP-RNA complex

1. 2.5. Mu.l, 20. Mu.l, 200. Mu.l, 1000. Mu.l pipette tips, 10, 200, 1000. Mu.l non-ribozyme tips, 0.6 ml centrifuge tubes, PCR octant tubes, 15ml centrifuge tubes, 50ml centrifuge tubes, 15ml100kDa ultrafiltration centrifuge tubes, DEPC water, 20XPBS, sodium citrate buffer (50 mmol, pH=4), ribozyme scavenger, 75% alcohol, absolute ethanol, PCR octant rack, 1.5 ml centrifuge rack, 15/50 ml centrifuge rack, 50ml syringe, 0.22 μm needle filters (all of the above mentioned liquids, tubes, etc. are non-ribozyme sterile).

2. Working solutions of cholesterol, DSPC, SM-102, DOTAP and DMG-PEG2000 are prepared by absolute ethyl alcohol in advance, the concentration of the working solutions is 2 mg/ml, and the working solutions are sealed and stored in a refrigerator to prevent the concentration from being influenced by the volatilization of the ethyl alcohol.

3. The operating environment was sterilized with 75% alcohol and then treated with a ribozyme scavenger to create a sterile, ribozyme-free environment (super clean bench).

4. 1xPBS was prepared with 20xPBS and DEPC water, then filtered with a syringe into a 50ml centrifuge tube for use, and then 6 ml PBS was loaded into a 15ml centrifuge tube.

5. Cholesterol, DSPC, SM-102, DOTAP, DMG-PEG were removed and mixed in one well of a PCR octant as described in example 1; then another 147 μl sodium citrate buffer was added to another well, then 2.5 μg Cas9mRNA and sgRNA 0.5 μg was added and incubated for 2 min.

6. Absolute ethanol was added to the organic phase of the lipid to a volume of 50 μl and mixed well. 50. Mu.l of the organic phase and 150. Mu.l of the aqueous phase were aspirated (both hands combined) with two guns, respectively, and added rapidly at the same time to a 0.6 ml centrifuge tube, which was then placed in a vortex mixer (2000 rpm,10 min).

7. An equal volume (200. Mu.l total of organic and aqueous) of PBS solution was added (step 4 formulation) and placed in a vortex mixer (2000 rpm,10 min).

8. The mixed liquid from step 7 was added to the 15ml centrifuge tube from step 4.

9. The LNP-mRNA mixture was concentrated by ultrafiltration centrifuge (3000 rpm, 5 min), and after completion of the concentration, the PBS prepared in step 4 of 2 ml was added and the LNP was washed 3 times by centrifugation.

10. The concentrated LNP was transferred to a PCR octant, and the total amount was 50-100. Mu.l, and stored at 2-4 ℃.

Example 11: LNP-RNA complex delivery of nucleic acids to ARPE-19 cells

293T cells and ARPE-19 cells were inoculated and cultured in 24-well plates with 10% FBS-containing DMEM high-sugar culture solution (HyClone, SH30022.01B), and 1X10 cells were plated on each well⁵ Individual cells containing penicillin (100U/ml) and streptomyin (100 μg/ml). Two hours prior to transfection, the medium was changed to antibiotic-free medium. Then 100. Mu.l (equivalent to 1. Mu.g EGFPmRNA) of the LNP-RNA complex prepared in example 9 was added to each well, and cultured for 24-48 hours, and the cells were subjected to fluorescence analysis detection by a flow cytometer to obtain the transfection efficiency of the LNP-RNA complex, and the results are shown in FIGS. 4-5.

Example 12: LNP-RNA complexes deliver CE-CBE mRNA to ARPE-19 cells

293T cells and ARPE-19 cells were inoculated and cultured in 24-well plates with 10% FBS-containing DMEM high-sugar culture solution (HyClone, SH30022.01B), and 1X10 cells were plated on each well⁵ Individual cells containing penicillin (100U/ml) and streptomyin (100 μg/ml). Two hours prior to transfection, the medium was changed to antibiotic-free medium. Then 100. Mu.l (corresponding to 1.25. Mu.g CE-CBE mRNA and 0.25. Mu.g sgRNA) of the LNP-RNA complex prepared in example 10 was added to each well and incubated for 48 hours. The cells are then lysed and PCR amplified by appropriate primers. Finally, the PCR amplified product was subjected to Sanger sequencing, and the base editing efficiency was analyzed, and the results are shown in FIG. 6.

Example 13: LNP-RNA complex delivers Cas9mRNA to ARPE-19 cells

ARPE-19 cells were inoculated and cultured in 24-well plates with 10% FBS-containing DMEM high-glucose culture medium (HyClone, SH30022.01B), 1X10 cells were plated per well⁵ Individual cells containing penicillin (100U/ml) and streptomyin (100 μg/ml). Two hours prior to transfection, the medium was changed to antibiotic-free medium. LNP-RNA complexes prepared in example 10 were then added per well100. Mu.l (equivalent to 1.25. Mu.g Cas9mRNA and 0.25. Mu.g sgRNA) and incubated for 48 hours. The cells are then lysed and PCR amplified by appropriate primers. Finally, the PCR amplified products were subjected to Sanger sequencing, and the gene editing efficiency was analyzed, and the results are shown in FIG. 7.

In the present application, the LNP-RNA complex of the corresponding EGFP mRNA was prepared by the method of example 9, and in vitro cell experiments were performed by the method of example 11 to examine the transfection efficiency of the LNP-RNA complex (see fig. 4-5), in a preferred embodiment six, ten, twenty-one, and twenty-two. The four preferred approaches approach 100% efficiency for EGFP mRNA delivery. Description protocol six, protocol ten, protocol twenty-one, protocol twenty-two all have efficiencies approaching 100% for gene delivery.

In the present application, the LNP-RNA complex of the corresponding CECBE mRNA was prepared by the method of example 10, and the in vitro cell experiment was performed by the method of example 12, and the base editing efficiency of the LNP-RNA complex was detected to be 56% (see fig. 6), with preference given to the sixth, tenth, twenty-one, and twenty-two schemes.

In the present application, preferred protocol six, protocol ten, protocol twenty-one, protocol twenty-two, LNP-RNA complexes of the corresponding Cas9mRNA were prepared by the method of example 10, and in vitro cell experiments were performed by the method of example 13, detecting that the gene editing efficiency of the LNP-RNA complexes reached 78% (see fig. 7).

While the basic concepts have been described above, it will be apparent to those skilled in the art that the foregoing detailed disclosure is by way of example only and is not intended to be limiting. Although not explicitly described herein, various modifications, improvements, and adaptations to the present disclosure may occur to one skilled in the art. Such modifications, improvements, and modifications are intended to be suggested within this specification, and therefore, such modifications, improvements, and modifications are intended to be included within the spirit and scope of the exemplary embodiments of the present invention.

Meanwhile, the specification uses specific words to describe the embodiments of the specification. Reference to "one embodiment," "an embodiment," and/or "some embodiments" means that a particular feature, structure, or characteristic is associated with at least one embodiment of the present description. Thus, it should be emphasized and should be appreciated that two or more references to "an embodiment" or "one embodiment" or "an alternative embodiment" in various positions in this specification are not necessarily referring to the same embodiment. Furthermore, certain features, structures, or characteristics of one or more embodiments of the present description may be combined as suitable.

In some embodiments, numbers describing the components, number of attributes are used, it being understood that such numbers being used in the description of embodiments are modified in some examples by the modifier "about," approximately, "or" substantially. Unless otherwise indicated, "about," "approximately," or "substantially" indicate that the number allows for a 20% variation. Accordingly, in some embodiments, numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and employ a method for preserving the general number of digits. Although the numerical ranges and parameters set forth herein are approximations that may be employed in some embodiments to confirm the breadth of the range, in particular embodiments, the setting of such numerical values is as precise as possible.

Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments of this specification. Other variations are possible within the scope of this description. Thus, by way of example, and not limitation, alternative configurations of embodiments of the present specification may be considered as consistent with the teachings of the present specification. Accordingly, the embodiments of the present specification are not limited to only the embodiments explicitly described and depicted in the present specification.

Claims (20)

1. A lipid nanoparticle suitable for RPE cell transfection, wherein the lipid nanoparticle comprises a starting material comprising a steroid lipid, a cationic lipid, a helper lipid, and a pegylated lipid in a molar ratio of 37:10:51 to 51.5:1.5 to 2; the steroid lipid is selected from at least one of cholesterol or beta-sitosterol, the auxiliary lipid is selected from at least one of cardiolipin or DSPC, the cationic lipid is selected from at least two of SM102, DOTAP or ALC-0315, the polyethylene glycol lipid is selected from at least one of DSPE-PEG-Biotin, DMG-PEG or PEG-folic acid, the cationic lipid comprises ionizable cationic lipid and permanent cationic lipid, and the mole ratio of the ionizable cationic lipid to the permanent cationic lipid is 25-50: 10 to 25.

2. The lipid nanoparticle of claim 1, wherein the mole ratio of the ionizable cationic lipid to the permanent cationic lipid is from 30 to 41.5:10 to 25.

3. The lipid nanoparticle of claim 1, wherein the mole ratio of the ionizable cationic lipid to the permanent cationic lipid is from 41 to 41.5:10.

4. the use of the lipid nanoparticle according to any one of claims 1 to 3 as a delivery vehicle for nucleic acids.

5. The use according to claim 4, wherein the nucleic acid is a nucleic acid for gene editing.

6. A lipid nanoparticle-nucleic acid complex, comprising a nucleic acid and the lipid nanoparticle of any one of claims 1-3, wherein the nucleic acid is loaded in the lipid nanoparticle.

7. The lipid nanoparticle-nucleic acid complex of claim 6, wherein the molar ratio of the lipid nanoparticle to the nucleic acid is 4-8;

and/or the nucleic acid comprises at least one of DNA or RNA.

8. The lipid nanoparticle-nucleic acid complex of claim 6, wherein the molar ratio of the lipid nanoparticle to the nucleic acid is 6.

9. The lipid nanoparticle-nucleic acid complex of claim 7, wherein the RNA comprises one or more of antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, sgRNA, tsRNA.

10. The lipid nanoparticle-nucleic acid complex of claim 9, wherein the mRNA comprises Cas9 nuclease mRNA or cytosine base editor mRNA, and the sgRNA is TIGIT gene sgRNA.

11. The lipid nanoparticle-nucleic acid complex of claim 9, wherein the mRNA further comprises an mRNA of a reporter molecule.

12. The lipid nanoparticle-nucleic acid complex of claim 9 or 10, wherein the mass ratio of mRNA to sgRNA is 1-5:1-5.

13. The lipid nanoparticle-nucleic acid complex of claim 9 or 10, wherein the mass ratio of mRNA to sgRNA is 5:1.

14. Use of a lipid nanoparticle-nucleic acid complex according to any one of claims 6 to 13 for gene editing.

15. The use of claim 14, wherein the lipid nanoparticle-nucleic acid complex has at least one of the following effects:

a, improving the transfection efficiency of nucleic acid;

b, improving the gene editing efficiency;

and c, improving the base editing efficiency.

16. Use of a lipid nanoparticle-nucleic acid complex according to any one of claims 6 to 13 in the manufacture of a medicament for the treatment of an eye-related disorder.

17. The use according to claim 16, wherein the eye-related disease is an acquired retinal degenerative disease, which is an age-related macular degeneration disease, including dry age-related macular degeneration disease and wet age-related macular degeneration disease, the lipid nanoparticle-nucleic acid complex being used for retinal pigment epithelial cell transfection, gene editing, base editing.

18. A method for delivering a nucleic acid to a cell comprising contacting the lipid nanoparticle-nucleic acid complex of any one of claims 6-13 with a cell.

19. A method of gene editing comprising contacting the lipid nanoparticle-nucleic acid complex of any one of claims 6 to 13 with a cell containing a gene to be edited.

Use of dspe-PEG-Biotin for the preparation of a lipid nanoparticle according to any one of claims 1 to 3 and a lipid nanoparticle-nucleic acid complex according to any one of claims 6 to 13.

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