Application of IL-4 receptor activator or M-CSF receptor activator in enhancing the efficiency of mRNA editing in macrophages

Abstract

The present invention provides an application of an IL-4 receptor activator or an M-CSF receptor activator in enhancing the efficiency of mRNA editing of macrophages, and relates to the field of biotechnology. IL-4 is an activator of the IL-4 receptor, and M-CSF is an activator of the M-CSF receptor. The inventors have found that IL-4, M-CSF or lipid nanoparticles loaded with their mRNA can significantly improve the in vivo and in vitro editing efficiency of lipid nanoparticles loaded with target mRNA for macrophages. Among them, the effect of M-CSF is better, and because M-CSF itself plays an important role in the differentiation, maturation and proliferation of macrophages, M-CSF can significantly improve the editing efficiency of macrophages by the LNP-mRNA system on the basis of improving the function of macrophages, which will greatly expand the future application value of the LNP-mRNA system in in vivo editing of macrophages in various disease fields.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the use of an IL-4 receptor activator or an M-CSF receptor activator in enhancing the efficiency of mRNA editing in macrophages. Background Art

[0002] Macrophages are innate immune cells that play a vital role in defending against pathogen infection and maintaining tissue homeostasis, and are widely involved in the development and progression of numerous diseases. Targeting macrophages has become an emerging therapeutic approach for treating a variety of diseases. For example, CAR-enhanced macrophages offer unique advantages in solid tumor treatment and have garnered widespread attention both domestically and internationally. Therefore, engineering macrophages is crucial for these therapies. However, due to their innate immune properties, editing macrophages has been a challenge both domestically and internationally. In recent years, thanks to the rapid development of LNP-mRNA (mRNA-loaded lipid nanoparticles), in vivo editing of immune cells using LNP-mRNA has become a hot topic in disease treatment. There have been reports of in vivo editing of LNP-mRNA-enhanced macrophages with CAR for the treatment of tumors and bacterial infections. However, overall editing efficiency is relatively low, particularly in macrophages. Macrophages can sense exogenous RNA through TLR3 and TLR7 receptors, triggering an immune response that leads to its degradation. Currently, researchers have reduced the immunogenicity of mRNA to a certain extent through nucleotide modification. However, for macrophages, exogenous mRNA can still stimulate the immune response of macrophages, resulting in limited expression efficiency. Therefore, for LNP-mRNA system editing of macrophages, a method that can significantly improve the editing efficiency of macrophages in vitro and in vivo is urgently sought.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The first object of the present invention is to provide the use of an IL-4 receptor activator or an M-CSF receptor activator in enhancing the efficiency of mRNA editing in macrophages, so as to solve the above technical problems.

[0005] The second object of the present invention is to provide a method for editing macrophages in vitro.

[0006] The third object of the present invention is to provide a method for editing macrophages in vivo.

[0007] In order to achieve the above objectives, the following technical solutions are adopted:

[0008] In a first aspect, the present invention provides the use of an IL-4 receptor activator or an M-CSF receptor activator in enhancing the efficiency of mRNA editing in macrophages, wherein the mRNA is loaded into liposome nanoparticles.

[0009] As a further embodiment, the IL-4 receptor activator includes IL-4;

[0010] The M-CSF receptor activator includes M-CSF.

[0011] In a second aspect, the present invention provides a method for editing macrophages in vitro, comprising the following steps: culturing macrophages in a culture medium, then adding at least one of IL-4, M-CSF, or lipid nanoparticles loaded with M-CSF mRNA and lipid nanoparticles loaded with target mRNA for transfection, and completing the in vitro editing of macrophages by the lipid nanoparticles loaded with the target mRNA after transfection.

[0012] As a further technical solution, the final concentration of IL-4 added is 5-40 ng / ml;

[0013] The final concentration of M-CSF added is 5-40 ng / ml;

[0014] The transfection concentration of the target mRNA is 1-20ug / ml;

[0015] The mass ratio of M-CSF mRNA to target mRNA in the lipid nanoparticles loaded with M-CSF mRNA and the lipid nanoparticles loaded with target mRNA is (1-2):1.

[0016] As a further technical solution, the target mRNA includes the mRNA of a chimeric antigen receptor;

[0017] The intracellular domain of the chimeric antigen receptor is the CD3 intracellular domain.

[0018] In a third aspect, the present invention provides a method for in vivo editing of macrophages, comprising the following steps: intraperitoneal injection of IL-4 or M-CSF into an animal, or intravenous injection of lipid nanoparticles loaded with M-CSF mRNA, followed by intravenous injection of lipid nanoparticles loaded with target mRNA, to complete the in vivo editing of macrophages by lipid nanoparticles loaded with target mRNA.

[0019] As a further technical solution, the animal includes a mouse.

[0020] As a further technical solution, the injection amount of IL-4 is 5ug / head.

[0021] The injection volume of M-CSF was 5 μg / mouse.

[0022] As a further technical solution, the injection amount of the M-CSF mRNA is 3ug / head;

[0023] The injection amount of the target mRNA was 3ug / mouse.

[0024] As a further technical solution, the target mRNA includes the mRNA of a chimeric antigen receptor;

[0025] The intracellular domain of the chimeric antigen receptor is the CD3 intracellular domain.

[0026] As a further technical solution, the CD3 intracellular domain sequence is as follows:

[0027] RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR (SEQ ID NO. 1).

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] IL-4 is an activator of the IL-4 receptor, and M-CSF is an activator of the M-CSF receptor. The inventors have found that IL-4, M-CSF, or lipid nanoparticles loaded with their mRNA can significantly improve the in vivo and in vitro editing efficiency of lipid nanoparticles loaded with target mRNA for macrophages. Among them, M-CSF has a better effect, and because M-CSF itself plays an important role in the differentiation, maturation, and proliferation of macrophages, M-CSF can significantly improve the editing efficiency of macrophages by the LNP-mRNA system on the basis of improving macrophage function. This will greatly expand the future application value of the LNP-mRNA system in in vivo editing of macrophages in various disease fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 The structures are G1, G2, G3 and G4;

[0032] Figure 2 To compare the expression rate of GPC3-CAR structure in macrophages after macrophages were infected with LNPs containing different CAR structures;

[0033] Figure 3To detect the effect of different cytokines on the efficiency of LNP-GFP infection of macrophages by flow cytometry;

[0034] Figure 4 Fluorescence photography of macrophages infected with LNP-GFP for 24 hours under stimulation of different cytokines;

[0035] Figure 5 The effect of co-transfection of M-CSF mRNA on target mRNA expression in macrophages;

[0036] Figure 6 The results show the proportion of GFP-positive macrophages after co-transfection of M-CSF mRNA into macrophages;

[0037] Figure 7 The effect of M-CSF signaling on the efficiency of LNP-GPC3 CAR transfection into macrophages;

[0038] Figure 8 Improve the editing efficiency of the LNP-mRNA system in macrophages for M-CSF specificity in vivo;

[0039] Figure 9 M-CSF had no significant effect on the expression of GFP in T cells in vivo. DETAILED DESCRIPTION

[0040] Below in conjunction with embodiment and example, embodiment of the present invention is described in detail, but those skilled in the art will appreciate that the following embodiment and example are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present invention. Unspecified conditions are carried out according to the conditions of normal conditions or manufacturer's recommendations. Reagents used or instruments not specified by the manufacturer are conventional products that can be purchased commercially.

[0041] Explanation of terms:

[0042] Lipid nanoparticles (LNPs) are usually composed of four components: ionizable cationic lipids, phospholipids, cholesterol and PEG-lipids. They can encapsulate mRNA by electrostatic adsorption, protecting mRNA from degradation by nucleases and avoiding clearance by the blood mononuclear phagocyte system.

[0043] Macrophages (abbreviated as mø) are tissue-resident white blood cells that originate from monocytes, which in turn originate from precursor cells in the bone marrow. Both macrophages and monocytes are phagocytic cells that participate in both nonspecific defense (innate immunity) and specific defense (cellular immunity) in vertebrates. Their primary function is to phagocytose (i.e., engulf and digest) cellular debris and pathogens, either as fixed or free cells, and to activate lymphocytes and other immune cells to respond to pathogens.

[0044] Macrophage colony-stimulating factor (M-CSF), also known as CSF-1, forms homodimers and heterodimers linked by disulfide bonds. M-CSF is a major regulator of macrophage survival, proliferation, and differentiation. M-CSF promotes monocyte survival, monocyte-to-macrophage differentiation, and macrophage proliferation. It also activates and enhances macrophage cytotoxicity against tumor cells and microorganisms, regulates the release of cytokines and other inflammatory mediators, and stimulates pinocytosis.

[0045] Interleukin-4 (IL-4): Its gene is located on chromosome 5 and consists of 129 amino acids. IL-4 is primarily produced by activated T cells, monocytes, mast cells, and basophils. Its primary biological function is to stimulate the proliferation of activated B and T cells and the differentiation of CD4+ T cells into type II helper T cells.

[0046] L929 cells are an immortalized cell line derived from mouse fibroblasts and are adherent connective tissue cells. L929 cells can secrete a variety of cytokines, particularly macrophage colony-stimulating factor.

[0047] L929 cell culture supernatant: The culture supernatant of L929 cells contains a large amount of macrophage colony-stimulating factor.

[0048] In a first aspect, the present invention provides the use of an IL-4 receptor activator or an M-CSF receptor activator in enhancing the efficiency of mRNA editing in macrophages, wherein the mRNA is loaded into liposome nanoparticles.

[0049] The inventors have discovered that lipid nanoparticles loaded with IL-4, M-CSF, or their mRNAs can significantly enhance the efficiency of macrophage editing in vivo and in vitro using lipid nanoparticles loaded with target mRNAs. This significantly enhances the application value of the LNP-mRNA system for in vivo editing of macrophages in various disease areas.

[0050] Those skilled in the art know that IL-4R stands for interleukin-4 receptor, which encodes a protein that binds to interleukin-4 (IL-4) and transmits signals. IL-4R is composed of two subunits, α and γ. IL-4Rα is a specific receptor that is highly sensitive to IL-4, while the γ chain (also known as the common γ chain) is shared by multiple cytokine receptors.

[0051] The interaction between M-CSF (Macrophage Colony-Stimulating Factor) and its receptor, CSF1R (Colony-Stimulating Factor 1 Receptor), plays a central role in regulating macrophage development, survival, proliferation, differentiation, and function. Upon binding, M-CSF dimerizes the receptor and activates downstream signaling pathways (such as PI3K / Akt, MAPK / ERK, and JAK / STAT), promoting cell survival and proliferation.

[0052] Therefore, the effect of IL4 and M-CSF on improving the efficiency of LNP-mRNA editing in macrophages is actually through activating the corresponding receptors on the surface of macrophages. For example, IL4 activates IL4R, and M-CSF activates CSF1R, which leads to the activation of downstream signaling pathways, thereby improving editing efficiency. Therefore, other activators of IL4R or CSF1R can also enhance the efficiency of mRNA editing in macrophages by activating IL4R or CSF1R.

[0053] In a second aspect, the present invention provides a method for editing macrophages in vitro, comprising the following steps: culturing macrophages in a culture medium, then adding at least one of IL-4, M-CSF, or lipid nanoparticles loaded with M-CSF mRNA and lipid nanoparticles loaded with target mRNA for transfection, and completing the in vitro editing of macrophages by the lipid nanoparticles loaded with the target mRNA after transfection.

[0054] The in vitro editing method of macrophages provided by the present invention significantly improves the in vitro editing efficiency of macrophages by lipid nanoparticles loaded with target mRNA.

[0055] In some optional embodiments, the final concentration of IL-4 added can be, for example, but not limited to, 5 ng / ml, 20 ng / ml or 40 ng / ml;

[0056] The final concentration of M-CSF added may be, for example, but not limited to, 5 ng / ml, 20 ng / ml or 40 ng / ml.

[0057] The inventors have found that, in the method of the present invention, a cytokine concentration of 5-40 ng / ml can be used.

[0058] In some optional embodiments, the transfection concentration of the target mRNA can be, for example, but not limited to, 0.1 ug / ml, 0.5 ug / ml, 1 ug / ml, 10 ug / ml or 20 ug / ml;

[0059] The inventors have found that in the method of the present invention, the transfection concentration of the target mRNA can be used in the range of 5-40 ng / ml.

[0060] The mass ratio of M-CSF mRNA to target mRNA in the lipid nanoparticles loaded with M-CSF mRNA and the lipid nanoparticles loaded with target mRNA is (1-2):1.

[0061] In some optional embodiments, the target mRNA includes but is not limited to mRNA of a chimeric antigen receptor, or mRNA of other proteins.

[0062] The intracellular domain of the chimeric antigen receptor is the CD3 intracellular domain.

[0063] The inventors have discovered that the length of exogenous mRNA and the structural domain of the protein it translates are key factors affecting mRNA expression. The expression of exogenous mRNA in macrophages gradually decreases with increasing length, with good expression observed for mRNAs around 1500bp, while expression is significantly restricted for mRNAs approaching 2000bp. Furthermore, the structural domain of the protein translated by the mRNA of an exogenous chimeric antigen receptor, such as the intracellular domain of TLR4, is a classic macrophage inflammatory signaling pathway that can strongly activate the inflammatory response of macrophages, thereby restricting the expression of subsequent exogenous mRNA. Compared to the intracellular domain of TLR4, the mRNA of the intracellular domain of CD3 has a higher expression level.

[0064] In a third aspect, the present invention provides a method for in vivo editing of macrophages, comprising the following steps: intraperitoneal injection of IL-4 or M-CSF into an animal, or intravenous injection of lipid nanoparticles loaded with M-CSF mRNA, followed by intravenous injection of lipid nanoparticles loaded with target mRNA, to complete the in vivo editing of macrophages by lipid nanoparticles loaded with target mRNA.

[0065] The in vitro macrophage editing method provided by the present invention significantly improves the in vivo editing efficiency of macrophages by lipid nanoparticles loaded with target mRNA.

[0066] In some alternative embodiments, the animal includes but is not limited to a mouse.

[0067] In some optional embodiments, the injection amount of IL-4 is 5ug / head.

[0068] The injection volume of M-CSF was 5 μg / mouse.

[0069] In some optional embodiments, the injection amount of the M-CSF mRNA is 3ug / head;

[0070] The injection amount of the target mRNA was 3ug / mouse.

[0071] In some optional embodiments, the target mRNA includes mRNA of a chimeric antigen receptor;

[0072] The intracellular domain of the chimeric antigen receptor is the CD3 intracellular domain.

[0073] As a further technical solution, the CD3 intracellular domain sequence is as follows:

[0074] RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQEGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLAPR (SEQ ID NO. 1).

[0075] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0076] The sequence of the CD3 intracellular domain in the following examples is shown in SEQ ID NO.1.

[0077] Example

[0078] 1. Exploring factors affecting the editing efficiency of the LNP-mRNA system in macrophages

[0079] 1) Design and synthesize GPC3-CAR (chimeric antigen receptor targeting GPC3) gene fragments containing different intracellular domains, and then clone the CAR gene fragments into in vitro transcription vectors containing T7 promoters. Use in vitro transcription kits to prepare mRNAs of different CAR structures (G1 / G2 / G3 / G4). The structures are as follows: Figure 1 shown.

[0080] The extracellular domains of these CAR structures all use sc-Fv of GPC3 monoclonal antibody targeting GPC3 antigen, among which the intracellular domain of G1 uses CD3 and TLR4 intracellular domain (TIR) ​​in series, G2 is a single CD3 intracellular domain, G3 is a single TIR intracellular domain, and G4 is a cytokine IL-21 in series after the CD3 intracellular domain. These mRNAs were used to prepare LNP-CAR mRNA (mRNA of chimeric antigen receptor loaded by lipid nanoparticles). Bone marrow-derived macrophages (BMDM) were then induced in vitro and infected with different LNP-CARmRNA (1ug / ml), and a blank control group (Control) was set up to detect the expression level of GPC3-CAR by flow cytometry at 24 and 48 hours. The results showed that the CAR structures of G1 and G3 were hardly expressed in macrophages, while the CAR structures of G2 and G4 were significantly expressed in macrophages ( Figure 2 This indicates that the intracellular domain of TLR4 can significantly inhibit mRNA expression in macrophages. Furthermore, since the expression efficiency of G4 is significantly lower than that of G2, this suggests that mRNA length is also an important factor affecting mRNA expression in macrophages.

[0081] 2. In vitro investigation of the effects of cytokines on the efficiency of LNP-mRNA editing in macrophages

[0082] GFP mRNA was prepared by in vitro transcription, and then GFP mRNA-loaded lipid nanoparticles (LNP-GFP) were prepared using GFP mRNA.

[0083] Bone marrow-derived macrophages were selected as the research subjects. Bone marrow-derived macrophages were plated and cultured in a 37°C incubator. The culture medium used was DMEM culture medium (MeilunBio, MA0212-Nov-01J). Then IL-4 (final concentration of 20ng / ml), IL-13 (final concentration of 20ng / ml), TGFβ (final concentration of 20ng / ml), and M-CSF (final concentration of 20ng / ml) were added respectively. After 24 hours, LNP-GFP (final concentration of 1ug / ml) was added for transfection. The control group was a group without any treatment, and the PBS group was a group that only added LNP-GFP for transfection. 24 hours after transfection, the proportion of GFP-positive cells in the macrophages of each group was detected by flow cytometry. Figure 3(Top: Percentage of GFP+ macrophages in each treatment group; Bottom: Statistical results of the percentage of GFP+ macrophages in each treatment group, three biological replicates per group) As shown, compared with the PBS group, both the IL-4 and M-CSF treatment groups significantly increased the percentage of GFP+ macrophages, while the IL-13 treatment group had no significant effect. However, the TGFβ treatment group significantly decreased the percentage of GFP+ macrophages. M-CSF had the greatest effect, increasing the GFP expression rate to over 90%.

[0084] Repeat the above experiment again. 24 hours after transfection, take pictures using a fluorescence microscope. Figure 4 As shown in the flow cytometry results, compared with the PBS group, both the IL-4 and M-CSF treatment groups significantly increased GFP expression in macrophages, the IL-13 treatment group had no significant effect, and the TGFβ treatment group significantly decreased GFP expression. This clearly demonstrates that TGFβ can inhibit the expression of exogenous mRNA in macrophages, while IL-4 and M-CSF can significantly increase the expression of exogenous mRNA in macrophages, with M-CSF having the best effect.

[0085] 3. Investigating the effect of co-delivery of M-CSF mRNA in macrophages on target mRNA expression

[0086] GFP and M-CSF mRNAs were generated, and then mRNA-loaded lipid nanoparticles (LNP-GFP and LNP-M-CSF) were prepared. Macrophages were plated and transfected with LNP-GFP and LNP-M-CSF at a ratio of 1:1 or 2:1. A blank control group (PBS) was also established. After transfection, the macrophages were cultured in an incucyte live cell imaging system, and the GFP signal was continuously monitored for 48 hours. After the 48-hour culture, GFP expression in the macrophages of each group was measured by flow cytometry.

[0087] The results showed that during the 48-hour continuous monitoring, M-CSF mRNA could significantly increase the expression efficiency of GFP in macrophages, and the expression efficiency was further increased with the increase of the M-CSF mixing ratio (e.g. Figure 5 At the end of 48 hours of culture, the positive rate of GFP in macrophages of each group was detected by flow cytometry. The results showed that M-CSF mRNA significantly increased the average fluorescence intensity of GFP in macrophages ( Figure 5 B (left: green fluorescence intensity in macrophages of different treatment groups; right: average fluorescence intensity of green fluorescence in macrophages of different treatment groups, 3 biological replicates in each group) and the proportion of GFP-positive macrophages ( Figure 6(Top: Proportion of GFP+ macrophages in each treatment group; Bottom: Statistical results of the proportion of GFP+ macrophages in each treatment group, three biological replicates per group). Similarly, increasing the incorporation ratio of M-CSF further enhanced GFP expression in macrophages. This demonstrates the feasibility of simultaneously delivering M-CSF mRNA to increase target mRNA expression.

[0088] 4. Exploring the Effect of M-CSF Signaling on the Expression of Transfected Long mRNA in Macrophages

[0089] GPC3-CAR (G2) mRNA was prepared, and the corresponding lipid nanoparticles (LNP-GPC3CAR) were prepared. Macrophages were plated and pretreated with 20% L929 supernatant by volume or M-CSF or LNP-M-CSF (the same mass as LNP-GPC3-CAR) at a final concentration of 20 ng / ml. Twenty-four hours after pretreatment, LNP-GPC3-CAR (1 ug / ml) was added for transfection. 24 hours after transfection, the expression rate of GPC3-CAR in macrophages in each group was determined by flow cytometry. The control group was untreated, and the LNP-GPC3 CAR group was transfected with LNP-GPC3-CAR alone.

[0090] like Figure 7 (Top: The proportion of GPC3-CAR macrophages in each treatment group; Bottom: Statistical results of the proportion of GPC3-CAR macrophages in each treatment group, 3 biological replicates per group) The results show that M-CSF, L929 supernatant, and LNP-M-CSF can significantly enhance the expression rate of GPC3 CAR in macrophages. This fully demonstrates that even when transfected with longer mRNA, M-CSF signaling can still strongly enhance the expression efficiency of exogenous mRNA in macrophages.

[0091] 5. Investigating the Effect of M-CSF Signaling on the Efficiency of Macrophage Editing by the LNP-mRNA System in Vivo

[0092] Fifteen C57 mice were selected and divided into five groups of three mice each. Three of the groups were pretreated with an intraperitoneal injection of IL-4 (5ug / mouse), an intraperitoneal injection of M-CSF (5ug / mouse), and an intravenous injection of LNP-M-CSF (3ug / mouse). 24 hours later, four groups of mice were intravenously injected with LNP-GFP (3ug / mouse), and one group was injected with PBS as a blank control (PBS group). The results are shown in Figure 2. Figure 8-Figure 9The control group was injected with LNP-GFP alone. Twenty-four hours after LNP-GFP injection, mice were dissected and liver single-cell suspensions were prepared. Flow cytometry was used to analyze GFP expression in macrophages and T cells in the livers of mice in each group.

[0093] The results showed that compared with the control group, all pretreatment groups could significantly increase the expression of GFP in macrophages (e.g. Figure 8 (Top: The proportion of GFP+ macrophages in the livers of mice in each treatment group; Bottom: Statistical results of the proportion of GFP+ macrophages in the livers of mice in each treatment group, 3 mice in each group). IL-4 had a limited effect on increasing GFP expression in macrophages, while M-CSF had the most significant effect, increasing the GFP-positive rate in macrophages to approximately 20%. Figure 8 It is worth noting that pre-intravenous injection of LNP-M-CSF can further increase the proportion of GFP+ macrophages to nearly 30% ( Figure 8 This indicates that local delivery and expression of M-CSF is more conducive to the in vivo editing of macrophages by the LNP-mRNA system. In addition, we analyzed the positive rate of GFP in T cells and found that the treatment groups had no significant effect on the expression of GFP in T cells ( Figure 9 (Top: Proportion of GFP+ T cells in the livers of mice in each treatment group; Bottom: Statistical results of the proportion of GFP+ T cells in the livers of mice in each treatment group, 3 mice in each group). This clearly demonstrates that M-CSF signaling can specifically and significantly enhance the editing efficiency of the LNP-mRNA system in macrophages in vivo.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of IL-4 or M-CSF in enhancing the expression of target mRNA in macrophages via mRNA-loaded lipid nanoparticles, wherein the application is for purposes other than disease diagnosis and treatment.

2. A method for editing macrophages in vitro, characterized in that: The following steps are involved: Macrophages are cultured in culture medium and then pretreated with at least one of IL-4, M-CSF, or lipid nanoparticles loaded with M-CSF mRNA. 24 hours later, lipid nanoparticles loaded with the target mRNA are added for transfection. After transfection, the in vitro editing of macrophages by lipid nanoparticles loaded with the target mRNA is completed.

3. The method according to claim 2, characterized in that The final concentration of IL-4 added is 5-40 ng / ml; the final concentration of M-CSF added is 5-40 ng / ml; The transfection concentration of the target mRNA is 0.1-20ug / ml; The mass ratio of M-CSF mRNA to target mRNA in the lipid nanoparticles loaded with M-CSF mRNA and the lipid nanoparticles loaded with target mRNA is (1-2):

1.

4. The method according to claim 2, characterized in that The target mRNA includes the mRNA of the chimeric antigen receptor; The intracellular domain of the chimeric antigen receptor is the CD3 intracellular domain.

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