Small interfering RNA targeting tumor-associated macrophage IRG1 and application thereof

Abstract

The invention provides a small interfering RNA (Ribonucleic Acid) targeting a tumor-associated macrophage IRG1 (Interferring Regulated Glycoprotein 1) and application thereof. Specifically, the invention provides a group of siRNA of a targeted metabolic enzyme IRG1, a siRNA-loaded LNP preparation and application of the siRNA-loaded LNP preparation. The siRNA disclosed by the invention can be used for remarkably down-regulating Irg1 gene expression and effectively inhibiting tumor growth, and has an application prospect in the anti-tumor field.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to small interfering RNA targeting tumor-associated macrophage IRG1 and uses thereof. Background Art

[0002] As a cancer treatment option following surgery, chemoradiotherapy, and targeted therapy, tumor immunotherapy has become one of the hottest areas in basic research, clinical translation, and application. T cell-based immune checkpoint blockade and chimeric antigen receptor T cell immunotherapy (CAR-T) offer hope for a cure for patients with hematologic malignancies. However, in solid tumors, their primary target, immunotherapy often exhibits a narrow window of response or even no response.

[0003] Macrophages are key effector cells of innate immunity and also play an important role in antigen presentation in adaptive immunity. Under different environmental and signaling stimuli, macrophages can polarize into subsets with distinct molecular and functional characteristics, such as the pro-inflammatory M1 and anti-inflammatory M2 subtypes. Within the tumor microenvironment, macrophages comprise 30–50% of infiltrating immune cells, and their phenotypes and functions are highly plastic and complexly heterogeneous. M1 tumor-associated macrophages (TAMs) are classically activated macrophages that participate in type I helper T (Th1) cell responses and produce pro-inflammatory and anti-tumor effects; M2 TAMs are selectively activated macrophages that participate in Th2 responses, produce anti-inflammatory mediators, and promote tumor progression. In tumor immunotherapy, circulating monocytes are the primary source of tumor-infiltrating macrophages, and the availability of peripheral blood monocytes makes macrophage-based therapeutic strategies clinically feasible.

[0004] Existing approaches to macrophage-targeted intervention can be summarized into five categories: blocking monocyte recruitment, depleting macrophages, inhibiting macrophage phagocytosis-related CD47-SIRPα signaling, adding chimeric antigen receptor (CAR-MAC), and inducing macrophage reprogramming. It is important to note that macrophages in the tumor microenvironment are a double-edged sword, serving as both a crucial component of tumor stromal cells and capable of phagocytosing tumor cells. Single-cell macrophage depletion can easily cause severe liver damage and may also increase the risk of infection, among other side effects. Using CCL2 antibodies to block monocyte recruitment or CD47 antibodies to inhibit phagocytosis has yielded limited success in multiple preclinical trials. Inducing TAM reprogramming to the pro-inflammatory M1 polarization while simultaneously preventing the polarization to the anti-inflammatory M2 polarization is a key breakthrough in macrophage-targeted intervention and enhancing anti-tumor efficacy.

[0005] Numerous studies have shown that macrophages polarize toward either the M1 or M2 phenotype under different environmental and signaling stimuli, with significant differences in their metabolic profiles. Itaconate (ITA) is a small molecule metabolite recently discovered to exhibit significant anti-inflammatory activity in M1 macrophages. In bacterially infected and activated macrophages, the mitochondrial metabolic enzyme ACOD1 (Aconitate decarboxylase 1; also known as IRG1) is rapidly induced, catalyzing the decarboxylation of aconitic acid and producing high concentrations of itaconate. Genetic studies in Irg1 knockout mice have shown that itaconate suppresses inflammatory gene expression and exerts anti-inflammatory effects. Previous studies have reported that itaconate inhibits the activity of the metabolic enzyme SDH, leading to the accumulation of succinate and affecting the aerobic electron transport respiratory chain. Itaconate, with its α,β-unsaturated carboxylic acid structure, can covalently modify cysteine ​​residues in proteins through Michael addition reactions, affecting KEAP1 and activating the KEAP1-NRF2 pathway. It also affects the activity of ALODA and GAPDH, inhibiting glycolysis. Previous studies have identified itaconate as a novel metabolite that regulates epigenetic regulation. Itaconate inhibits the DNA dioxygenase TET by competing with α-ketoglutarate (α-KG), regulating the expression of numerous inflammatory genes in the NF-kB and JAK-STAT pathways. In animal models of endotoxemia, ITA inhibits Tet2 activity, suppressing cytokine storms, alleviating liver and lung damage, and prolonging survival in mice.

[0006] In the tumor immune microenvironment, Irg1 is induced to express in TAMs and produces high levels of ITA. Irg1 gene knockout alters the composition of tumor-associated macrophage (TAM) subsets within the tumor microenvironment and significantly inhibits tumor growth in animal models of melanoma, colon cancer, breast cancer, and pancreatic cancer. Mechanistically, Irg1 / ITA inhibits the activity of the DNA dioxygenase Tet2 in monocytes and TAM subsets, regulating inflammatory gene expression, promoting TAM reprogramming, and altering their immune function phenotype, including antigen presentation, CD8+ T cell recruitment, and vascular remodeling. Studies have confirmed that IRG1 / ACOD1 is an important regulator of TAM reprogramming, suggesting that this metabolic enzyme may become a potential new target for anti-tumor drugs.

[0007] Since Bangham et al. first reported lipid nanostructures in 1965, they have become one of the most promising drug delivery systems due to their excellent biocompatibility and modifiable targeted delivery. In 1995, the US Food and Drug Administration (FDA) approved doxorubicin liposomes (Doxil), becoming the first marketed nanodrug. Subsequently, the FDA approved several lipid nanodrugs for clinical use. Over nearly half a century of development, traditional liposomes have evolved into numerous novel lipid nanocarriers. Among them, lipid nanoparticles (LNPs), composed of neutral lipids, ionizable cationic lipids, cholesterol, and PEGylated lipids, are currently the most advantageous nucleic acid drug delivery system with established clinical applications. In 2018, the FDA approved Onpattro (Patisiran), the first small interfering RNA drug based on the LNP delivery platform, for the treatment of hereditary transthyretin amyloidosis with polyneuropathy, marking the advent of the RNAi era. Currently, several siRNA drugs have been approved for marketing or are in clinical trials. The application of LNP technology for siRNA delivery is of great significance for its clinical translation. In addition, the clinical application of mRNA vaccines based on LNP delivery technology has greatly promoted the global pharmaceutical market's attention to LNP and nucleic acid drugs / vaccines.

[0008] Therefore, there is a need in the art to develop a tumor therapeutic drug that targets tumor-associated macrophage IRG1. Summary of the Invention

[0009] The purpose of the present invention is to provide a tumor therapeutic drug targeting tumor-associated macrophage IRG1.

[0010] In a first aspect of the present invention, an siRNA for inhibiting Irg1 gene expression is provided, wherein the nucleotide sequence of the siRNA is shown in any one of SEQ ID NOs: 1-23.

[0011] In another preferred embodiment, the nucleotide sequence of the siRNA is shown in any one of SEQ ID NO: 1, 2, 6, 7, 8, 11, 12, 14, 16, 17, 18, 21, and 22.

[0012] In another preferred embodiment, the siRNA is modified or unmodified siRNA.

[0013] In another preferred embodiment, the modification method is selected from the following group: methylation modification, hydrocarbon modification, glycosylation modification, nucleic acid modification, peptide modification, lipid modification, halogen modification, and nucleic acid modification.

[0014] In another preferred embodiment, the efficiency of the siRNA in inhibiting the expression of cellular Irg1 protein is ≥50%, preferably ≥70%, and more preferably ≥80%.

[0015] In another preferred embodiment, the cells are macrophages, preferably macrophages differentiated from THP1 cells.

[0016] In another preferred embodiment, the cells are activated by the tumor microenvironment.

[0017] In a second aspect of the present invention, a siRNA precursor is provided, wherein the 5' to 3' ends of the siRNA precursor have a structure as shown in Formula I:

[0018]

[0019] wherein B1 is the sense strand sequence of the siRNA as described in the first aspect of the present invention;

[0020] B2 is a sequence that is substantially complementary or completely complementary to B1, and B2 is not complementary to C;

[0021] C is a stem-loop structure sequence;

[0022] A1 and A2 are each independently an RNA sequence consisting of no or optional bases;

[0023] Wherein, the precursor sequence can be processed in the host to form the siRNA as described in the first aspect of the present invention.

[0024] In another preferred embodiment, the siRNA sequence is shown in any one of SEQ ID NOs: 1-23.

[0025] In another preferred embodiment, the siRNA sequence is shown as SEQ ID NO: 1.

[0026] In the third aspect of the present invention, a polynucleotide is provided, which encodes the siRNA or its complementary sequence as described in the first aspect of the present invention, or can be transcribed by a host to form the siRNA precursor as described in the second aspect of the present invention.

[0027] In the fourth aspect of the present invention, an expression vector is provided, wherein the expression vector contains the polynucleotide according to the third aspect of the present invention, or expresses the siRNA precursor according to the second aspect of the present invention.

[0028] In another preferred embodiment, the expression vector includes a viral vector and a non-viral vector.

[0029] In another preferred embodiment, the expression vector is a plasmid.

[0030] In another preferred embodiment, the expression vector contains a promoter, a replication origin and a marker gene.

[0031] In the fifth aspect of the present invention, a host cell is provided, wherein the host cell contains the expression vector according to the fourth aspect of the present invention.

[0032] In another preferred embodiment, the host cell is a mammalian cell.

[0033] In the sixth aspect of the present invention, a method for producing siRNA is provided, comprising the steps of culturing the host cell according to the fifth aspect of the present invention under conditions suitable for producing siRNA, thereby producing siRNA in the host cell.

[0034] In another preferred embodiment, the method further comprises obtaining the produced siRNA from the host cell.

[0035] In another preferred embodiment, the method is non-therapeutic in vitro.

[0036] In a seventh aspect of the present invention, a pharmaceutical preparation is provided, comprising:

[0037] (a) an active ingredient selected from the group consisting of: the siRNA according to the first aspect of the present invention, the siRNA precursor according to the second aspect of the present invention, the polynucleotide according to the third aspect of the present invention, and the expression vector according to the fourth aspect of the present invention; and

[0038] (b) a pharmaceutically acceptable carrier.

[0039] In another preferred embodiment, the pharmaceutical preparation is in liquid dosage form.

[0040] In another preferred embodiment, the pharmaceutical preparation is an injection.

[0041] In another preferred embodiment, the pharmaceutical preparation is a liposome preparation, which includes the active ingredient and liposomes loaded with the active ingredient. Preferably, the active ingredient is siRNA.

[0042] In another preferred embodiment, the liposome comprises a component selected from the group consisting of a cationic lipid, cholesterol, an auxiliary lipid, a PEG-modified lipid, or a combination thereof.

[0043] In another preferred embodiment, the liposome comprises or consists of the following components: cationic lipid DLin-MC3, cholesterol, auxiliary lipid DSPC and PEG2000-DMG-OH lipid.

[0044] In another preferred embodiment, the liposome comprises or consists of the following components in molar ratio:

[0045] 45-55% cationic lipid DLin-MC3;

[0046] 5-15% cholesterol;

[0047] 35%-40% auxiliary lipid DSPC; and

[0048] 1-2% PEG2000-DMG-OH lipid.

[0049] In another preferred embodiment, in the liposome preparation, the molar ratio of lipid to siRNA is 1-5:1, preferably 2-3:1.

[0050] In another preferred embodiment, the pharmaceutical preparation further comprises other drugs for preventing or treating tumors.

[0051] In another preferred embodiment, the other drugs for preventing or treating tumors are selected from the following group: cytotoxic chemotherapy drugs, immunosuppressive drugs, targeted drugs, or a combination thereof.

[0052] In an eighth aspect, the present invention provides use of the siRNA according to the first aspect of the present invention, the siRNA precursor according to the second aspect of the present invention, the polynucleotide according to the third aspect of the present invention, the expression vector according to the fourth aspect of the present invention, or the host cell according to the fifth aspect of the present invention in preparing a composition or preparation, wherein the composition or preparation is used for:

[0053] (i) preventing and / or treating cancer; and / or

[0054] (ii) Inhibition of IRG1 gene expression.

[0055] In another preferred embodiment, the cancer is selected from the group consisting of colon cancer, breast cancer, melanoma, granulocytic leukemia, lymphocytic leukemia, kidney cancer, pancreatic cancer, gastric cancer, and liver cancer.

[0056] In another preferred embodiment, the composition or preparation is used to inhibit macrophage Igr1.

[0057] In the ninth aspect of the present invention, a method for preventing and / or treating cancer in a subject in need is provided, the method comprising the steps of administering a therapeutically effective amount of the siRNA as described in the first aspect of the present invention, the siRNA precursor as described in the second aspect of the present invention, the polynucleotide as described in the third aspect of the present invention, the expression vector as described in the fourth aspect of the present invention, or the host cell as described in the fifth aspect of the present invention, or the pharmaceutical preparation as described in the seventh aspect of the present invention to a subject in need, thereby preventing and / or treating tumors.

[0058] In another preferred embodiment, the subject in need thereof is a human or a non-human mammal.

[0059] In another preferred embodiment, the non-human mammal is a mouse, a rat or a monkey.

[0060] In another preferred embodiment, the method further comprises simultaneously administering chemotherapy, radiotherapy or magnetic induction therapy to the subject in need.

[0061] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention defined by the claims.

[0063] Figure 1 This figure shows that knockdown of macrophage Irg1 using siRNA, stimulated with tumor-conditioned medium, promotes CD8+ T cell migration. (AB) After Irg1 was knocked down in BMDM cells by electroporation of siRNA, the BMDMs were stimulated with conditioned medium from B16-F10 cells for 12 hours. The knockdown efficiency of Irg1 at the mRNA and protein levels was determined by RT-PCR and Western blot, respectively. (C) The expression of the chemokine Cxcl9 was assessed by RT-PCR in the sample shown in A. (DE) After Irg1 was knocked down in BMDM cells by LNP-delivered siRNA, the BMDMs were stimulated with conditioned medium from B16-F10 cells and E0771 cells for 12 hours. The knockdown efficiency of Irg1 and the expression of chemokines Cxcl9 / 10 were assessed by RT-PCR. (F) A chamber experiment using primary CD8+ T cells and BMDMs was used to examine the effect of B16-F10 cell-conditioned medium on T cell migration.

[0064] Figure 2 This study demonstrates that lipid nanoparticles (LNPs) can effectively target tumor-associated macrophages. (A-B) The proportion of TAMs targeted by fluorescently labeled LNPs at different time points in the MC38 mouse subcutaneous tumor system. (C-E) The proportion of TAMs targeted by fluorescently labeled LNPs at different time points in the 4T1 mouse orthotopic breast cancer and lung metastasis model was measured in the orthotopic breast cancer (D) and lung metastasis (E).

[0065] Figure 3LNP-siIrg1 formulations inhibit tumor growth in immunocompetent mice. (AC) LNP-siIrg1 formulations and controls were injected into the tail vein of MC38 mice with subcutaneous tumors. TAMs were then separated by magnetic beads (A) to assess the removal of the metabolite ITA (B) and the knockdown efficiency of Irg1 transcripts (C). (DF) Tumor growth curves (D) and tumor weights (EF) were observed at the indicated time points in the above tumor models.

[0066] Figures 4A-4C Shown is the knockdown of IRG1 in human macrophages using siRNA. Figure 4A The siRNA sequence design targeting human IRG1 and the knockdown efficiency at the mRNA and protein levels are shown. Figure 4B The figure shows that human THP1 cells were differentiated into macrophages, transfected with reagents to deliver the indicated siRNA sequences, and then the macrophages were stimulated with conditioned medium from MDA-MB-231 cells. The knockdown efficiency of IRG1 transcript levels was detected by RT-PCR. Figure 4C The figure shows that human THP1 cells were differentiated into macrophages, transfected with reagents to deliver the specified siRNA sequences, and then the macrophages were stimulated with conditioned medium from MDA-MB-231 cells. The knockdown efficiency of IRG1 protein levels was detected by western blotting.

[0067] Figure 5 This figure shows that knocking down IRG1 in human macrophages using siRNA accelerates T cell migration when stimulated with tumor-conditioned medium. (A) Macrophages were differentiated from human THP1 cells, and after knocking down IRG1, the macrophages were stimulated with conditioned medium from MDA-MB-231 cells. Chemokine mRNA levels were detected by RT-PCR. (B) Macrophages were differentiated from human THP1 cells, and after knocking down IRG1, the macrophages were stimulated with conditioned medium from MDA-MB-231 cells. A chamber experiment using Jurkat T cells and activated human macrophages was used to examine the effect of macrophages on T cell migration when stimulated with tumor-conditioned medium. DETAILED DESCRIPTION

[0068] After extensive and in-depth research, the inventors have developed for the first time a small interfering RNA (siRNA) targeting IRG1 in tumor-associated macrophages and its uses. This invention provides a set of siRNAs that target the metabolic enzyme IRG1 and effectively inhibit Irg1 gene expression. By utilizing a transmembrane amino acid (LNP) delivery system targeting TAMs (tumor-associated macrophages), the siRNAs of this invention can be delivered to TAMs, significantly downregulating Irg1 gene expression, thereby inducing TAM reprogramming and enhancing anti-tumor immunity. In vivo experimental results demonstrate that the LNP-siRNAs of this invention effectively inhibit the growth of colon cancer in mice.

[0069] the term

[0070] In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can change. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0071] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0072] As used herein, the terms "comprise," "include," and "contain" are used interchangeably to include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of," "consisting essentially of."

[0073] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reaction), ie, has a reasonable benefit / risk ratio.

[0074] As used herein, the term "therapeutically effective amount" refers to an amount that produces a function or activity in humans and / or animals and is tolerated by humans and / or animals. Those skilled in the art will appreciate that the "therapeutically effective amount" may vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and combination therapy with other drugs.

[0075] RNA interference

[0076] RNA interference (siRNA) is a post-transcriptional gene silencing technique. siRNA is unzipped into sense and antisense strands by intracellular RNA helicases. The antisense strand then binds to enzymes in the cell (including endonucleases, exonucleases, and helicases) to form the RNA-induced silencing complex (RISC). RISC specifically binds to homologous regions of mRNA expressed by exogenous genes. RISC, acting as a nuclease, cleaves the mRNA at the binding site, leading to specific gene silencing. shRNA is an RNA molecule that can be cloned into an expression vector and expressed as a double-stranded siRNA. It consists of two short inverted repeats separated by a stem-loop sequence, forming a hairpin structure. In contrast to siRNA, shRNA is synthesized in the cell nucleus, further processed, and transported to the cytoplasm, where it enters the RISC for activity.

[0077] As used herein, the term "siRNA" (small interfering RNA, siRNA) refers to a small RNA molecule (approximately 21-25 nucleotides) that can be processed by Dicer (an enzyme in the RNase III family that is specific for double-stranded RNA) from precursors (such as dsRNA and shRNA). It can also be chemically synthesized or produced by processing other proteins. siRNA is a key member of the siRISC (siRISC) complex, stimulating the rapid cleavage and degradation of target RNAs with complementary sequences, leading to silencing of target genes. Therefore, it is a key functional molecule in RNAi.

[0078] The siRNA sequence of the present invention is shown in any one of SEQ ID NOs: 1 to 23. For example, the siRNA sequence of the present invention can be shown in any one of 1, 2, 6, 7, 8, 11, 12, 14, 16, 17, 18, 21, and 22, all of which can achieve an IRG1 protein knockdown efficiency of more than 80%.

[0079] As used herein, the term "shRNA" is an abbreviation for short hairpin RNA. shRNA consists of two short reverse-complementary sequences separated by a loop sequence, forming a hairpin structure. Transcription is typically controlled by an endogenous RNA polymerase III promoter. Five to six T residues are attached to the end of the shRNA coding sequence, serving as an RNA polymerase III transcription terminator. shRNA can also be transcribed from promoters of other RNA polymerases.

[0080] As used herein, the term "precursor siRNA" refers to an RNA molecule that can be processed in mammalian cells to produce siRNA, specifically, it is selectively processed by Dicer or other similar proteins to produce mature siRNA, thereby implementing RNAi.

[0081] In the present invention, the precursor siRNA is an artificially synthesized precursor siRNA, and the precursor siRNA has the structure shown in Formula I:

[0082]

[0083] Wherein B1 is the required first ribonucleic acid sequence, which includes the sense strand sequence of the siRNA as described in the first aspect of the present invention;

[0084] B2 is a sequence that is substantially complementary or completely complementary to B1, and B2 is not complementary to C;

[0085] C is a stem-loop structure sequence;

[0086] A1 is a sticky end sequence with no or no restriction endonuclease cleavage site;

[0087] A2 is a transcription termination sequence with no or 5-6 consecutive Us.

[0088] The precursor siRNA shown can be processed in the host to form IRG1 siRNA.

[0089] In Formula I, B2 and B1 are substantially complementary. As used herein, "substantially complementary" means that the sequences of nucleotides are sufficiently complementary to interact in a predictable manner, such as to form a secondary structure (e.g., a stem-loop structure). Typically, two "substantially complementary" nucleotide sequences have at least 70% of their nucleotides complementary to each other; preferably, at least 80% of their nucleotides complementary; more preferably, at least 90% of their nucleotides complementary; further preferably, at least 95% of their nucleotides complementary; such as 98%, 99%, or 100%. Generally, two sufficiently complementary molecules can have a maximum of 40 mismatched nucleotides; preferably, a maximum of 30 mismatched nucleotides; more preferably, a maximum of 20 mismatched nucleotides; further preferably, a maximum of 5 mismatched nucleotides, such as 1, 2, 3, 4, or 5 mismatched nucleotides.

[0090] As used in this application, a "stem-loop" structure, also known as a "hairpin" structure, refers to a nucleotide molecule that can form a secondary structure including a double-stranded region (stem), wherein the double-stranded region is formed by two regions of the nucleotide molecule (located on the same molecule), the two regions being located on either side of the double-stranded portion; and it also includes at least one "loop" structure, which includes non-complementary nucleotide molecules, i.e., single-stranded regions. Even if the two regions of the nucleotide molecule are not completely complementary, the double-stranded portion of the nucleotide can remain double-stranded. For example, insertions, deletions, substitutions, etc. may result in a small region being non-complementary or the small region itself forming a stem-loop structure or other form of secondary structure. However, the two regions may still be substantially complementary and interact in a predictable manner to form a double-stranded region of the stem-loop structure. Stem-loop structures are well known to those skilled in the art. Generally, after obtaining a nucleic acid with a nucleotide sequence having a primary structure, those skilled in the art can determine whether the nucleic acid can form a stem-loop structure.

[0091] In the present invention, a "stem-loop structure" may exist at the end of the precursor siRNA shown in Formula I. For example, after B1 and B2 form substantial complementarity, C will form a fixed terminal stem-loop structure. The "stem-loop structure" may also exist within the precursor siRNA shown in Formula I. For example, since B1 and B2 are not fully complementary, the bases of B1 or B2 that are not complementary will form an internal stem-loop.

[0092] The present invention also includes siRNA variants and derivatives. In addition, siRNA derivatives in a broad sense may also include siRNA variants. Those skilled in the art can use common methods to modify siRNAs that inhibit tyrosine kinases. Modification methods include (but are not limited to): methylation modification, alkyl modification, glycosylation modification (such as 2-methoxy-glycosyl modification, alkyl-glycosyl modification, sugar ring modification, etc.), nucleic acid modification, peptide modification, lipid modification, halogen modification, nucleic acid modification (such as "TT" modification), etc.

[0093] Polynucleotide (construct) and expression vector

[0094] According to the siRNA sequence provided by the present invention, a specific polynucleotide (construct) can be designed that can be processed into the siRNA that can affect the expression of the corresponding mRNA after being imported. The polynucleotide construct can raise the amount of the corresponding siRNA in vivo. Therefore, the invention provides a kind of isolated polynucleotide (construct), the polynucleotide (construct) encoding the siRNA of the present invention, or can be transcribed into precursor RNA by human cells, and the precursor RNA can be sheared and expressed into the siRNA by human cells.

[0095] As a preferred embodiment of the present invention, the polynucleotide construct comprises the structure shown in Formula II from the 5' to the 3' end:

[0096] Seq 正向 -X-Seq 反向 , Formula II

[0097] In Formula II,

[0098] Seq 正向 is a nucleotide sequence that can be expressed in cells to form the siRNA that inhibits IRG1, Seq 反向 For Seq 正向 Substantially complementary nucleotide sequences; or, Seq 反向 is a nucleotide sequence that can be expressed in cells as the siRNA, Seq 正向 For Seq 正向 Substantially complementary nucleotide sequences; X is located at Seq 正向 and Seq 反向 The spacer sequence between the two sequences is the same as Seq 正向 and Seq 反向 Not complementary.

[0099] After the structure shown in Formula II is transferred into cells, it forms the secondary structure shown in Formula III:

[0100]

[0101] In Formula III, Seq 正向 、Seq 反向 and X is defined as above;

[0102] || represents the complementary base pairing relationship formed between Seq forward and Seq reverse.

[0103] Typically, the polynucleotide construct is located on an expression vector. Therefore, the present invention also includes an expression vector containing the siRNA or the polynucleotide construct. The expression vector typically also contains a promoter, a replication origin, and / or a marker gene. Methods well known to those skilled in the art can be used to construct the expression vector required for the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, and the like. The expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting transformed host cells, such as karatomycin, gentamicin, hygromycin, and ampicillin resistance.

[0104] In the present invention, the expression vector is not particularly limited and includes commercially available or conventionally prepared expression vectors. In another preferred embodiment, in the expression vector, the promoter operably linked to the polynucleotide expressing the precursor siRNA includes a constitutive promoter or a tissue-specific promoter, preferably a promoter specifically activated in liver tissue. In other words, these promoters are used to drive the expression of the precursor siRNA.

[0105] Representative promoters include, but are not limited to, Pcmv promoter, U6, H1, CD43 promoter, CD45 (LCA) promoter, CD68 promoter, Endoglin (CD105) promoter, Fibronectin promoter, Flt-1 (VEGFR-1) promoter, GFAP promoter, GPIIb (IntegrinαIIb) promoter, ICAM-2 (CD102) promoter, MB (Myoglobin) promoter, NphsI (Nephrin) promoter, SPB promoter, SV40 / hAlb promoter, SYN1 promoter, WASP promoter, or a combination thereof.

[0106] Pharmaceutical compositions and methods of administration

[0107] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0108] As used herein, the term "pharmaceutically acceptable" refers to a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, including various excipients and diluents.

[0109] The pharmaceutical compositions of the present invention contain a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to) saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be compatible with the mode of administration. The pharmaceutical compositions of the present invention are available in the form of injections, oral preparations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. For example, they can be prepared using conventional methods using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions are preferably manufactured under sterile conditions.

[0110] The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, etc. Generally, when the active ingredient of the present invention is administered at a dose of about 0.00001 mg-50 mg / kg animal body weight (preferably 0.0001 mg-10 mg / kg animal body weight) per day, a satisfactory effect can be obtained. For example, depending on the urgency of the treatment condition, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0111] The pharmaceutically acceptable carriers of the present invention include, but are not limited to, water, saline, liposomes, lipids, microparticles, microvesicles, exosomes, shedding vesicles, nanocapsules (nanoparticles), β-cyclodextrin capsules (β-cyclodextriniclusion compounds), proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be compatible with the mode of administration, as is well known to those skilled in the art.

[0112] In the present invention, the expression vector can be directly administered to a subject, or the expression vector can be prepared into a pharmaceutical combination with a pharmaceutically acceptable carrier and then administered. The administration includes intravenous injection.

[0113] Liposomal preparations

[0114] The pharmaceutical preparation of the present invention may be a liposome preparation. In the present invention, the terms "LNP-siRNA" and "LNP-siIRG1" are used interchangeably, both referring to a liposome preparation loaded with the siRNA targeting IRG1 of the present invention.

[0115] Nanoliposomal drugs have excellent targeting properties for the liver. Previous studies have shown that IRG1 expression in the microenvironment of liver cancer patients receiving anti-PD-1 immunotherapy is negatively correlated with CD8+ T cell infiltration and the patient's immune response. In a mouse liver cancer model, Irg1 deficiency can also significantly inhibit liver cancer growth. LNP-siIRG1 formulations may have greater applicability for liver cancer patients. In addition to the liver, intravenous LNPs also accumulate in the lungs, which also contain a large number of macrophages. Previous studies have found that IRG1 expression is increased in tumor tissue compared to adjacent tissue in lung cancer patients. Therefore, LNP-siIRG1 treatment may also be suitable for lung cancer patients. Due to the phagocytic function of macrophages, their uptake rate of LNPs is higher than that of other cells, such as B cells and T cells. Therefore, when modifying LNPs, one can start by improving organ targeting. For example, current studies have improved the targeting of LNPs to organs such as the lungs and kidneys.

[0116] Since IRG1 is widely expressed in various solid tumors, such as colon cancer, breast cancer, kidney cancer, pancreatic cancer, gastric cancer, etc., the LNP loaded with the siRNA of the present invention can be appropriately modified to improve the targeting rate to different organs, thereby widely applying LNP-siIRG1 to the intervention of the above solid tumors.

[0117] Furthermore, since targeting Irg1 in TAMs inhibits tumor growth by increasing T cell infiltration in the tumor immune microenvironment, and Irg1 deficiency enhances the efficacy of anti-PD-(L)1 immunotherapy, LNP-siIRG1 may also enhance the efficacy of anti-PD-(L)1 immunotherapy. The LNP-siIRG1 of the present invention can also be used in conjunction with conventional chemoradiotherapy, potentially helping to reduce the dose of chemoradiotherapy and enhance anti-tumor efficacy.

[0118] Treatment

[0119] The present invention also provides a method for treating cancer, that is, administering a safe / therapeutically effective amount of the siRNA as described in the first aspect of the present invention, the precursor as described in the second aspect of the present invention, the polynucleotide as described in the third aspect of the present invention, the expression vector as described in the fourth aspect of the present invention, the host cell as described in the fifth aspect of the present invention, or the pharmaceutical composition as described in the eighth aspect of the present invention to a subject in need, thereby treating cancer.

[0120] The main advantages of the present invention include:

[0121] 1) The present invention provides a group of siRNAs that can effectively inhibit the expression of the Irg1 gene.

[0122] 2) The present invention uses the metabolic enzyme IRG1 as the intervention target, utilizes a TAM-targeted LNP delivery system to carry siRNA, delivers it to TAMs and downregulates Irg1 gene expression, which can induce TAM reprogramming and enhance anti-tumor immunity.

[0123] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0124] Experimental methods

[0125] Construction of tumor model in immunocompetent mice

[0126] Subcutaneous tumor implantation experiments were performed using 8-10 week old female C57B / 6 mice. MC38 tumor cells were diluted to 2*10 5 25 μL of matrix gel was added and mixed. The mixture was placed on ice and the tumor was injected subcutaneously as soon as possible. The skin of each mouse was disinfected with a 75% alcohol cotton ball and 50 μL of the mixture was injected subcutaneously on the right side of the back of the mouse using an insulin needle. The mouse breast cancer orthotopic model was established using the 4T1 mouse breast cancer cell line and BALBC mice. The 4T1 tumor cells were diluted to 1*10 5 Add 25 μL of Matrigel to the mixture. Place the mixture on ice and inject into the fat pad as soon as possible. Inject the mixture into the fat pad of the female mouse via an insulin needle. Measure the volume every 2-3 days after tumor growth. Calculate volume using the formula: V = (width x width x length) / 2.

[0127] Preparation of LNP-siRNA and tumor treatment in mice

[0128] The LPN-siIrg1 preparation was commissioned to Suzhou Kris Pharmaceutical Co., Ltd. for synthesis. The siRNA-loaded LNPs used commercial ionizable lipids DLin-MC3 / CHOL / DSPC / PEG2000-DMG-OH with a molar ratio of 50 / 10 / 38.5 / 1.5. Each lipid was dissolved in ethanol and mixed in the organic phase to achieve the specified molar ratio. The siRNA in the aqueous phase was dissolved in 25mM sodium acetate buffer pH 5 to reach 1mg / ml, and the organic phase and the aqueous phase were mixed through a microfluidic chip using a microfluidic preparation instrument MPE-L2 to keep the NP lipid:siRNA molar ratio constant at 3. The outflowing LNPs were diluted into DEPC water, concentrated using a 100kDa ultrafiltration tube, centrifuged at 1500g for 2 minutes, and resuspended in PBS after ultrafiltration twice. After the mice were inoculated with tumors, they were allowed to grow to 200mm. 3 The LNP preparation was injected into the tail vein once every two days for a total of three times, and the tumor growth of the mice was observed at designated time points during the period.

[0129] LNP fluorescent labeling

[0130] The preparation method of LNP was as described above. When synthesizing LNP, DiI was added to the lipid solution (organic phase) at a concentration of 0.5 mol% (total lipids) to fluorescently label the LNP.

[0131] Cell culture

[0132] Table 1 Immortalized cells used in the examples

[0133]

[0134] During cell culture, the medium needs to be changed and the cell growth status needs to be checked every day. Cells need to have good morphology, normal growth rate and no contamination before they can be passaged or subjected to subsequent experiments. Cells need to be passaged when they grow to a density of about 90%. First, aspirate the cell culture medium and add 10mL of preheated PBS for washing. After aspirating the PBS, add 1-2mL of trypsin to a 100mm culture dish and transfer the cells to a 37℃ incubator for digestion. Stop digestion when some cells fall off. Remove the culture dish and add 5mL of DMEM culture medium containing 10% FBS to evenly distribute the cells. Then use a 1mL pipette to transfer the cells to a 15mL BD tube and centrifuge at 1000rpm for 5 minutes. After removing the supernatant, resuspend the cells in new DMEM culture medium containing 10% FBS and count them. Plate the cells according to the required cell amount, shake gently to evenly distribute them, and then transfer them to a 37℃ incubator for culture. Check the adhesion after 12 hours.

[0135] Culture and differentiation induction of THP1 cells

[0136] THP1 cells are a human leukemia monocytic cell line and are a type of suspension cell. THP1 cells are density-dependent and have high requirements for serum. During culture, care must be taken to prevent cell agglomeration and activation. The recovery and freezing of THP1 are similar to other immortalized cells, but the requirements for culture are different. THP1 is cultured in 1640 medium containing 10% FBS, and 0.05mM β-mercaptoethanol is added to the medium exogenously. Cells are replenished and counted every 2-3 days. The density of THP1 cells must not be less than 5*10 5 cells / ml culture medium, when the cell density exceeds 1*10 6 After the cells have been homogenized, they can be passaged. After blowing them evenly, transfer them to a 15 mL BD tube using a 1 mL pipette and centrifuge at 1000 rpm for 5 minutes. After removing the supernatant, count the cells in 1640 medium containing 10% FBS and transfer the cells to a new culture dish. Note that the cell density should not be less than 5*10 5 cells / mL culture medium. After shaking the cells, place them in 37℃ culture medium for culture. After 12 hours, observe the cell aggregation and growth. If the cells are obviously aggregated or the cell growth slows down, they need to be discarded. When it is necessary to induce THP1 to differentiate into macrophages, count the THP1 and calculate the number of cells per 10 6 Plate cells at a density of 100 cells / well in a 6-well plate. Add 10 mM PMA to the culture medium to induce THP1 differentiation. Incubate the cells in a 37°C incubator. Remove the cells after 24-48 hours and observe whether they have adhered to the wall and become spindle-shaped. If these cells are spindle-shaped, induction is successful.

[0137] Jurkat cell culture

[0138] Jurkat cells are suspension cells, and their thawing and freezing procedures are similar to those for immortalized adherent cells. For cell passaging, transfer the cultured Jurkat cells to a 15mL BD tube using a 1mL pipette. Centrifuge at 1000 rpm for 3 minutes. After centrifugation, remove the supernatant and add fresh 1640 medium supplemented with 10% FBS. Mix thoroughly, transfer the culture to a 100mm dish, shake well, and incubate in a 37°C incubator. After 12 hours, inspect the cells for clumping and growth.

[0139] Table 2 Primary cells used in the examples

[0140] Cell name Cell type BMDM Mouse bone marrow-derived macrophages <![CDATA[CD8 + T cells]]> <![CDATA[CD8 T cells derived from mouse spleen + >

[0141] Isolation and culture of mouse bone marrow-derived macrophages (BMDM)

[0142] Reagent and equipment preparation:

[0143] Reagents: pre-chilled PBS, 10% FBS DMEM medium, red blood cell lysis buffer, MCSF.

[0144] Consumables: 0.45 mm needle, 10 ml syringe, 40 μm filter membrane, 100 mm culture dish, surgical scissors, curved forceps.

[0145] After the reagents and consumables are prepared, proceed with the BMDM isolation and culture experiment. First, place the necessary consumables in a clean bench under UV light for half an hour. Sterilize surgical scissors and curved forceps in a beaker of 75% alcohol for half an hour. Pre-cool the centrifuge to 4°C. Grasp the base of the tail of a wild-type mouse and lift it. Place it on the lid of its cage. Use your thumb and index finger to firmly press down on the head and neck, and use your other hand to pull the tail until the mouse dies by cervical dislocation. Secure the deceased mouse with pins on a foam board, abdomen facing up, and spray it with 75% alcohol for disinfection. Spray the mouse's legs with alcohol and dry the alcohol-soaked surgical scissors and forceps over an alcohol burner. Lift the mouse's thigh with forceps in one hand. With the other hand, carefully cut the tibia and femur of both lower limbs along the direction of muscle growth with surgical scissors. Trim away the outer fur and excess muscle, taking care not to break the tibia and femur. Wrap the tibia and femur with sandpaper and remove them by hand, or use forceps to remove the attached muscle. Soak the peeled tibia and femur in 75% alcohol for further disinfection.

[0146] Take a 100mm culture dish and fill it with pre-chilled PBS. Rinse the tibia and femur once in PBS and place in a fresh PBS culture dish. Cut the ends of the leg bone with scissors. Use a 0.45mm needle and a 10mL syringe to draw up 7mL of pre-chilled PBS. Insert the needle into the bone marrow cavity and repeatedly flush the cavity until the PBS is clear and the cavity turns white.

[0147] Gently pipette the PBS removed until the cells are dispersed. Place a 40μm filter on a 50mL BD tube. Pipette the cell suspension obtained in the previous step through the filter and filter. After filtration, add 3mL of fresh PBS to the original tube to wash, then pipette the suspension back into the filter. After filtration, cap the BD tube and remove it from the laminar flow hood. Centrifuge at 400g for 5 minutes at 4°C.

[0148] After centrifugation, aspirate the supernatant. The cells at the bottom will be light red. Add 4 mL of red blood cell lysis buffer and mix thoroughly using a 1 mL pipette. Place the BD tube in a 37°C incubator for 10 minutes.

[0149] After red blood cell digestion is complete, add PBS to the cell suspension to 8 mL, cap the BD tube, and remove it from the clean bench. Centrifuge at 400g for 5 minutes.

[0150] Remove the supernatant; the cells at the bottom should be white. Add 2 mL of DMEM and mix thoroughly. Plate the isolated bone marrow cells in a 100 mm dish and culture in DMEM supplemented with 100 ng / mL MCSF and 10% FBS. Change the medium on the third and fifth days, gently rinsing with pre-chilled PBS.

[0151] On day 7, BMDMs are fully mature. Digest the cells with Versene digestion buffer at 37°C for 10 minutes. Once the cells are partially free of adhesion, add 10 mL of DMEM (10% FBS) and gently pipette to dissociate into single cells. Transfer the cells to a BD tube. Centrifuge at 400g for 5 minutes at 4°C.

[0152] After centrifugation, the cells were counted using a cell counter and resuspended in DMEM medium containing 100 ng / mL MCSF and 10% FBS. 5*10 5 BMDMs / well were plated into 6-well plates and subjected to subsequent experiments.

[0153] Isolation and culture of CD8+ T cells

[0154] Reagents and equipment:

[0155] Reagents: pre-chilled PBS, 10% FBS-containing 1640 culture medium, red blood cell lysis buffer, anti-CD3 antibody, anti-CD28 antibody, and recombinant mouse IL-2.

[0156] Consumables: Mouse naive CD8a+ T cell isolation kit, magnetic stand, MACS column for magnetic cell separation, 10 mL syringe, 40 μm filter membrane, 100 mm culture dish, surgical scissors, curved forceps.

[0157] Once the reagents and consumables are ready, proceed with the CD8+ T cell culture experiment. First, place the consumables in a clean bench and irradiate them with UV light for half an hour. Sterilize the surgical scissors and curved forceps in a beaker of 75% alcohol for half an hour. Pre-cool the centrifuge to 4°C.

[0158] Execute the mouse by cervical dislocation. Secure the dead mouse to a foam board with pins, abdomen facing up, and spray with 75% alcohol for disinfection. Spray the mouse's abdomen with alcohol and dry alcohol-soaked surgical scissors and forceps over an alcohol lamp. Make a small incision on the right side of the mouse's abdomen. Use forceps to pull up the peritoneum and use another pair of forceps to remove the mouse's spleen. Place the spleen in 75% alcohol for further disinfection. After disinfection, quickly remove the spleen and place it in a 100mm dish filled with PBS. Rinse once and soak in pre-chilled PBS.

[0159] Place a 40 μm filter in the mouth of a 50 mL BD tube and place the spleen on the 40 μm filter. Triturate the spleen using the soft tip of a 10 mL syringe and rinse with PBS until most cells have passed through the filter. Collect the suspension and centrifuge at 400 g for 5 minutes in a 4°C centrifuge.

[0160] Aspirate the supernatant, which will now be a dark red cell pellet. Add 5 mL of red blood cell lysis buffer and resuspend the cells. Place the cells in a 37°C incubator for 5 minutes to lyse. After five minutes, remove the cells and centrifuge at 400 g for 5 minutes at 4°C.

[0161] Remove the supernatant by suction, and the cell pellet will appear off-white. Resuspend in PBS and count according to 1*10 7 Add CD8+ T cell separation magnetic beads to 10 cells / 20μL. Mix thoroughly with a pipette and incubate at 4℃ for 10 minutes in the dark. Remove the LS magnetic column and place it on the magnetic stand. Rinse with PBS. Each magnetic column can be loaded with up to 2*10 9 After 10 minutes of binding, gently resuspend the cells in 3 mL of PBS and add them to the magnetic column for binding. Wash twice with 5 mL of PBS, remove the magnetic column, place it in a 15 mL BD tube, add 3 mL of PBS to rinse the cells, centrifuge at 4°C, resuspend in 1640 medium containing 10% FBS, and count at 400 g for 5 minutes.

[0162] The separated cells can be detected by flow cytometry for separation efficiency and stained for Cd3 and Cd8.

[0163] The separated CD8 + T cells were cultured in 24-well plates, 1*10 6 Cells were cultured in 1640 medium supplemented with 10% FBS, supplemented with 5 μg / mL anti-Cd3 monoclonal antibody and 2.5 μg / mL anti-Cd28 monoclonal antibody. In some experiments, 1 μg / mL recombinant IL-2 was added for stimulation. Anti-Cd3 monoclonal antibody and anti-Cd28 monoclonal antibody were replenished every three days.

[0164] Preparation of tumor-conditioned medium (TCM)

[0165] Culture B16-F10, E0771, and MDA-MB-231 tumor cells to 80% confluency. Replace the culture medium with serum-free DMEM. Add 10 mL of DMEM per 100 mm dish. After 24 hours, collect the culture supernatant and filter through a 0.22 μm or 0.45 μm filter to remove cell debris. Mix the supernatant with 10% DMEM in a 1:1 ratio and store at -20°C. Use within one week.

[0166] Cell electroporation

[0167] For cells in the middle of logarithmic growth, trypsinize them and stop digestion with culture medium. Centrifuge at 1000 rpm for 3 minutes on a tabletop centrifuge, remove the supernatant, and resuspend the cells with electroporation buffer. Add 100 pmol of siRNA per million cell suspension, mix thoroughly, and transfer it to the electroporation cup after evenly dispersing. Use the Lonza instrument for electroporation. After completion, add 0.5 mL of ordinary culture medium to the electroporation cup, transfer the cell mixture to a culture dish, and add some culture medium to make the final culture medium volume appropriate. Then, place the sample cells back into the incubator to grow. After 6-8 hours, observe the cell status, change the medium, and continue culturing until the cells are harvested at 48 hours.

[0168] Cell transfection experiments

[0169] When the cell density reaches about 50%-80%, start preparing for transfection. For cells in a six-well plate, use 150 μL Dilute with 9 μL of Medium RNAiMAX and 30 pmol siRNA were mixed at a 1:1 ratio and incubated at room temperature for 15 minutes. The cells were then added to the cell culture medium and replaced with fresh medium after 5-6 hours. The cells were harvested after 48 hours.

[0170] LC-MS

[0171] Prepare an 80% methanol solution. Prepare 80% methanol solution using chromatographic or mass spectrometry grade pure methanol solution with ultrapure water with a resistivity of 18 MΩ*cm (25°C) and store in a -80°C refrigerator.

[0172] Use a cell counter to record the cell number and cell diameter. After gently washing the cells with 5 mL of PBS, add 500 μL of 80% methanol pre-cooled at -80°C overnight and shake at 4°C for half an hour. Remove the EP tube, centrifuge at 14,000 rpm at 4°C for 30 minutes, and aspirate 350 μL of the supernatant. Transfer the aspirated supernatant to a new EP tube and freeze-dry it using a vacuum concentrator. After freeze-drying, reconstitute it with 55 μL of 80% methanol and insert the EP tube into a 4°C turntable overnight. Centrifuge the dissolved suspension at 4°C. Centrifuge at 14,000 rpm for 30 minutes.

[0173] Finally, 40 μL of the supernatant was transferred to a V-bottom loading tube for detection. The cell extract was analyzed by ultra-high performance liquid chromatography (Acquity UPLC I-Class, Waters) coupled to a triple quadrupole mass spectrometer (Xevo TQ-XS, Waters).

[0174] Assume that cells are spherical and use an external standard curve to calculate the intracellular metabolite concentration. Consider the cell diameter (d, micrometers) and cell number. The calculation formula is as follows: [metabolite] = amount of metabolite (moles) / ((4 / 3000)π(d / 2) 3 ).

[0175] Real-time quantitative PCR (qRT-PCR)

[0176] RNA was extracted from the cell samples using the EZ-press RNA Rapid Extraction Kit. After washing the 6-well plate once with PBS, the supernatant was discarded and 350 μL of lysis buffer was added. The plate was lysed on a horizontal shaker for 10 minutes. After adding 350 μL of anhydrous ethanol, the lysate was thoroughly mixed and transferred to a spin column using a pipette. The plate was centrifuged at 6000 rpm for 1 minute at room temperature. After removing the waste liquid, 10 μL of ddH2O and 2 μg of DNA Remover were added and the plate was allowed to stand for 5 minutes to remove DNA contamination. 500 μL of wash buffer was added and the plate was centrifuged at 8000 rpm for 1 minute at room temperature. After removing the waste liquid, the plate was emptied for 1 minute. The column was uncapped and allowed to stand at room temperature for 5 minutes until the ethanol evaporated completely. The column was transferred to a new EP tube and 30 μL of RNase-free water was added. The plate was allowed to stand at room temperature for 5 minutes. The column was then centrifuged at 13000 rpm for 3 minutes at room temperature.

[0177] After calculating the RNA concentration using a spectrophotometer, take a portion of the RNA for reverse transcription. The reaction system is as follows:

[0178] Total RNA 1 μg

[0179] 4x EZscript RT Mix II*1 5μL

[0180] Add water to 20 μL.

[0181] After shaking and mixing, reverse transcription is performed. The reaction procedure is as follows:

[0182] 42℃15min

[0183] 85℃30s

[0184] 4℃10min

[0185] The cDNA sample was diluted 10-fold for real-time fluorescence quantitative PCR. The reaction system is as follows:

[0186] SYBR premix EX taq (2x) 5 μl

[0187] cDNA 1 μL

[0188] Forward primer 0.2 μL

[0189] Reverse primer 0.2 μL

[0190] ddH2O 3.6mL

[0191] After vortexing and mixing, the cells were added to a 384-well plate for qPCR. After verifying the suitability of the primers using a melting curve, relative quantification of the gene was performed using β-actin or 18sRNA as an internal reference.

[0192] Western Blot

[0193] Western blotting includes the following steps: gel electrophoresis, membrane transfer, blocking, incubation with primary and secondary antibodies, and color development.

[0194] First, prepare two 10% separation gels: 4 mL H₂O, 3.3 mL 30% acrylamide solution, 2.5 mL 1.5 M Tris-HCl + 10% SDS (pH 8.8), 100 μL 10% ammonium persulfate, and 5 μL TEMED. Mix thoroughly and add the mixture to the gel plates. Then, add 1 mL of anhydrous ethanol to seal the liquid surface. After the separation gel solidifies, pour off the anhydrous ethanol and invert the gel plates to dry. Next, prepare the stacking gel: 2.7 mL H₂O, 0.67 mL 30% acrylamide solution, 0.5 mL 1 M Tris-HCl + 10% SDS (pH 6.8), 40 μL 10% ammonium persulfate, and 4 μL TEMED. Mix thoroughly and add to the gel plates. Quickly insert the sample loading comb. Mount the protein gel in the electrophoresis tank, add running buffer, and apply the sample to the wells. Set the electrophoresis instrument program to 80V for 20 minutes, then 135V for 60 minutes. Observe the protein markers for separation and the smooth, even migration of bromophenol blue to the bottom of the separation gel. Prepare transfer buffer and pre-chill it at 4°C. Transfer the membrane immediately after the run. Moisten the NC membrane, two sponges, and two pieces of filter paper with pre-chilled transfer buffer. Place the sponge, filter paper, protein gel, NC membrane, filter paper, and sponge in the transfer cassette and tighten the cassette. Avoid air bubbles during stacking. Once the cassette is secure, insert the transfer tank. Set the electrophoresis instrument program to 330mA for 1 hour. Place the electrophoresis tank in an ice-water mixture. After transfer, remove the NC membrane and stain with Ponceau red to check for air bubbles and smooth bands. Wash with water, then incubate with 5% BSA at room temperature for 1 hour. After incubation, rinse with TBST and prepare the primary antibody using primary antibody diluent and incubate at 4°C overnight. Recover the primary antibody and wash three times with TBST for 10 minutes each. The secondary antibody was prepared in 5% BSA and incubated at room temperature for 1 hour.

[0195] Recover the secondary antibody and wash three times with TBST for 10 minutes each. Prepare the colorimetric solution and add it dropwise to the membrane. Develop in the dark for 1-3 minutes, then terminate the reaction with ddH2O. Scan and visualize using a Typhoon scanner.

[0196] T cell invasion and migration assay

[0197] The invasion chamber experiment was performed using a 24-well plate as the bottom plate and a 5.0 μm pore size polycarbonate membrane chamber. 5 BMDM or THP1-derived macrophages were stimulated with tumor-conditioned medium for 12 h, and lymphocytes were added to the upper chamber. The cells were incubated in a 37°C cell culture incubator for 4 h, after which the number of lymphocytes that migrated to the bottom chamber was counted.

[0198] Flow cytometry

[0199] Tumor-bearing mice were killed by cervical dislocation and fixed on a foam board with steel pins. The tumor part was sprayed with 75% alcohol, and then the tumor was removed using surgical scissors and curved forceps and placed in a 100 mm culture dish filled with PBS.

[0200] Prepare the digestion solution: 4 mL 1640 medium + 400 μL fetal bovine serum + 200 μL collagenase IV + 40 μL Dispase I + 16 μL DNAse. Finely mince the tumor with surgical scissors and place it in an EP tube. Add 1 mL of digestion solution to each tumor and shake in a 37°C shaker for 0.5-1 hour. Remove the tube and pipette with a 1 mL syringe to aid digestion. Transfer the digested tumor to a 50 mL BD tube fitted with a 40 μm filter. Triturate the tumor using the soft tip of a 10 mL syringe and pipette with PBS to filter as many cells as possible. Centrifuge the filtered cells at 400 g for 5 minutes in a 4°C precooled centrifuge. Remove the supernatant and add 2 mL of red blood cell lysis buffer to the cell pellet, then lyse the pellet in a 37°C incubator for 5 minutes. Centrifuge the lysed cells at 400 g for 5 minutes in a 4°C precooled centrifuge. The pellet will appear off-white. Remove the supernatant and resuspend in PBS. Count the cells and take about 5*10 6Cells were dissolved in 50 μL of PBS for staining. First, stain the membrane surface with antibodies. Pre-mix the antibodies and add 0.3 μL of flow cytometry antibody and 0.05 μL of fixable reactive dye to each sample. Mix thoroughly and stain for half an hour at 4°C in the dark. After staining, resuspend the cells in 1 mL of PBS and centrifuge at 400 g for 5 minutes in a pre-cooled 4°C centrifuge. Aspirate the supernatant. Wash once with PBS. Next, stain the intracellular antibodies. Add 50 μL of permeabilization fixative to each sample. Mix thoroughly and stain for half an hour at 4°C in the dark. Resuspend the cells in 1 mL of PBS and centrifuge at 400 g for 5 minutes in a pre-cooled 4°C centrifuge. Aspirate the supernatant. Wash once with PBS. Next, mix the intracellular antibodies and add 0.3 μL of flow cytometry antibody to each sample. Mix thoroughly and stain for half an hour at 4°C in the dark. After staining, resuspend the cells in 1 mL of PBS and centrifuge at 400 g for 5 minutes in a pre-cooled 4°C centrifuge. Remove the supernatant. Wash once more with PBS. Finally, resuspend the cells in 300 μL of PBS and immediately transfer to the centrifuge, or fix with 300 μL of paraformaldehyde. Store at 4°C in the dark.

[0201] Example 1: Targeting mouse macrophage Irg1 with siRNA to promote T cell migration

[0202] Using electroporation technology, Irg1 was knocked down in mouse bone marrow-derived macrophages (BMDM), and the knockdown efficiency of Irg1 was detected by RT-PCR and western blot. The siRNA sequence 5'-CAGGUUUACCAAUAUCUAAUU-3' (SEQ ID NO: 23) can eliminate more than 90% of Irg1 at the transcriptional level. Figure 1 A), and the protein level in activated macrophages also decreased by 70% ( Figure 1 B) Previous studies have shown that tumor cell culture supernatant (TCM) can activate macrophages and activate the expression of Irg1 in macrophages, and Irg1 knockout can upregulate CD8 + Transcription of T cell recruitment-related genes Cxcl9 / 10. Knockdown of Irg1 in BMDM by electroporation of siRNA with the above sequence can also upregulate Cxcl9 expression under TCM stimulation. Using the LNP delivery system to carry siRNA with the above sequence targeting Irg1 in BMDMs can also effectively knockdown Irg1 in activated macrophages ( Figure 1 D). In BMDM activated by melanoma B16-F10 culture supernatant (B16-F10-TCM), the knockdown efficiency was approximately 61.02%, and in BMDM activated by breast cancer cell E0771 culture supernatant (E0771-TCM), the knockdown efficiency was approximately 57.48% ( Figure 1D) Correspondingly, LNP-siIrg1 treatment can also upregulate CD8 in B16-F10-TCM-activated macrophages. + Expression of T cell recruitment-related genes Cxcl9 / 10 ( Figure 1 E) In macrophages and primary CD8 + In an in vitro T cell chamber experiment, knocking down Irg1 in macrophages using siIrg1 promoted CD8 + T cell migration ( Figure 1 F).

[0203] Example 2: Using LNP to deliver siIrg1 can be used for anti-tumor therapy

[0204] A mouse colon cancer model was established by subcutaneous injection of MC38 colon cancer cells. Fluorescently labeled LNPs (1 mg / kg) were injected into the tail vein. The proportion of LNPs in tumor macrophages was detected at different time points after injection, and the targeting rate in TAMs after LNP injection was calculated ( Figure 2 A). The results showed that after only one injection of LNP, about 15% of TAMs could be targeted by LNP after 48 hours ( Figure 2 B). In BALB / c white mice, 4T1 cell-mediated breast cancer model ( Figure 2 C), a single injection of LNP can not only effectively target TAMs (about 15%) of in situ breast cancer ( Figure 2 D) The targeting rate of TAMs in the lung metastasis microenvironment is close to 10% ( Figure 2 E) These results indicate that LNPs can effectively deliver siRNA to TAMs.

[0205] Next, in the MC38 subcutaneous injection mouse colon cancer model, LNP-siIrg1 and its control preparation were injected into the tail vein at a dose of 3 mg siRNA / mouse, once every two days. After three rounds of treatment, the tumor size and immune microenvironment were measured ( Figure 3 A). The results showed that the mRNA and ITA content of TAMs Irg1 in the tumor immune microenvironment of mice treated with siIrg1 were effectively suppressed (decreased by about 50%) ( Figure 3 B and 3C). Furthermore, the growth of colon cancer in mice treated with LNP-siIrg1 was significantly suppressed, and the tumor size was significantly reduced ( Figure 3 D-3F).

[0206] Example 3 Screening and Verification of Small Interfering RNA Sequences Targeting Human Macrophage IRG1 for Promoting Anti-tumor Immunity

[0207] Targeting the IRG1 mRNA sequence (NM_001258406.2), 22 small interfering RNA sequences were designed ( Figure 4A Human leukemia-derived monocytes were induced to differentiate into macrophages in vitro, and then the culture supernatant of the MDA-MB-231 human breast cancer cell line was used to activate THP1-differentiated macrophages to induce IRG1 expression. Before the above cell induction, siIRG1 with different sequences in Figure A was transiently transfected using a transfection reagent, and the transcriptional level of IRG1 after activation was detected by RT-PCR ( Figure 4B ), and protein expression levels were detected by protein immunohistochemistry ( Figure 4C ) and verified the knockdown efficiency of siIRG1 with different sequences. The results showed that these siRNAs with different sequences could effectively knock down IRG1 in human macrophages ( Figure 4A -C). Based on the protein knockdown efficiency, the siRNA sequence effects are classified. Protein knockdown efficiency of more than 80% is classified as Class A, more than 70% is classified as Class B, and less than 70% is classified as Class C ( Figure 4A To further verify the function of these siRNAs, sequence 1# (category A) and sequence 2# (category C) were selected for verification. The results showed that in activated human macrophages, knockdown of IRG1 by siRNA of the screening sequence could promote the binding of IRG1 to CD8 in macrophages. + The expression of T cell recruitment-related genes CXCL10 and CXCL11 ( Figure 5 A). And as the efficiency of IRG1 knockdown increases, chemokine expression also increases further. Furthermore, in the in vitro chamber experiment of Jurkat T cells and human macrophages, the intervention of macrophage IRG1 can significantly promote the migration of T cells to the culture medium below ( Figure 5 B) This result suggests that if the above siRNA sequence that effectively targets human IRG1 can be effectively delivered into human TAMs, it may be used for anti-tumor therapy.

[0208] Table 3 siRNA sequences

[0209]

[0210]

[0211] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An siRNA for inhibiting Irg1 gene expression, characterized in that: The nucleotide sequence of the siRNA is shown in any one of SEQ ID NOs: 1-23.

2. The siRNA according to claim 1, wherein The nucleotide sequence of the siRNA is shown in any one of SEQ ID NO: 1, 2, 6, 7, 8, 11, 12, 14, 16, 17, 18, 21, and 22.

3. The siRNA according to claim 1, wherein The efficiency of the siRNA in inhibiting the expression of cell Irg1 protein is ≥80%.

4. The siRNA according to claim 3, wherein The cells are macrophages differentiated from THP1 cells.

5. An siRNA precursor, wherein the 5' to 3' ends thereof have the structure shown in Formula I: Wherein B1 is the sense strand sequence of the siRNA according to claim 1; B2 is a sequence that is substantially complementary or completely complementary to B1, and B2 is not complementary to C; C is a stem-loop structure sequence; A1 and A2 are each independently an RNA sequence consisting of no or optional bases; in, The precursor sequence can be processed in a host to form the siRNA as claimed in claim 1.

6. The siRNA precursor according to claim 5, wherein The siRNA sequence is shown in SEQ ID NO:

1.

7. A polynucleotide encoding the siRNA according to claim 1 or its complementary sequence, or capable of being transcribed by a host to form the siRNA precursor according to claim 5.

8. An expression vector comprising the polynucleotide according to claim 7, or expressing the siRNA precursor according to claim 5.

9. A host cell comprising the expression vector according to claim 8.

10. A pharmaceutical preparation comprising: (a) an active ingredient selected from the group consisting of: The siRNA according to claim 1, the siRNA precursor according to claim 5, the polynucleotide according to claim 7, the expression vector according to claim 8; and (b) a pharmaceutically acceptable carrier.

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