An IL1RN mRNA lipid nanoparticle drug and its application

Abstract

The application relates to the fields of biological medicine and gene therapy, and discloses an IL1RN mRNA lipid nanoparticle drug and application thereof. The drug comprises a lipid nanoparticle and IL1RN mRNA encapsulated in the lipid nanoparticle; the IL1RN mRNA encodes a human IL1RN protein; and the lipid nanoparticle comprises ionizable lipids, phospholipids, cholesterol and pegylated lipids. The application encapsulates IL1RN-201 / 203 mRNA in conventional LNP for the first time, completes preparation characterization and in-vivo expression verification, and proves significant antitumor effect in a high-pressure tail vein transposon-induced in-situ KRAS mutant iCCA mouse model, and partial animals achieve complete tumor regression, thereby filling the blank in the field of IL-1 related inflammatory disease gene therapy and having outstanding originality and broad clinical conversion prospect.

Description

Technical Field

[0001] This application relates to the fields of biomedicine and gene therapy, and more specifically, to an IL1RN mRNA lipid nanoparticle drug and its application. Background Technology

[0002] KRAS is one of the most common mutated oncogenes in human cancers, frequently appearing in pancreatic cancer, colorectal cancer, non-small cell lung cancer, and intrahepatic cholangiocarcinoma (iCCA). KRAS mutations not only drive tumor cell proliferation by continuously activating signaling pathways such as MAPK and PI3K, but more importantly, they reshape the tumor microenvironment, inducing an "inflammatory tumor" phenotype. Recent studies have shown that KRAS mutations promote the production and release of IL-1 (interleukin-1) family cytokines through multiple mechanisms: on the one hand, KRAS mutations upregulate the expression of IL-1α / β in tumor cells; on the other hand, KRAS-driven recruitment and activation of myeloid cells further amplify IL-1β secretion, forming a positive feedback inflammatory loop. The resulting abnormal activation of the IL-1 pathway not only promotes angiogenesis and recruits myeloid-derived suppressor cells (MDSCs), but also inhibits cytotoxic T lymphocyte infiltration, ultimately leading to immune escape and treatment resistance. Therefore, KRAS-mutant tumors are essentially a type of "IL-1-driven inflammation-associated tumor."

[0003] IL-1 is a core mediator of innate immunity and inflammatory responses. IL-1α and IL-1β initiate downstream NF-κB and MAPK signaling by binding to IL-1 receptor 1 (IL-1R1), inducing a series of pro-inflammatory cascade responses. Aberrant activation of the IL-1 pathway has been shown to participate in the pathogenesis of various diseases: in autoimmune diseases such as rheumatoid arthritis, gout, and inflammatory bowel disease, IL-1β is a key driver of inflammation of the synovial membrane and intestinal mucosa; in cardiovascular diseases such as atherosclerosis and myocarditis, IL-1 promotes endothelial activation and plaque instability; and in acute inflammation such as sepsis-associated cytokine storms, IL-1 is a core component of the cytokine storm. Therefore, the IL-1 pathway has been recognized as a common therapeutic target for "inflammation-related diseases."

[0004] IL1RN (IL-1 receptor antagonist) is the only endogenous negative regulator of IL-1 signaling in the human body. It blocks the pro-inflammatory effects of IL-1α / β by competitively binding to IL-1R1. Recombinant IL1RN protein (analexin) has been approved for use in autoimmune diseases such as rheumatoid arthritis, but its clinical application is severely limited: the protein has a half-life of only 4-6 hours, requiring daily subcutaneous injection; systemic exposure is high while local concentration at the lesion site is insufficient, resulting in weak and unsustainable efficacy. For chronic diseases requiring long-term, stable inhibition of the IL-1 pathway, especially solid tumors, recombinant protein therapy falls far short of clinical needs.

[0005] The applicant previously conducted a multi-omics analysis of clinical samples and cell lines from KRAS-mutant iCCA, and for the first time discovered that alternative splicing of IL1RN has a previously unrecognized immunoregulatory function in this tumor subtype. Specifically, IL1RN-201 and IL1RN-203 splice variants exhibit specific expression profiles in KRAS-mutant iCCA and are associated with features such as decreased intratumoral neutrophils and increased CD8+ T cell infiltration. This finding suggests that the expression level of endogenous IL1RN is closely related to the immune status of KRAS-mutant tumors, and IL1RN supplementation may disrupt the KRAS-driven IL-1 inflammatory circuit and reverse immunosuppression.

[0006] Messenger RNA (mRNA) therapy has developed rapidly in recent years, and lipid nanoparticles (LNPs) are currently the most clinically validated in vivo mRNA delivery vector. Conventional intravenous injection of LNPs exhibits natural passive enrichment in the liver—a unique therapeutic advantage for liver-originating iCCA (inflammation-related cancers); for other inflammation-related diseases, the modifiability and targeting strategies of LNPs also offer the possibility of local delivery to the lesion. Encapsulating IL1RN mRNA in LNPs allows for sustained in vivo expression of the IL1RN protein, overcoming the short half-life limitation of recombinant proteins.

[0007] Therefore, if IL1RN mRNA, especially the IL1RN-201 and IL1RN-203 splice variants with immunomodulatory functions, can be efficiently encapsulated in optimized lipid nanoparticles (LNPs) to construct a novel IL1RN mRNA lipid nanoparticle drug, it is expected to achieve long-term and stable expression of IL1RN at the lesion site. Summary of the Invention

[0008] This invention aims to provide an IL1RN-201 / 203 mRNA lipid nanoparticle drug composition. By verifying its in vivo expression efficiency and anti-tumor efficacy in a KRAS mutant iCCA mouse model, its application in the preparation of drugs for treating KRAS mutant iCCA is established. Based on the core driving role of the IL-1 pathway in various inflammation-related diseases, the application scope of this drug composition is further expanded to other IL-1-mediated inflammation-related tumors and inflammatory diseases, providing a novel treatment strategy for a wider range of patients.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] In a first aspect, this application provides a pharmaceutical composition comprising lipid nanoparticles and IL1RN mRNA encapsulated in the lipid nanoparticles; The IL1RN mRNA encodes the human IL1RN protein; The lipid nanoparticles include ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids.

[0011] Furthermore, the coding sequence of the IL1RN mRNA is selected from the nucleotide sequence of IL1RN-201 (ENST00000259206.9) or IL1RN-203 (ENST00000361779.7).

[0012] Furthermore, the lipid nanoparticles have a particle size of 80-100 nm, a polydispersity index ≤0.1, and an mRNA encapsulation efficiency ≥95%.

[0013] Furthermore, the ionizable lipid is SM-102; the phospholipid is DSPC; and the PEGylated lipid is DMG-PEG2000.

[0014] Furthermore, the molar ratio of the ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids is 50:10:38.5:1.5.

[0015] Secondly, this application provides the use of the pharmaceutical composition described herein in the preparation of a medicament for treating IL-1-mediated inflammation-related diseases or conditions.

[0016] Furthermore, the inflammation-related disease or condition is selected from at least one of inflammation-related tumors, autoimmune diseases, or inflammatory diseases.

[0017] Furthermore, the inflammation-related tumors include intrahepatic cholangiocarcinoma, pancreatic cancer, colorectal cancer, non-small cell lung cancer, melanoma, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, and other solid tumors.

[0018] Furthermore, the autoimmune or inflammatory diseases include rheumatoid arthritis, osteoarthritis, gout, inflammatory bowel disease, psoriasis, systemic lupus erythematosus, multiple sclerosis, asthma, chronic obstructive pulmonary disease, atherosclerosis, myocarditis, sepsis, graft-versus-host disease, novel coronavirus infection-related cytokine storm, and other diseases with abnormal activation of the IL-1 pathway.

[0019] Thirdly, this application provides a method for preparing the pharmaceutical composition, comprising the following steps: (1) In vitro transcription to synthesize IL1RN-201 / 203 mRNA; (2) Ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids are dissolved in the organic phase; (3) Dissolve IL1RN-201 / 203 mRNA in the aqueous phase; (4) Rapidly mix the organic phase with the aqueous phase to form lipid nanoparticles; (5) Dialysis to remove organic solvents, ultrafiltration concentration, and sterile filtration.

[0020] This invention is based on the following scientific logic: starting from a clinical challenge (the lack of effective treatments for KRAS-mutant tumors), combining the mechanism (KRAS-driven abnormal activation of the IL-1 pathway), targeting the specific site (IL1RN splice variants), selecting the technology (mRNA-LNP delivery system), conducting validation (in vivo expression, in situ iCCA efficacy), and, based on the universality of the IL-1 pathway, rationally extending to a wider range of inflammation-related diseases. This invention is the first to encapsulate IL1RN-201 / 203 mRNA in a conventional LNP, systematically completing formulation characterization and in vivo expression validation, and demonstrating significant anti-tumor efficacy in a high-pressure tail vein transposon-induced in situ KRAS-mutant iCCA mouse model, with some animals achieving complete tumor regression. This marks the establishment of a complete translational chain from "KRAS-driven inflammation mechanism" to "IL1RN-mRNA therapy," filling a gap in the field of gene therapy for IL-1-related inflammatory diseases, and possessing outstanding originality and broad clinical translational prospects.

[0021] In summary, compared with the prior art, the present invention has the following significant advantages: (1) Based on the theoretical basis of KRAS mutation driving abnormal activation of the IL-1 pathway, IL1RN mRNA therapy was applied for the first time to block the pathway, realizing the transformation from mechanism to treatment; (2) By utilizing the passive enrichment characteristics of the liver after intravenous administration of LNP, mRNA can be effectively delivered and expressed in liver tumors in situ. (3) In vivo, it was confirmed that IL1RN protein was continuously expressed in liver tissue and tumor local area for ≥24 hours, overcoming the short half-life defect of recombinant protein; (4) The significant tumor-suppressing effect was verified by the in situ KRAS mutation iCCA model of high-pressure tail vein, and the data are reliable; (5) Based on the core role of the IL-1 pathway in various inflammation-related diseases, the pharmaceutical composition of the present invention can be widely used in inflammation-related tumors and autoimmune / inflammatory diseases, and has significant platform technology attributes and broad clinical translation prospects. Attached Figure Description

[0022] Figure 1 Agarose gel electrophoresis identification of in vitro transcription products of IL1RN-201 / 203 mRNA.

[0023] Figure 2 Figure: Particle size distribution and encapsulation efficiency of IL1RN-201 / 203 mRNA-LNP.

[0024] Figure 3Agarose gel electrophoresis identification of LNP-IL1RN-201-HA and LNP-IL1RN-203-Flag.

[0025] Figure 4 : Protein expression kinetics analysis of IL1RN-201 / 203 mRNA-LNP drugs in normal liver tissue (A) and tumor tissue (B) of mice; Figure 5 Tumor inhibitory effect of IL1RN-201 / 203 mRNA-LNP on KRAS-mutant iCCA orthotopic mouse model. A: Schematic diagram of experimental grouping and administration regimen. After the orthotopic KRAS-mutant iCCA mouse model was established, mice were randomly divided into several groups and treated with PBS (blank control), empty vector LNP (negative control), or IL1RN-201 / 203 mRNA-LNP, respectively. The administration route was tail vein injection, and the administration was continuous for 2 weeks; B: Representative in vivo bioluminescence imaging images (left) and tumor burden quantitative analysis (right) of mice in each group on day 14 after modeling; C: Representative in vivo bioluminescence imaging images (left) and tumor burden quantitative analysis (right) of mice in each group on day 21 after modeling. Detailed Implementation

[0026] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings. Experimental methods in the following embodiments without specific conditions are performed according to conventional methods and conditions, or according to the product instructions; the materials and reagents used, unless otherwise specified, are all commercially available products.

[0027] Example 1: In vitro synthesis and purification of IL1RN mRNA Using plasmids containing the IL1RN-201 / 203 coding sequence (ENST00000259206.9 / ENST00000361779.7) as templates, the DNA was linearized with restriction endonucleases and then purified. Using the linearized DNA as a template, in vitro transcription was performed using the T7 High Yield RNA Synthesis Kit (Aibotek, RK30220). The reaction system contained ATP, GTP, CTP, and UTP, and the mixture was incubated at 37°C for 2–4 hours. After the reaction, DNase I was added to digest the template DNA, and mRNA was purified by lithium chloride precipitation. The precipitate was washed with 70% ethanol and dissolved in DEPC water. The obtained mRNA was quantified by UV spectrophotometry, and its integrity was assessed by agarose gel electrophoresis. The mRNA was stored at -80°C for later use.

[0028] The results are as follows Figure 1As shown, both IL1RN-201-HA and IL1RN-203-Flag mRNAs exhibited clear, single target bands in the swimming lanes, with sizes consistent with expectations, and no diffusion or tailing, indicating that the synthesized mRNAs were of good integrity and had not undergone degradation.

[0029] Example 2: Preparation and characterization of IL1RN mRNA-LNP Ionizable lipids (SM-102; Avitol, O02010), DSPC (Avitol, S01005), cholesterol (Avitol, O01001), and DMG-PEG2000 (Jiankai, A5435) were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5. IL1RN-201 / 203 mRNA was dissolved in 50 mM citrate buffer (pH 4.0) to a final concentration of 0.2 mg / mL. The resulting LNP suspension was mixed with the ethanol phase at a ratio of 1:3 (v / v) and dialyzed overnight in PBS (pH 7.4) at 4°C. The suspension was then concentrated using an Amicon ultrafiltration tube (MWCO 100 kDa) and sterilized by filtration through a 0.22 μm filter membrane. The particle size and polydispersity index (PDI) of the LNPs were determined using dynamic light scattering (DLS), and the mRNA encapsulation efficiency (EE%) was determined using the RiboGreen method.

[0030] The results are as follows Figure 2 As shown, the LNP-Blank particle size was 91.37±5.799 nm, and the PDI was 0.023±0.009; the LNP-IL1RN-201 mRNA particle size was 87.76±4.225 nm, and the PDI was 0.036±0.030, with an encapsulation efficiency of 99%; the LNP-IL1RN-203 mRNA particle size was 88.25±5.229 nm, and the PDI was 0.023±0.019, with an encapsulation efficiency of 99%. These results indicate that the LNP-mRNA particles prepared in this invention are all within the range of 80-100 nm, with PDIs ≤0.1 and encapsulation efficiencies ≥95%, meeting the requirements for in vivo delivery.

[0031] The results are as follows Figure 3 As shown, clear and single target bands were observed in both LNP-IL1RN-201-HA and LNP-IL1RN-203-Flag lanes, with the size as expected, and no diffusion or degradation tailing, indicating that the LNP-encapsulated mRNA remained intact during the preparation process and did not undergo degradation.

[0032] Example 3: In vivo expression verification To evaluate the in vivo protein expression capacity of IL1RN-201 / 203 mRNA-LNP, validation was performed in wild-type C57 mice and KRAS-mutant iCCA orthotopic tumor-bearing mouse models. Wild-type C57 mice underwent a single tail vein injection of IL1RN-201 / 203 mRNA-LNP (1 mg / kg or 2 mg / kg), with liver tissue collected at 6, 12, and 24 hours post-administration. KRAS-mutant iCCA orthotopic mice underwent a single tail vein injection of IL1RN-201 / 203 mRNA-LNP (2 mg / kg), with tumor tissue collected 24 hours post-administration. IL1RN protein expression levels were detected using Western blot.

[0033] The results are as follows Figure 4 As shown, IL1RN protein levels significantly increased in normal liver tissue 6 hours after administration and remained elevated for more than 24 hours; definite IL1RN protein expression was also detected in tumor tissue 24 hours after administration. This indicates that the LNP-mRNA described in this invention can achieve efficient and sustained IL1RN protein expression in liver and tumor tissues in vivo.

[0034] Example 4: Construction of an in situ KRAS mutant iCCA mouse model Using the Sleeping Beauty transposon system: 15μg pT3-EF1a-YAP S127A , 10 μg pT3-EF1α-HA-myr-Akt, 2.5 μg pT3-EF1a-KRAS G12D Mix with 2.75 μg SB13-Luciferase plasmid, dissolve in 2 mL of physiological saline, and rapidly inject via the tail vein of mice within 6-8 seconds (high-pressure tail vein method).

[0035] Example 5: In vivo efficacy evaluation A KRAS mutant iCCA orthotopic mouse model was constructed using the method described in Example 4. On day 7 post-modeling, tumor-bearing mice were randomly divided into 5 groups (n=5 per group): PBS control group, empty LNP control group (encapsulated control mRNA), IL1RN-201 mRNA-LNP treatment group, IL1RN-203 mRNA-LNP treatment group, and IL1RN-201+203 mRNA-LNP treatment group. All groups were subsequently treated via tail vein injection for 2 weeks. In vivo bioluminescence imaging was performed weekly after administration, and tumor burden was quantified using the average fluorescence value of the region of interest (ROI) (p / sec / cm² / sr).

[0036] The results are as follows Figure 5As shown, on day 14 after modeling, in vivo imaging revealed that the tumor burden in mice in the IL1RN-201 mRNA-LNP group, IL1RN-203 mRNA-LNP group, and IL1RN-201+203 mRNA-LNP group was significantly lower than that in the empty LNP control group, indicating that the tumor-suppressing effect was already evident in the early stages of treatment. On day 21 after modeling, the tumor-suppressing effect of each treatment group was further enhanced, and the tumor burden remained lower than that of the control group, with the difference further widening.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention. Based on the core technical principle disclosed in the present invention (KRAS mutation drives inflammation through the IL-1 pathway, and IL1RN can antagonize this pathway), those skilled in the art can reasonably foresee the application of the pharmaceutical composition described in the present invention in a wider range of IL-1-mediated inflammation-related diseases, and these applications all fall within the protection scope of the present invention.

[0038] sequence: IL1RN-201 plasmid (SEQ ID NO:1):

[0039] IL1RN-203 plasmid (SEQ ID NO:2):

Claims

1. A pharmaceutical composition, characterized in that, Contains lipid nanoparticles and IL1RN mRNA encapsulated within the lipid nanoparticles; The IL1RN mRNA encodes the human IL1RN protein; The lipid nanoparticles include ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids.

2. The pharmaceutical composition according to claim 1, characterized in that, The coding sequence of the IL1RN mRNA is selected from the nucleotide sequence of IL1RN-201 (ENST00000259206.9) or IL1RN-203 (ENST00000361779.7).

3. The pharmaceutical composition according to claim 1, characterized in that, The lipid nanoparticles have a particle size of 80-100 nm, a polydispersity index ≤0.1, and an mRNA encapsulation efficiency ≥95%.

4. The pharmaceutical composition according to claim 1, characterized in that, The ionizable lipid is SM-102; the phospholipid is DSPC; and the PEGylated lipid is DMG-PEG2000.

5. The pharmaceutical composition according to claim 1, characterized in that, The molar ratio of the ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids is 50:10:38.5:1.

5.

6. Use of the pharmaceutical composition according to any one of claims 1-5 in the preparation of a medicament for treating IL-1-mediated inflammation-related diseases or conditions.

7. The application according to claim 6, characterized in that, The inflammation-related disease or condition is selected from at least one of inflammation-related tumors, autoimmune diseases, or inflammatory diseases.

8. The application according to claim 7, characterized in that, The inflammation-related tumors include intrahepatic cholangiocarcinoma, pancreatic cancer, colorectal cancer, non-small cell lung cancer, melanoma, breast cancer, ovarian cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, liver cancer, and other solid tumors.

9. The application according to claim 7, characterized in that, The autoimmune or inflammatory diseases mentioned include rheumatoid arthritis, osteoarthritis, gout, inflammatory bowel disease, psoriasis, systemic lupus erythematosus, multiple sclerosis, asthma, chronic obstructive pulmonary disease, atherosclerosis, myocarditis, sepsis, graft-versus-host disease, novel coronavirus infection-related cytokine storm, and other diseases with abnormal activation of the IL-1 pathway.

10. A method for preparing the pharmaceutical composition according to any one of claims 1-5, characterized in that, Includes the following steps: (1) In vitro transcription to synthesize IL1RN-201 / 203 mRNA; (2) Ionizable lipids, phospholipids, cholesterol, and polyethylene glycol-modified lipids are dissolved in the organic phase; (3) Dissolve IL1RN-201 / 203 mRNA in the aqueous phase; (4) Rapidly mix the organic phase with the aqueous phase to form lipid nanoparticles; (5) Dialysis to remove organic solvents, ultrafiltration concentration, and sterile filtration.

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