An engineered macrophage for regulating uric acid and inflammation, and a preparation method and application thereof

Abstract

The present application relates to a kind of engineering macrophage for regulating uric acid and inflammation and its preparation method and application, belong to the field of biological medicine, solve the scheme of " uric acid degradation " and " myeloid inflammation inhibition " two kinds of treatment functions in prior art cannot be integrated simultaneously, MSU crystalline deposit cannot be removed from root, block gout repeated attack, need long-term administration, easily induce the problem of drug resistance antibody and infusion related adverse reactions.The genome of the engineering macrophage provided by the present application contains the coding gene of exogenous uricase UOX And the coding gene of exogenous interleukin-1 receptor antagonist IL-1Ra The engineering macrophage provided by the present application can tolerate high uric acid environment, can reduce MSU crystallization formation or continuous stimulation, block IL-1 / NF-κB inflammation amplification path, to realize sustained, synergistic treatment effect in gout inflammation local.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an engineered macrophage that regulates uric acid and inflammation, its preparation method, and its application. Background Technology

[0002] Gout is a metabolic-inflammatory disease driven by persistent hyperuricemia and the deposition of monosodium urate (MSU) crystals. Elevated serum uric acid levels (hyperuricemia) are the core risk factor for gout; however, not all individuals with hyperuricemia develop acute gout, suggesting that gout is not solely caused by elevated uric acid levels but is also influenced by other regulatory conditions. Recent studies have shown that MSU crystals can activate NF-κB preactivation and the NLRP3 inflammasome pathway in monocytes / macrophages, promoting the release of inflammatory mediators such as IL-1β, TNF-α, and CXCL8, and inducing neutrophil recruitment, thereby triggering an acute synovial inflammatory response. This pathological effect, along with hyperuricemia, exerts a synergistic pro-inflammatory effect, jointly driving acute gout attacks.

[0003] Currently, clinical treatment mainly includes two strategies: one is uric acid-lowering therapy, such as xanthine oxidase inhibitors, uricosuric drugs, and uricase replacement therapy; the other is acute inflammation control therapy, such as nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine, glucocorticoids, and IL-1 pathway inhibitors. However, for uric acid-lowering treatments, conventional uric acid-lowering drugs lack the ability to directly, rapidly, and locally regulate existing urate deposits or the amplification of acute inflammation. Uricase (UOX) can oxidize uric acid into allantoin, which is more soluble and excreted, and is an important way to rapidly reduce uric acid load; humans lost functional uricase during evolution and need to supplement exogenous uricase. However, existing exogenous recombinant uricase preparations still have drawbacks such as limited half-life, the need for repeated dosing, and the potential to induce anti-drug antibodies and infusion-related adverse reactions. For acute inflammation control therapy, inflammatory factor inhibitors or anti-inflammatory factors are often used to control inflammation in patients with acute gout attacks. However, this type of method still has some problems. For example, IL-1 pathway blocking mainly targets the inflammation amplification process and cannot directly reduce uric acid load or reduce the source of MSU crystals. It can only relieve inflammatory symptoms in the short term and cannot eradicate the root cause of the disease. In addition, existing technologies also have the problem of high dosing frequency and the need for multiple administrations.

[0004] Therefore, there is an urgent need for a treatment that can simultaneously achieve uric acid degradation and myeloid inflammation suppression, integrating both "uric acid degradation" and "myeloid inflammation suppression" therapeutic functions, thereby eliminating MSU crystal deposits from the root cause, blocking recurrent gout attacks, and achieving a therapeutic agent that does not require long-term high-frequency administration and is less likely to induce anti-drug antibodies and infusion-related adverse reactions. Summary of the Invention

[0005] Based on the above analysis, the embodiments of the present invention aim to provide an engineered macrophage that regulates uric acid and inflammation, as well as its preparation method and application, to solve the problem that the existing technology cannot simultaneously integrate the two therapeutic functions of "uric acid degradation" and "myeloid inflammation suppression", thereby eliminating the problems of MSU crystal deposition, blocking recurrent gout attacks, requiring long-term high-frequency drug administration, and easily inducing anti-drug antibodies and infusion-related adverse reactions from the root cause.

[0006] In a first aspect, the present invention provides an engineered macrophage that regulates uric acid and inflammation, wherein the genome of the engineered macrophage contains a gene encoding an exogenous uricase. UOX Genes encoding exogenous interleukin-1 receptor antagonists IL-1Ra .

[0007] Specifically, the gene encoding uricase. UOX The nucleotide sequence is shown in SEQ ID NO:3.

[0008] Specifically, the gene encoding interleukin-1 receptor antagonists IL-1Ra The nucleotide sequence is shown in SEQ ID NO:4.

[0009] Specifically, engineered macrophages are obtained from macrophage precursor cells through directed induction differentiation and gene recombination. Preferably, the macrophage precursor cells are derived from at least one of bone marrow, peripheral blood, spleen, and stem cells.

[0010] In a second aspect, the present invention provides a method for preparing the engineered macrophages described in the first aspect, the method comprising: introducing an exogenous gene encoding uricase into the macrophages. UOX Genes encoding exogenous interleukin-1 receptor antagonists IL-1Ra .

[0011] Specifically, the method for preparing macrophages includes stimulating macrophage precursor cells with stimulating factors.

[0012] Specifically, macrophage precursor cells are derived from at least one of the following: bone marrow, peripheral blood, spleen, and stem cells.

[0013] Specifically, the stimulating factor is selected from at least one of M-CSF, GM-CSF, PMA and interleukin, preferably M-CSF.

[0014] Specifically, methods for introducing exogenous genes into macrophages include: viral vector-mediated transduction, transposon systems, CRISPR / Cas systems, and any one of base editing.

[0015] Thirdly, the present invention provides the use of engineered macrophages of the first aspect in the preparation of medicaments for the prevention and / or treatment of gout.

[0016] Fourthly, the present invention provides the application of engineered macrophages of the first aspect in reducing uric acid levels and / or reducing inflammatory responses in vitro.

[0017] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects: 1. The engineered macrophages provided by this invention reduce uric acid load and decrease MSU crystal formation or continuous stimulation by stably expressing uricase in gout lesions; block IL-1 / NF-κB inflammation amplification by stably expressing IL-1Ra; and achieve a continuous and synergistic therapeutic effect in the local gout inflammation, which can promote inflammation resolution, reduce neutrophil recruitment and synovial tissue damage, and reduce the risk of recurrence after acute gout attacks. 2. The gene encoding uricase in engineered macrophages provided by this invention. UOX It can express proteins with uric acid degradation activity, promote uric acid degradation, thereby reducing uric acid concentration and inhibiting the formation of monosodium urate crystals; 3. The engineered macrophages provided by this invention continuously express and secrete the interleukin-1 receptor antagonist IL-1Ra, which can competitively bind to the IL-1 receptor on the cell membrane surface, effectively blocking the binding of IL-1β to the receptor and the subsequent transduction of inflammatory signals, thereby inhibiting the continuous activation of the NF-κB pathway and gradually reducing NF-κB inflammatory activity. This negative feedback regulatory loop can timely counterbalance the over-activated inflammatory response, effectively preventing the inflammatory response from being continuously amplified and out of control, and maintaining the body's inflammatory immune homeostasis. 4. The engineered macrophages provided by this invention retain the inherent microenvironmental adaptability and chemotactic properties of macrophages. They can adapt to the special microenvironment of high MSU crystal load, inflammatory factor enrichment and enhanced oxidative stress in gout lesions. Furthermore, they can further reduce uric acid load by contacting, surrounding or phagocytosing MSU crystals in this microenvironment through a continuous chain of action of phagocytosis of crystals-local degradation of uric acid-blocking of IL-1 inflammation amplification.

[0018] In this invention, the above-described technical solutions can be combined with each other to achieve more preferred combinations. Other features and advantages of this invention will be set forth in the following description, and some advantages may become apparent from the description or be learned by practicing the invention. The objects and other advantages of this invention can be realized and obtained from what is particularly pointed out in the description and drawings. Attached Figure Description

[0019] The accompanying drawings are for illustrative purposes only and are not intended to limit the scope of the invention. Figure 1 This is a schematic diagram illustrating the entire process of constructing, characterizing, and validating engineered macrophages in this embodiment of the invention.

[0020] Figure 2 Results of public single-cell transcriptome data mining and analysis of monocytes associated with acute gout inflammation.

[0021] Figure 3 Results for the F4 / 80 surface marker of differentiated and undifferentiated macrophages.

[0022] Figure 4 The results show the GFP fluorescence of BMDM-mi.

[0023] Figure 5 The results of GFP fluorescence analysis for BMDM-mi and iBMDM-0 are shown.

[0024] Figure 6 The results show the FITC-MSU and mCherry fluorescence of BMDM-mic.

[0025] Figure 7 Cell counts and uric acid reduction results for the BMDM-mi and BMDM-0 groups.

[0026] Figure 8 The results show the changes in IL-1Ra content over time for the BMDM-mi and BMDM-0 groups.

[0027] Figure 9 The expression of the biomarkers CD86, CD206, iNOS and Arg1 in each group of mice was shown.

[0028] Figure 10 The results show the changes in swelling rate over time for each group of mice.

[0029] Figure 11 The images show knee swelling in mice from each group 6 hours after injection.

[0030] Figure 12 The results show the uric acid concentrations in each group of mice.

[0031] Figure 13 The images show the HE staining results of joint tissues from each group of mice and a schematic diagram of the expression of TNF-α and Ly6G in the synovial tissue.

[0032] Figure 14 This is a schematic diagram showing the total radiation efficiency of the liver, inflamed joints, and healthy joint areas of each group of live mice.

[0033] Figure 15 This is a schematic diagram showing the total radiation efficiency of isolated livers, inflamed joints, and healthy joint regions of mice in each group. Detailed Implementation

[0034] Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which form part of this application and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not intended to limit the scope of the present invention.

[0035] During an acute gout attack, classic / intermediate monocytes in PBMCs exhibit systemic inflammatory characteristics. NLRP3 , IL1B , TNF , PTGS2 The expression of genes and related regulatory networks is enhanced in monocytes. The inventors of this invention conducted quality control, integration, cell annotation, differential expression, pathway enrichment, and module scoring analysis based on gout patients, finding enhanced NF-κB signaling, inflammasome pathways, cytokine signaling, chemokine responses, and oxidative stress-related pathways in peripheral blood monocytes during acute attacks. Simultaneously, transcriptional features related to uric acid / purine metabolism and transport were coupled with inflammatory modules, suggesting a pre-activated state of uric acid-sensitive inflammatory monocytes in acute gout. Furthermore, studies have shown the presence of myeloid / macrophage compartments more closely resembling local joint inflammation in the synovial fluid of acute gout patients, and inflammatory communication features such as IL-1 and TNF can be observed. In other words, acute gout attacks are not simply due to elevated serum uric acid, but rather a uric acid-sensitive innate immune activation process centered on monocytes / macrophages.

[0036] From a disease mechanism perspective, although combining the uric acid-lowering effect mediated by uricase with the anti-inflammatory effect mediated by IL-1 pathway blockade seems to be able to simultaneously intervene in the uric acid load and inflammatory amplification process of gout, there are still multiple technical obstacles in the actual construction of the treatment system. It is not simply a matter of constructing a system that expresses both simultaneously to achieve the combined effect: First, uricase, as an exogenous enzyme protein, is prone to inducing anti-drug antibodies and infusion-related reactions when continuously exposed or repeatedly administered in vivo. Meanwhile, inflammatory blocking molecules such as IL-1Ra require sufficient local concentrations around MSU crystal deposition, synovial inflammation, or tophi. Conventional systemic administration makes it difficult to achieve simultaneous enrichment and sustained action of both in the same lesion area. Second, uricase mainly acts on the uric acid metabolism process, while IL-1Ra mainly acts on the IL-1β-mediated inflammatory amplification process. Their target sites, effective time windows, tissue distribution, and pharmacokinetic characteristics are all different. Simple combined administration cannot guarantee the spatiotemporal synergy of the two in the urate crystal-induced inflammatory microenvironment. Third, if the uricase gene and... IL-1RaIntegrating genes into the same delivery system simultaneously requires addressing challenges such as bifunctional gene expression cassette design, expression ratio control, maintenance of uricase activity, effective secretion of IL-1Ra, maintenance of engineered cell activity, functional stability under inflammatory stimulation, and in vivo safety. Specifically, excessive uricase expression may cause local metabolic stress or oxidative stress, while insufficient IL-1Ra expression makes it difficult to effectively block the IL-1β-mediated inflammatory cascade. Figure 1 As shown, the inventors of this invention, through extensive cell construction, gene optimization, and functional verification studies, optimized the synergistic expression ratio of dual genes and cell modification processes, and finally successfully constructed an engineered macrophage delivery system. The secretory uricase expression module and the IL-1Ra expression module are integrated into the same lentiviral expression vector, enabling engineered macrophages to maintain their ability to induce inflammation, phagocytose or accumulate MSU crystals at the deposition site, while simultaneously exerting uric acid degradation and IL-1 signal blocking effects at the lesion site. This achieves synergistic intervention on the continuous pathological process of "increased uric acid load - MSU crystal deposition - NLRP3 inflammasome activation - IL-1β inflammation amplification," solving problems such as half-life mismatch, weak tissue tropism, insufficient local sustained effect, immunogenicity risk, and burden of repeated administration that exist when free uricase and free anti-inflammatory factors are simply combined.

[0037] Based on the above findings, in a first aspect, the present invention provides an engineered macrophage that regulates uric acid and inflammation, wherein the genome of the engineered macrophage contains a gene encoding an exogenous uricase. UOX Genes encoding exogenous interleukin-1 receptor antagonists IL-1Ra .

[0038] In this invention, italic characters represent gene names, and regular characters represent the proteins encoded by those genes. For example, characters represented in italic form... IL-1Ra The gene encoding an interleukin-1 receptor antagonist is IL-1Ra, which is represented in regular font as an interleukin-1 receptor antagonist.

[0039] Small molecule uric acid-lowering drugs such as allopurinol and febuxostat mainly rely on systemic circulation and sustained inhibition of uric acid production. They can exert their effects regardless of the location of inflammation, resulting in low specificity. Moreover, allopurinol xanthine oxidase inhibitors mainly lower blood uric acid levels by inhibiting uric acid production, but they lack direct clearance and local anti-inflammatory effects on existing MSU crystal deposits, the local microenvironment of tophi, and the amplification process of acute inflammation. At the same time, some patients may not achieve ideal therapeutic effects due to abnormal renal function, drug intolerance, allergic reactions, poor medication adherence, or insufficient control of inflammation during acute attacks.

[0040] This invention integrates the control of uric acid metabolism at its source and the regulation of myeloid inflammation into a single cell carrier, resulting in an engineered macrophage with dual therapeutic functions. This engineered macrophage can chemotactically attract and reside in urate crystal deposition areas or synovial inflammation areas to achieve local enrichment. It reduces uric acid load, decreases MSU crystal formation, or inhibits continuous stimulation by expressing uricase. Furthermore, it blocks the IL-1 / NF-κB inflammatory amplification pathway by expressing the anti-inflammatory factor IL-1Ra, achieving a sustained and synergistic therapeutic effect at the site of gout inflammation. The engineered macrophages provided by this invention have good biocompatibility, are non-toxic to the body, do not irritate or damage the inflamed site, and can be used for extended periods and multiple times without significant cytotoxicity or immune rejection risks. While achieving similar or comparable effects in reducing serum uric acid, alleviating joint swelling, decreasing inflammatory factors, or improving histopathology, the engineered macrophages provided by this invention can reduce systemic drug dosage, frequency of administration, and systemic drug exposure, solving the problems of systemic toxicity and immune sensitization caused by repeated administration of free drugs. Therefore, the engineered macrophages provided by this invention are particularly suitable for patients or model scenarios where conventional uric acid-lowering drugs are ineffective, recurrent acute attacks occur, tophi burden is high, inflammatory response is obvious, or local continuous anti-inflammatory treatment is required.

[0041] Conventional natural macrophages can recognize sodium urate monohydrate crystals and sense the local inflammatory microenvironment, possessing basic chemotactic migration, foreign body phagocytosis, lesion retention, and inflammatory factor regulation capabilities. The engineered macrophages constructed in this invention fully retain the inherent chemotactic properties and microenvironmental adaptability of macrophages. They can actively migrate and accumulate towards arthritic lesions under the induction of inflammatory signals; simultaneously, in response to local inflammatory stimulation, they internalize uric acid while specifically secreting therapeutic-level functional anti-inflammatory factors, effectively alleviating local inflammatory infiltration and reducing the severity of joint lesions.

[0042] The gene encoding uricase in engineered macrophages provided by this invention UOX The protein can continuously express and secrete uric acid-degrading proteins, catalyzing the conversion of uric acid to allantoin, thereby reducing uric acid concentration and inhibiting the formation of monosodium urate crystals. Furthermore, the uricase secreted by the engineered macrophages of this invention has a longer in vivo half-life than that of free-administered uricase, which can prolong the duration of enzyme activity in vivo, thereby reducing the frequency and dosage of administration.

[0043] When excessive uric acid levels in the body lead to an inflammatory response, the expression level of the pro-inflammatory factor IL-1β in the body significantly increases. This activates the nuclear factor NF-κB inflammatory signaling pathway, causing NF-κB to remain continuously activated. This, in turn, induces synovial cells and macrophages to secrete large amounts of pro-inflammatory mediators, amplifying local inflammatory infiltration, tissue edema, and bone damage, leading to pathological damage such as joint redness, swelling, and pain, forming an adverse inflammatory cascade. The engineered macrophages provided by this invention can express the interleukin-1 receptor antagonist IL-1Ra in the inflammatory region, competitively binding to the IL-1 receptor on the cell membrane surface. This effectively blocks the binding of IL-1β to the receptor and the subsequent transduction of inflammatory signals, thereby inhibiting the continuous activation of the NF-κB pathway. This gradually reduces NF-κB inflammatory activity and downregulates the levels of pro-inflammatory cytokines such as TNF-α and IL-6, as well as neutrophil chemokines such as CXCL1 and CXCL8, in the lesion area. It also reduces the recruitment and infiltration of Ly6G-positive neutrophils to the inflammatory site, further alleviating acute joint inflammation induced by MSU crystals. This negative feedback regulatory loop can promptly counterbalance the over-activated inflammatory response, effectively preventing the inflammatory response from continuing to amplify and become uncontrollable, and maintaining the body's inflammatory immune homeostasis.

[0044] It is understandable that uricase amino acid sequences, protein structures, and catalytic activities vary from species to species. The catalytic efficiency and substrate compatibility of uricase also differ for different organisms. Those skilled in the art can adaptively screen, modify, or replace homologous uricase sequences according to actual experimental needs, application scenarios, and host characteristics. Uricase variants modified with low immunogenicity, humanization, half-life optimization, or secretion optimization can be used.

[0045] Similarly, IL-1Ra from different species may differ in their coding gene sequence, amino acid composition, protein conformation, signal peptide sequence, secretion efficiency, and splicing / translation isoforms, leading to variations in IL-1 receptor binding capacity, competitive antagonistic efficacy, protein stability, tissue half-life, and in vivo anti-inflammatory activity. IL-1Ra primarily inhibits IL-1-dependent inflammatory amplification by competitively binding to the IL-1 receptor, blocking the activation of downstream inflammatory signaling pathways such as MyD88-IRAK-TRAF6-NF-κB / MAPK mediated by IL-1α or IL-1β binding to their receptors. Those skilled in the art can adaptively select, optimize, or homologously replace the coding sequence of IL-1Ra from corresponding species based on the model species, cell source, expression vector, secretion requirements, and therapeutic application scenarios, or use coding sequences of functional variants of IL-1Ra that have undergone secretion enhancement, stability optimization, low immunogenicity modification, half-life optimization, or receptor binding capacity optimization.

[0046] Preferably, the amino acid sequence of the uricase is shown in SEQ ID NO:1.

[0047] Preferably, the amino acid sequence of the interleukin-1 receptor antagonist is shown in SEQ ID NO:2.

[0048] Specifically, the gene encoding uricase. UOX The nucleotide sequence is shown in SEQ ID NO:3.

[0049] Specifically, the gene encoding interleukin-1 receptor antagonists IL-1Ra The nucleotide sequence is shown in SEQ ID NO:4.

[0050] MAHYHDNYGKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLRSKKDYLHGDNSDIIPTDTIKNTVHVLAKLRGIRNIETFAMNICEHFLSSFNHVTRAHVYVEEVPWKRFEKNGIKHVHAFIHTPTGTHFCEVEQMRNGPPVI HSGIKDLKVLKTTQSGFEGFLKDQFTTLPEVKDRCFATQVYCKWRYQRRDVDFEAIWGAVRDIVLQKFAGPYDKGEYSPSVQKTLYDIQVLSLSQLPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLPSRL (SEQ ID NO:1) MEICWGPYSHLISLLLILLFHSEAACRPSGKRPCKMQAFRIWDTNQKTFYLRNNQLIAGYLQGPNIKLEEKIDMVPIDLHSVFLGIHGGKLCLSCAKSGDDIKLQLEEVNITDLSKNKEEDKRFTFIRSEKGPTTSFESAACPGWFLCTTLEADRPVSLTNTPEEPLIVTKFYFQEDQ (SEQ ID NO: 2) ATGGCCCATTACCATGACAACTACGGAAAGAATGATGAAGTGGAATTTGTCCGAACTGGCTATGGGAAAGACATGGTCAAAGTTCTCCATATTCAGAGAGATGGAAAATACCACAGCATCAAAGAGGTGGCAACTTCGGTCCAGTTGACTCTGAGGTCCAAGAAGGATTACCTACACGGTGATAATTCCGACATCATCCCTACAGACACCATCAAGAACACTGTGCACGTCCTGGCGAAGCTCAGAGGGATCAGAAACATCGAGACCTTTGCAATGAACATCTGTGAGCACTTCCTCTCTTCTTTTAACCATGTCACTCGAGCCCACGTCTACGTGGAAGAGGTCCCCTGGAAACGATTTGAAAAGAATGGGATCAAACACGTCCATGCATTCATCCACACCCCGACGGGAACGCACTTCTGTGAGGTGGAGCAGATGAGAAACGGACCTCCCGTCATTCACTCTGGAATCAAAGACCTCAAGGTCTTGAAAACAACCCAGTCTGGGTTTGAAGGGTTCCTCAAGGACCAGTTCACTACCCTCCCTGAGGTGAAGGACCGATGCTTTGCCACTCAAGTGTACTGCAAGTGGCGCTACCAGAGACGGGACGTGGACTTCGAGGCTATCTGGGGCGCTGTCCGGGACATTGTCCTGCAGAAATTTGCTGGGCCCTATGACAAAGGTGAATACTCACCTTCCGTGCAGAAGACCCTCTATGATATACAAGTGCTGTCCCTGAGCCAGCTTCCTGAGATAGAAGACATGGAAATCAGCCTTCCGAACATTCACTACTTTAACATCGACATGTCCAAAATGGGACTGATCAACAAGGAAGAGGTTTTGCTGCCTCTCGACAATCCCTACGGCAAAATAACAGGGACAGTCAAGAGGAAGCTGCCTTCCAGGCTG(SEQ ID NO:3) (SEQ ID NO:4).

[0051] It is understood that, due to codon degeneracy, the same amino acid sequence can be encoded by a variety of different nucleotide sequences. Therefore, the encoding genes of uricase and interleukin-1 receptor antagonists involved in this invention not only include the nucleotide sequences shown in SEQ ID NO: 3-4, but may also include any synonymous nucleotide variants arising from genetic code degeneracy.

[0052] The CCK-8 cell viability assay results showed that the cell activity of engineered macrophages was not significantly different from that of macrophages without the introduced exogenous gene, suggesting that the introduced exogenous gene would not have a significant adverse effect on the survival and proliferation of macrophages. Furthermore, the engineered macrophages provided by this invention, by introducing the genes shown in SEQ ID NO: 3-4, not only gained the ability to secrete uricase and IL-1Ra, but also unexpectedly enhanced their tolerance to high uric acid. Even in hyperuricemic lesions, they could still survive stably, proliferate continuously, and their cell number would not decrease with the uric acid degradation process. On the contrary, they could expand their cell count while performing uric acid-lowering functions, thereby enriching more engineered macrophages with uric acid decomposition functions at the inflammatory site, thus achieving a long-term therapeutic effect and obtaining technical advantages that are difficult to achieve with direct drug administration.

[0053] To promote the secretion of uricase into the extracellular microenvironment and thus exert a therapeutic effect, in some specific embodiments of the present invention, the engineered macrophages further include a gene encoding an exogenous mGM-CSF signal peptide. mGM-CSF Preferably, the amino acid sequence of the mGM-CSF signal peptide is shown in SEQ ID NO: 5, and the nucleotide sequence encoding the signal peptide is shown in SEQ ID NO: 6; this signal peptide can guide downstream uricase into the endoplasmic reticulum-Golgi apparatus secretion pathway, achieving efficient release of uricase into the extracellular space. In some specific embodiments, mGM-CSF Directly connected to UOX Upstream of.

[0054] MWLQNLLFLGIVVYSLS (SEQ ID NO:5) ATGTGGCTGCAGAATTTACTTTTCCTGGGCATTGTGGTCTACAGCCTCTCA (SEQ ID NO: 6) Specifically, in order to ensure cell proliferation activity, inflammatory chemotaxis and in vivo colonization stability, while improving the efficiency of engineering modification and the stability of anti-inflammatory function, the engineered macrophages can be obtained from macrophage precursor cells through directed induction differentiation and gene recombination.

[0055] According to some preferred embodiments of the present invention, the macrophage precursor cells may be derived from at least one of bone marrow, peripheral blood, spleen, and stem cells. The stem cells include at least one of hematopoietic stem cells, pluripotent stem cells, and induced pluripotent stem cells (iPSCs), and preferably, the macrophage precursor cells are derived from bone marrow.

[0056] According to some specific embodiments of the present invention, the gene recombination method includes, but is not limited to, any one of: viral vector-mediated transduction, transposon system, CRISPR / Cas system, and base editing.

[0057] Understandably, to avoid immune rejection caused by xenogeneic proteins or cells, reduce the risk of immune clearance after cell reinfusion, and improve the survival time and functional sustainability of cell preparations in the human body, UOX , IL-1Ra Both the engineered macrophages and the human macrophages can be derived from human sequences or human cells. Specifically, in human applications, the engineered macrophages are preferably human-derived or preferably designed with human characteristics. For example, macrophages obtained by directed differentiation of patient-derived peripheral blood mononuclear cells, healthy donor-derived peripheral blood mononuclear cells, artificial hematopoietic stem cells, or human induced pluripotent stem cells (hiPSCs) can be used as starting cells, and humanized UOX variants, human IL-1Ra coding sequences, or functionally equivalent sequences optimized for low immunogenicity can be introduced.

[0058] In applications involving allogeneic or universal cells, further gene regulation, immune checkpoint molecule expression regulation, introduction of safety switch genes, or low-immunogenicity engineering modifications can reduce the risk of host immune recognition and clearance. Therefore, this invention is not only applicable to animal models such as mice for validation, but can also be extended to humanized macrophages, hiPSC-derived macrophages, or universal engineered macrophage preparations for the human treatment of gout, hyperuricemia, tophi, and chronic inflammatory diseases related to urate crystals.

[0059] The present invention further provides a pharmaceutical composition comprising the engineered macrophages and / or cultures of engineered macrophages as described in the first aspect.

[0060] The results of the in vivo distribution experiment in mice showed that: (1) After intravenous administration, engineered macrophages were mainly enriched in organs related to the mononuclear-phagocytic system, such as the liver, lungs, and spleen; and the cell signal intensity in the inflamed joints was higher than that in the contralateral non-inflammatory joints. As the gout inflammation gradually subsided, the signal level of engineered macrophages in various tissues gradually decreased, and there was no long-term abnormal retention phenomenon; (2) After treatment with these engineered macrophages, neutrophil recruitment, inflammasome activation, and IL-1-mediated inflammatory cascade amplification effects in the joints were significantly inhibited. The results of RNA sequencing of synovial tissue, combined with qRT-PCR, Western blot, and immunohistochemical detection, further confirmed that the inflammasome pathway, NF-κB signaling pathway, chemokine signaling pathway, and transcriptional programs related to neutrophil recruitment were all significantly downregulated; (3) Moreover, the engineered macrophages provided by this invention reduced blood uric acid load in a dose-related manner, and at the same time alleviated synovial hyperplasia, inflammatory cell infiltration, and joint tissue damage.

[0061] Therefore, the engineered macrophages provided by this invention can simultaneously achieve the dual effects of lowering uric acid and anti-inflammatory repair of joints in a dose-dependent manner, effectively improving hyperuricemia and gouty joint pathological damage induced by hyperuricemia. The therapeutic effect is clear and controllable, and it has excellent application value and clinical translation prospects for the treatment of gout.

[0062] Furthermore, by monitoring changes in mouse body weight, general survival status, routine blood indicators, serum ALT, AST, BUN, and creatinine levels, and conducting histopathological examinations of the heart, liver, spleen, lungs, and kidneys, as well as the results of H2O2 and reactive oxygen species (ROS) generated by uricase catalysis, and oxidative stress-related markers such as Nrf2, Hmox1, Nqo1, Cat, and Sod2, it was demonstrated that within the dosage range that can produce an effective uric acid-lowering effect, the engineered macrophages provided by this invention do not induce significant liver and kidney dysfunction or damage to major organs, and the oxidative stress level caused by uricase expression is within a controllable range, without causing persistent tissue oxidative damage.

[0063] According to some specific embodiments of the present invention, the culture of engineered macrophages may include the supernatant obtained from culturing engineered macrophages. The supernatant can be prepared using methods conventional in the art, such as collecting the culture medium of the cell culture system after culturing the cells, removing the solid cell components, and then sterilizing it. The culture supernatant of engineered macrophages is rich in uricase and IL-1RA protein, and can be used alone for in vitro intervention or in vivo administration of hyperuricemia and joint inflammation.

[0064] In some embodiments, the pharmaceutical composition includes pharmaceutically acceptable excipients. These excipients include excipients, stabilizers, injection solvents, solubilizers, acid-base adjusters, lyophilization protectants, antioxidants, etc.

[0065] In some embodiments, the pharmaceutical composition can be prepared into common dosage forms, such as injections, granules, tablets, capsules, aerosols, etc. In some preferred embodiments, the pharmaceutical composition is administered by local injection, intravenous injection, or intravenous infusion. Administration techniques well known to those skilled in the art can also be used. According to some specific embodiments of the present invention, the injection delivery method can further enhance the enrichment capacity and tissue specificity of the aforementioned engineered macrophages at the gout site, further effectively overcoming the shortcomings of poor targeting and insufficient specificity in existing technologies.

[0066] In a second aspect, the present invention provides a method for preparing the engineered macrophages described in the first aspect, the method comprising: introducing an exogenous gene encoding uricase into the macrophages. UOX Genes encoding exogenous interleukin-1 receptor antagonists IL-1Ra .

[0067] Among them, the uricase encoding gene UOX Genes encoding interleukin-1 receptor antagonists IL-1Ra The definitions, sequences, and functions are consistent with those described in the first aspect of this invention.

[0068] Specifically, to obtain macrophages with stable activity, excellent chemotactic ability, and high transduction efficiency, the macrophages can be prepared using conventional methods such as primary isolation and culture, stem cell directed differentiation induction, and cell line expansion culture. According to some embodiments of the present invention, the method for preparing the macrophages may include: stimulating macrophage precursor cells with a stimulating factor. The stimulating factor enables the macrophage precursor cells to differentiate into mature functional macrophages.

[0069] Specifically, to ensure cell proliferation activity, inflammatory chemotaxis, and in vivo colonization stability, while simultaneously improving engineering efficiency and anti-inflammatory function stability, the engineered macrophages can be obtained from macrophage precursor cells through directed induction differentiation and gene recombination. Preferably, the macrophage precursor cells can be derived from at least one of bone marrow, peripheral blood, spleen, and stem cells, wherein the stem cells include at least one of hematopoietic stem cells, pluripotent stem cells, and induced pluripotent stem cells (iPSCs). Preferably, the macrophages are derived from bone marrow.

[0070] Specifically, the stimulating factor is a cytokine capable of inducing the directed differentiation of macrophage precursor cells and promoting the maturation and proliferation of macrophages. For example, the stimulating factor can be selected from at least one of macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), phorbol ester (PMA), and interleukin. Those skilled in the art can make the selection according to the actual situation. According to a preferred embodiment of the present invention, the stimulating factor is preferably M-CSF. Macrophages stimulated with M-CSF have better survival rate, adherent growth ability, phagocytic function, and tissue-resident-like characteristics, and usually exhibit a relatively mild and stable inflammatory baseline state, which is beneficial for subsequent lentiviral transduction, exogenous functional gene expression, and maintaining a controllable immune response under the stimulation of MSU crystals or inflammatory factors. Compared with GM-CSF-induced inflammatory macrophages, the M-CSF induction system is more conducive to obtaining a macrophage population with repair tendency, better tolerance, and stronger in vivo adaptability, thereby improving the functional stability and safety of engineered macrophages in the inflammatory microenvironment of gout.

[0071] The M-CSF can be a commercially available active product or prepared in-house through genetic engineering recombination expression, artificial purification, or other methods. According to some specific embodiments of the present invention, M-CSF is obtained by culturing fibroblasts (such as human or mouse fibroblasts) and collecting their supernatant. It is understood that, to further improve the biocompatibility and therapeutic suitability of the cells in vivo, cells homologous to the target species can be used for preparation. For example, when using mouse gouty arthritis as a research model, mouse-derived precursor cells can be used for induced differentiation and engineered modification; when applied to human gouty arthritis, human-derived precursor cells can be used for preparation and modification.

[0072] According to some specific embodiments of the present invention, the concentration of the stimulating factor can be flexibly adjusted according to cell type, differentiation state, culture system, and actual experimental needs to ensure efficient and stable directed differentiation of macrophage precursor cells. For example, the final concentration of the stimulating factor can be 1-100 ng / mL, preferably 10-50 ng / mL.

[0073] According to some specific embodiments of the present invention, the method for preparing the stimulating factor is as follows: Mouse fibroblast cell line L929 was seeded into culture flasks and cultured in high-glucose DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics, and incubated at 37°C and 5% CO2 saturated humidity. When L929 cells reached approximately 80%-90% confluence, the medium was replaced with fresh complete medium and cultured for another 4-6 days. After 4-6 days of culture, the culture supernatant of L929 cells was collected, centrifuged at 350-450g for 4-6 min to remove cell debris, and the supernatant was filtered through a 0.22 μm sterile filter to obtain a culture medium containing stimulating factors. The volume fraction of this culture medium in the induction culture system was 1-20 vol%.

[0074] Specifically, methods for introducing exogenous genes into macrophages may include any one of the following: viral vector-mediated transduction, transposon system, CRISPR / Cas system, or base editing, with viral vector transduction (e.g., lentivirus transduction) being preferred.

[0075] To enable engineered macrophages to achieve self-sustaining, directed differentiation, and enhanced function, and to reduce their dependence on exogenous stimulating factors, this invention preferably introduces genes encoding exogenous stimulating factors into macrophages. Those skilled in the art can select the genes encoding exogenous stimulating factors at their own discretion.

[0076] According to some specific embodiments of the present invention, the lentiviral expression vector uses pRRL- mUOX - mIL1Ra -Construction of expression cassette for GFP plasmid, in which pRRL- mUOX - mIL1Ra -The expression cassette of the GFP plasmid contains, in sequence,... EF-1α promoter, Kozak sequence, mGM-CSF , mUOX , GSG-P2A , mIL1Ra , GSG-T2A , GFP and WPRE Component. pRRL- mUOX - mIL1Ra In the expression cassette of the -GFP plasmid, besides mUOX and mIL1Ra Apart from that, the functions, roles, and sequences of the other components are well known to those skilled in the art, and they can be selected according to the actual situation, so they will not be described in detail here.

[0077] According to some preferred embodiments of the present invention, in order to promote the secretion of uricase, the gene encoding the mGM-CSF signal peptide may be introduced into the engineered macrophages. The definition, sequence, and function of the mGM-CSF signal peptide are consistent with those described in the first aspect of the present invention.

[0078] Thirdly, the present invention provides the use of the engineered macrophages described in the first aspect in the preparation of medicaments for the prevention and / or treatment of gout.

[0079] According to some preferred embodiments of the present invention, the symptoms of gout include hyperuricemia and / or joint inflammation.

[0080] The engineered macrophages provided by this invention not only possess (1) dual therapeutic functions of highly efficient uric acid reduction and potent anti-inflammatory effects, but also can continuously secrete uricase to degrade local uric acid, inhibit the formation of monosodium urate crystals, block the IL-1 inflammatory pathway by relying on IL-1Ra, downregulate pro-inflammatory factors such as TNF-α and IL-6, and reduce neutrophil infiltration; (2) possess excellent high uric acid tolerance and autonomous proliferation and differentiation capabilities, and can survive for a long time in gout lesions, continuously accumulate and maintain stable therapeutic activity, effectively improve hyperuricemia and joint inflammation damage, and achieve long-term prevention and treatment of gout. Therefore, the engineered macrophages provided by this invention achieve synergistic treatment from multiple dimensions, including removing pathogenic factors from the source, blocking the inflammatory cascade reaction, and maintaining long-term therapeutic activity, effectively overcoming the defects of traditional drugs such as short half-life, single action, need for repeated administration, and poor treatment sustainability. They have both safety and long-term efficacy, and have significant clinical application value. They can be used to prepare drugs for the prevention and / or treatment of gout.

[0081] Fourthly, the present invention provides the application of the engineered macrophages described in the first aspect in reducing uric acid levels and / or reducing inflammatory responses in vitro.

[0082] It is understood that the applications described in the fourth aspect of this invention are scientific research applications for non-disease treatment purposes and do not involve the treatment or diagnosis of diseases. For example, the engineered macrophages provided by this invention can be used to regulate uric acid levels and inflammatory states in experimental animals, constructing animal models with different uric acid contents and different degrees of inflammation to meet the research needs of studying the mechanisms of gout and hyperuricemia, drug screening, and efficacy evaluation.

[0083] The engineered macrophages for regulating uric acid and inflammation, their preparation method, and their applications are further illustrated below through specific embodiments.

[0084] Unless otherwise specified, all raw materials used are commercially available products.

[0085] Table 1

[0086] Example 1 This embodiment is used to conduct public single-cell transcriptome data mining and analysis on monocytes associated with acute gout inflammation.

[0087] 1. Acquisition of Public Single-Cell Transcriptome Data We analyzed the single-cell transcriptome dataset GSE211783 from the publicly available Gene Expression Omnibus (GEO) database. This dataset was derived from peripheral blood mononuclear cell (PBMC) samples from patients with acute gout, including samples from both acute attack and remission phases.

[0088] After downloading the original expression matrix and corresponding annotation files, subsequent single-cell transcriptome analysis was performed.

[0089] 2. Single-cell data preprocessing and cell cluster analysis Quality control, standardization, dimensionality reduction, and cluster analysis were performed on the GSE211783 dataset. Filtering conditions included: nFeature_RNA range of 300-6000 and mitochondrial gene proportion less than 15%. Batch correction was performed using the Harmony algorithm, and cell clustering and visualization analysis were conducted based on PCA and UMAP.

[0090] UMAP results showed that PBMCs mainly consisted of immune cell populations such as CD14 monocytes, CD16 monocytes, T cells, NK cells, B cells, dendritic cells, and platelets.

[0091] The above results are as follows Figure 2 As shown, single-cell transcriptomic analysis reveals circulating monocyte inflammatory remodeling associated with acute gout attacks. Figure 2 (a) shows the PBMC single-cell UMAP, displaying the main immune cell population. Figure 2 (b) shows that the proportion of inflammation-related mononuclear cells in all three paired samples showed an increasing trend in the acute phase, but there was heterogeneity among the samples. Figure 2 (c) Shows the expression patterns of representative inflammatory genes at the patient level. Figure 2 (d) Shows differential expression of paired pseudobulk and exploratory pathway analysis, in which CXCL8 and related procedures of innate immunity, interferon response, complement and scavenger receptors were altered. Figure 2 (e) shows that the IL-1 / NF-κB module scores were elevated in all three patients during the acute phase, suggesting that acute gout is accompanied by pre-activation of circulating mononuclear cell inflammation.

[0092] Example 2 This embodiment illustrates the process of constructing engineered macrophages.

[0093] 1. Segregation and Induced Differentiation of BMDM Preparation of supernatant from mouse fibroblast cell line (L929 cells): Mouse fibroblast cell line L929 cells were seeded into culture flasks and cultured in high-glucose DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics. The culture was incubated at 37°C in a 5% CO2 saturated humidity incubator. When the L929 cells reached approximately 80%–90% confluence, the medium was replaced with fresh complete medium and cultured for another 5 days. After 5 days of culture, the culture supernatant of L929 cells was collected, centrifuged at 400g for 5 min to remove cell debris, and the supernatant was filtered through a 0.22 μm sterile filter to obtain L929 conditioned medium (stimulating factor), aliquoted, and stored. Keep at 80℃ for later use.

[0094] Eight-week-old C57BL / 6J mice were euthanized by cervical dislocation. The femurs and tibias were aseptically separated, and the epiphyses were removed. The bones were centrifuged at 5000 rpm for 30 seconds to isolate bone marrow cells. Bone marrow-derived cells were obtained after erythrocyte lysis. The cells were resuspended in BMDM complete medium and the density was adjusted to 1.5 × 10⁻⁶. 6 Cells were seeded at a density of 1 / mL in culture dishes and incubated at 37°C in a 5% CO2 incubator. BMDM complete medium consisted of DMEM high-glucose basal medium supplemented with 10% FBS, 10% L929 conditioned medium, and 1% penicillin-streptomycin. By day 5, adherent cells differentiated into mature bone marrow-derived macrophages (BMDM).

[0095] Flow cytometry detection of surface markers: Bone marrow-derived macrophages obtained on day 5 of culture were collected, washed with pre-chilled PBS buffer, and single-cell suspensions were prepared. Cells were added to flow cytometry staining buffer, and fluorescently labeled anti-mouse CD11b antibody and anti-mouse F4 / 80 antibody were added, respectively. The cells were incubated at 4°C in the dark for 30 min to allow the fluorescent antibodies to bind to the corresponding antigens on the cell surface. After incubation, the cells were washed with flow cytometry staining buffer to remove unbound fluorescent antibodies, and then resuspended and analyzed by flow cytometry.

[0096] During flow cytometry analysis, the cell population was initially delineated based on forward scattered (FSC) and side scattered (SSC) signals to exclude cell debris and abnormal cell populations. Further single-cell population analysis was used to exclude cell aggregation, and the macrophage population was determined based on CD11b and F4 / 80 fluorescence signals. CD11b was used as the primary indicator. + F4 / 80 +The proportion of double-positive cells serves as an indicator of the differentiation efficiency of bone marrow-derived macrophages. The results of the macrophage surface marker F4 / 80 are as follows: Figure 3 As shown, where, Figure 3 (a) is the result of a mature BMDM. Figure 3 (b) shows the results of undifferentiated bone marrow-derived macrophages. The results indicate that CD11b is present in mature BMDM cells. + F4 / 80 + The proportion of double-positive cells exceeded 90%, indicating that the bone marrow-derived macrophages were successfully induced to differentiate and can be used for subsequent genetic engineering, inflammatory stimulation, and functional evaluation experiments.

[0097] 2. Lentiviral packaging Encoded mice UOX The full-length cDNA sequence ( mUOX (The sequence is shown in SEQ ID NO:3) and IL-1Ra ( mIL1Ra (The sequence is shown in SEQ ID NO:4) pRRL- was prepared according to conventional methods. mUOX - mIL1Ra -GFP lentiviral expression vector, in which mUOX and mIL1Ra Located downstream of the EF-1α promoter, mIL1Ra Through respectively GSG-P2A and GSG-T2A and mUOX and GFP The recombinant expression plasmid was obtained by ligation, and sequencing confirmed its correctness.

[0098] Take human kidney epithelial cell line (293T cells) in good growth condition, and administer at a rate of 2×10⁻⁶. 6 Inoculate each 10cm dish with DMEM complete medium (containing 10% FBS and 1% P / S (penicillin-streptomycin antibiotic)) and incubate overnight until 70%-80% confluence. pRRL- mUOX - mIL1Ra -The expression cassette of the GFP plasmid contains, in sequence,... EF-1α promoter, Kozak Sequence, mG-CSF signal peptide encoding gene (sequence shown in SEQ ID NO:5), mUOX , GSG-P2A , mIL1Ra , GSG-T2A , GFP and WPRE element.

[0099] Co-transfection was performed using linear polyethyleneimine (PEI, fast-dissolving) transfection: lentiviral backbone plasmid (pMD2.G), packaging plasmid (psPAX2), and recombinant expression plasmid were mixed at a mass ratio of 1:2:3 (total plasmid 15 μg / dish), dissolved in 200 μL serum-free DMEM, and gently mixed to obtain a DNA solution. Separately, 45 μg of the fast-dissolving PEI reagent was added to 200 μL serum-free DMEM, allowed to stand at room temperature for 5 min, and then slowly added to the DNA solution, gently mixed, and incubated at room temperature for 25 min to form a transfection complex. The complex was then evenly added dropwise to 293T cell culture dishes and gently shaken to mix. Eight hours after transfection, the medium was replaced with fresh DMEM complete medium (containing 10% FBS). Cells were cultured for 48 h and 72 h, and the cell supernatant was collected, filtered through a 0.45 μm filter, and the filtrates were combined to obtain the lentiviral supernatant. The lentivirus supernatant was concentrated using PEG8000, and then the concentrated lentivirus was resuspended in complete DMEM medium to obtain D-lentivirus, which was aliquoted and frozen at -80℃.

[0100] 3. Lentiviral transfection Add the aforementioned BMDM, D-lentivirus (final concentration to achieve MOI=20), and polybrene (final concentration 8 μg / mL) differentiated to DMEM complete medium on day 3, mix well, centrifuge at 800g for 2 h, incubate at 37℃ for 8-12 h, then replace with fresh medium. Observe green fluorescence after 72 hours, and use flow cytometry to confirm the positive rate is qualified, thus obtaining BMDM- msUOX - IL-1Ra (BMDM-mi).

[0101] In addition, control engineered macrophages were constructed using the same method, the difference being the recombinant expression plasmid used in the lentiviral transduction step: (1) BMDM-0: The lentiviral transfer plasmid used was pRRL-GFP, which does not contain mG-CSF , mUOX , GSG-P2A , mIL1Ra The gene expresses only the GFP reporter protein and does not express exogenous UOX and IL-1Ra; (2) BMDM-mUOX (BMDM-m): pRRL- mUOX -GFP, which does not contain GSG-P2A , mIL1Ra The gene expresses only GFP and UOX, but not exogenous IL-1Ra; (3) BMDM-IL-1Ra (BMDM-i): The recombinant expression plasmid used was pRRL- mIL1RA -GFP, which does not contain mG- CSF , mUOX , GSG-P2A The gene expresses only GFP and IL-1Ra, but not exogenous UOX.

[0102] Example 3 This embodiment is used to identify the phenotype of the engineered macrophages prepared in Example 2.

[0103] 1. Experimental Procedure (1) Infection efficiency Flow cytometry was used to detect GFP positivity: BMDM cells obtained on day 5 post-lentiviral infection were collected and washed twice with PBS buffer to remove culture medium and free fluorescent substances. The washed cells were prepared into single-cell suspensions and resuspended in appropriate amounts of PBS or flow cytometry buffer. Cell analysis was performed using a CytoFLEX flow cytometer. The cell population size was determined by forward scatter (FSC) and side scatter (SSC) signals, excluding cell debris and abnormal cell populations. Subsequently, transduction-positive cells were identified based on the intensity of the GFP fluorescence signal.

[0104] BMDM cells that had not undergone lentiviral infection were used as a negative control. The GFP positivity threshold was set based on the GFP fluorescence background signal of the negative control cells. The fluorescence results of BMDM-mi are as follows: Figure 4 As shown, the fluorescence analysis results of BMDM-mi and BMDM-0 are as follows: Figure 5 As shown, where, Figure 5 (a) and Figure 5 (b) The fluorescence analysis results of BMDM-mi and iBMDM-0 are shown respectively. The proportion of GFP-positive cells in the total effective detection cells was calculated, and the proportion of GFP-positive cells in engineered BMDM (GFP positivity rate) was obtained according to the following formula, which was used to evaluate the efficiency of lentivirus-mediated exogenous gene delivery.

[0105] GFP positivity rate (%) = (Number of GFP-positive cells / Total number of effectively detected cells) × 100% Among them, the number of GFP-positive cells is the number of cells whose GFP fluorescence signal is higher than the threshold set for the negative control in flow cytometry; the total number of effective cells is the total number of cells included in the analysis after cell population screening.

[0106] (2) Detection of UOX mRNA expression BMDM-mi, BMDM-0, BMDM-i, and BMDM-m cells were collected. Total RNA was extracted from each cell type using an RNA extraction kit and reverse transcribed into cDNA using standard methods. Real-time quantitative PCR was performed using the SYBR Green assay to detect UOX expression levels.

[0107] The primer sequences are: UOX forward 5'-TCACCTTCCGTGCAGAAGAC-3', reverse 5'-CGTAGGGATTGTCGAGAGGC-3'; the internal reference gene used is... GAPDH With 2^ -ΔΔCt The relative expression level was calculated to verify the expression effect of UOX.

[0108] (3) Detection of uricase activity BMDM-mi, BMDM-0, BMDM-i, and BMDM-m cells were collected and washed twice with pre-cooled PBS. Cells were counted and cultured in DMEM complete medium containing 10% L929 conditioned medium at 37°C and 5% CO2 for 24 h. The cell culture supernatant was collected, and uricase activity was measured according to the Solarbio uricase activity assay kit instructions. Enzyme activity was calculated using the standard curve method, expressed in mU / mL. The results are shown in Table 2.

[0109] (4) Detection of IL-1Ra expression level A standard curve for IL-1Ra concentration at OD450 nm was established; cell culture supernatant was prepared and collected according to method (3), and particles were removed by centrifugation. The mouse IL1RA ELISA kit was used, and the procedure was followed according to the instructions. The standard and the supernatant to be tested were added to an ELISA plate pre-coated with capture antibody, incubated, and then biotinylated detection antibody was added. After binding with streptavidin-HRP, the absorbance was measured at 450 nm after TMB color development. The concentration of IL-1Ra in the supernatant was calculated based on the standard curve, and the results are shown in Table 2.

[0110] (5) Engineered macrophages were prepared according to the method in Example 1, except that the recombinant expression plasmid used was pRRL- mUOX - mIL1Ra - mCherry Engineered macrophages expressing mCherry, BMDM-mic, were obtained to dynamically observe the interaction and uptake process of macrophages with fluorescently labeled MSU crystals. One day prior to the experiment, BMDM-mic cells were seeded in clear-bottomed, black-walled culture plates and allowed to adhere overnight. During the experiment, the medium was replaced with fresh medium containing FITC-labeled MSU crystals, and the cells were immediately placed in an Incucyte live-cell imaging system. Images were continuously acquired using bright-field, green fluorescence, and red fluorescence channels to characterize the BMDM-mic's ability to internalize uric acid. The results are as follows: Figure 6As shown, a significant spatial overlap can be observed between the green FITC-MSU signal and the red mCherry-labeled macrophage region, with some green fluorescent signals located within the outline of the red cells, suggesting that engineered macrophages can bind to and take up MSU crystals. Combined with the phenomenon of the green fluorescent signal gradually entering and moving with the red cell region during continuous dynamic imaging, this further supports the macrophages' ability to phagocytose / internalize MSU crystals.

[0111] Table 2

[0112] Example 4 This embodiment is used to verify the in vitro function of the engineered macrophages prepared in Example 2.

[0113] 1. Ability to lower uric acid and cell survival rate BMDM-mi, BMDM-0, BMDM-i, and BMDM-m were seeded in six-well plates and cultured in a CO2 incubator (37℃, 5% CO2) for 6 hours to stabilize the cells. Then, uric acid solution with a final concentration of 400-450 μmol / L was added to the six-well plates, and the cells were cultured for 96 hours. Cell status was photographed before the addition of uric acid solution and at 12h, 24h, and 48h after the addition. The results for BMDM-mi (engineered macrophages) and BMDM-0 (control macrophages) are shown below. Figure 7 As shown in (a), cell counting was performed at the end of the 96-hour experiment, and the results are as follows. Figure 7 (c) shows that 100 μL of supernatant was collected every 12 hours during the experiment, and the uric acid concentration was measured using a uric acid detection kit. The changes in uric acid concentration over time were similar for BMDM-0 and BMDM-i, and neither had the ability to lower uric acid. The changes in uric acid concentration over time for BMDM-m and BMDM-mi were similar. The results of the changes in uric acid concentration over time for BMDM-0 and BMDM-mi are shown in Figure 1. Figure 7 As shown in (b), the results show that BMDM-mi has excellent uric acid-lowering ability, which is significantly higher than that of BMDM-0 and BMDM-i.

[0114] 2. Anti-inflammatory effects of IL-1Ra A stock solution of monosodium urate (MSU) with a concentration of 3 mg / mL was prepared using sterile PBS and dispersed by sonication to obtain an MSU solution.

[0115] BMDM, BMDM-mi, BMDM-i, BMDM-m, and BMDM-0 are respectively set at 1×10 6Cells were seeded per well in 6-well plates (2 mL per well). After overnight adhesion, 100 μL of MSU solution was added to obtain the experimental groups: BMDM+MSU group (control macrophages + sodium urate crystal group), BMDM-mi+MSU group (engineered macrophages + sodium urate crystal group), BMDM-i+MSU group, BMDM-m+MSU group and BMDM-0+MSU group (control macrophages - GFP + sodium urate crystal group).

[0116] Additionally, the settings are as follows: (1) Blank group: M0 macrophage group, empty control cell group and engineered macrophage blank group were added to BMDM and BMDM-0 and BMDM-mi with equal volume of culture medium after overnight adhesion; (2) M1 macrophages with a final concentration of 100 mg / mL LPS and 20 ng / mL IFN-γ were added to BMDM after overnight adhesion, and M2 macrophages with 20 ng / mL IL-4 were added to BMDM after overnight adhesion. (3) The isotype control group (blank group) of the flow cytometry staining system is used to subtract non-specific fluorescence signals and define the threshold of positive cells.

[0117] After 24 hours of culture, staining was performed: cells were incubated at 4°C in the dark for 30 minutes using fluorescent PE-labeled anti-mouse CD86 antibody and APC-labeled anti-mouse CD206 antibody. Separately, cells were treated with the same grouping method, washed, and then treated with fixation / permeabilization buffer (BD Cytofix / Cytoperm). Intracellular staining was then performed: cells were incubated at 4°C in the dark for 45 minutes using PE-labeled anti-human inducible nitric oxide synthase (iNOS, M1 marker) and APC-labeled anti-human arginase 1 (Arg-1, M2 marker). After washing, the cells were resuspended and analyzed by flow cytometry, with at least 5 × 10⁶ cells collected from each sample. 3 Each cell.

[0118] A standard curve was established for the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) at OD450 nm. Cell culture supernatants were collected from each well at 2 h, 8 h, 12 h, and 24 h after treatment. The supernatants were centrifuged at 3000 rpm for 5 min to remove cell debris and any MSU crystals, yielding a clear supernatant. Using the corresponding ELISA kits and following the instructions, the OD450 nm levels of TNF-α, IL-6, and IL-1Ra in the clear supernatant were measured, and the levels were calculated based on the standard curve. Each sample was tested in duplicate.

[0119] The results of the changes in IL-1Ra content over time in the BMDM-0 and BMDM-mi groups are as follows: Figure 8 As shown, Figure 8The results showed that BMDM-0 could hardly express the IL-1 receptor antagonist IL-1Ra, while the expression level of IL-1Ra in BMDM-mi increased significantly over time.

[0120] The levels of TNF-α and IL-6 in each group after culture are shown in Table 3. The results show that engineered macrophages can reduce the expression levels of TNF-α and IL-6.

[0121] Table 3

[0122] The expression of markers CD86, CD206, iNOS, and Arg1 was detected by flow cytometry to assess the phenotypic stability and immunomodulatory capacity of engineered macrophages under inflammatory conditions. Results for each group are as follows: Figure 9 As shown.

[0123] Example 5 This embodiment is used to verify the in vivo function of the engineered macrophages prepared in Example 2.

[0124] All animal husbandry and experimental projects in this embodiment were approved by the Animal Protection and Ethics Committee of Peking University School of Medicine (ethics number: DLASBF0462) and strictly followed the Peking University Biomedical Ethics Guidelines.

[0125] SPF-grade male C57BL / 6J mice (8-10 weeks old, weighing 22±2g) were selected and acclimatized for 7 days before model initiation. Mice were randomly divided into five groups: four experimental groups (model group, positive drug group, control cell group (BMDM-0), and engineered cell group (BMDM-mi)) and one negative control group. The experimental groups were administered hypoxanthine (500 mg / kg, dissolved in 0.5% sodium carboxymethyl cellulose solution) and potassium oxonate (300 mg / kg, dissolved in sterile saline) orally by gavage daily for 7 consecutive days. Potassium oxonate is a uricase inhibitor that reduces uric acid breakdown; hypoxanthine is a uric acid precursor that increases uric acid production. The negative control group was injected with the same volume of solution.

[0126] During model establishment, blood was collected from the orbital venous plexus on days 1 (before drug administration), 3, 5, and 7. Serum uric acid levels were measured using a uric acid assay kit after serum separation. The hyperuricemia model was considered successfully established when the serum uric acid level in mice was significantly higher than that in the control group (administered with an equal volume of solvent) (P<0.05) and remained stable. The positive control group received allopurinol (2.5 mg / kg) daily by gavage starting from day 3, and colchicine (1 mg / kg) daily by gavage starting from day 5.

[0127] On the afternoon of the 7th day of the experiment, the control cell group and the engineered cell group were injected with control cells BMDM-0 and BMDM-mi via tail vein (injection volume of 5 × 10⁻⁶ cells / year). 6 (cells / mouse)

[0128] On day 8 of the experiment, acute gouty arthritis was induced. MSU crystal powder (MCE) was accurately weighed, added to sterile PBS, and sonicated to prepare a homogeneous suspension of 20 mg / mL. Before use, the suspension was sonicated again to ensure uniform crystal dispersion. Mice were anesthetized with isoflurane inhalation and fixed in a flexed knee position. Using a disposable insulin syringe (30G needle), the needle was inserted obliquely inward and downward from the lateral side of the patellar ligament into the right knee joint cavity. The experimental group was slowly injected with 50 μL of the MSU crystal suspension. After injection, the knee joint was gently moved several times to ensure uniform crystal distribution. Control group mice were injected with an equal volume of sterile PBS.

[0129] Starting from day 10 of the experiment, the following indicators were observed and recorded daily in the positive drug group at 0h before gavage injection and at 2h, 6h, 12h, 24h, 48h, and 72h after injection: Joint swelling: The knee joint diameter was measured using electronic calipers, and the swelling rate was calculated according to the following formula. The change in swelling rate over time is shown in the figure. Figure 10 As shown; among them, a photo of knee swelling 6 hours after injection is shown. Figure 11 As shown.

[0130] Swelling rate = (diameter after injection) (Pre-injection diameter) / (Pre-injection diameter) × 100% Figure 10 The results showed that the BMDM-mi group had the fastest swelling reduction and the most significant effect.

[0131] Figure 11 The results showed that the joint swelling on the affected side was significant in the model group; the joint swelling was reduced in both the positive drug group and the engineered cell group, with the engineered cell therapy group showing a more significant improvement trend than the control cell group.

[0132] Seventy-two hours after injection, one mouse from each group was sacrificed, and knee joint tissue was collected for HE staining. Microscopic observation revealed typical pathological changes in the synovial tissue, including neutrophil infiltration, vasodilation, and inflammatory exudation. The combined effects of these indicators—joint redness, swelling, heat, pain, and inflammatory cell infiltration—showed successful establishment of an acute gouty arthritis model.

[0133] Mice were sacrificed on day 10, and blood and joint tissues were collected for the following tests.

[0134] (1) Immediately after euthanizing the mice, collect approximately 0.5-1.0 mL of whole blood from the orbital venous plexus and collect it in a 1.5 mL EP tube. Allow it to stand at room temperature for 30 min to allow it to coagulate naturally. Then, centrifuge at 3000 r / min for 15 min, carefully aspirate the upper pale yellow serum, aliquot it into new EP tubes, and store them at -80℃ for later use. Uric acid (UA) level determination: Establish a uric acid concentration-absorbance standard curve; add 50 μL of the serum to be tested to the working solution of the uric acid detection kit, incubate at 37℃ for 30 min, and measure the absorbance value at a wavelength of 505 nm. Calculate the serum uric acid concentration (in μmol / L) according to the standard curve. Samples were prepared in duplicate, and the average value was taken.

[0135] The uric acid concentration results for the model group, positive drug group, control cell group, engineered cell group, and negative control group are as follows: Figure 12 As shown, the engineered cell group exhibited excellent uric acid-lowering effects, comparable to allopurinol, a first-line clinical drug.

[0136] (2) Joint tissue examination: The right knee joint and surrounding soft tissue of mice were completely excised, excess muscle and hair were removed, and the tissue was immediately fixed in 4% paraformaldehyde (pH 7.4) for 48 hours. After fixation, the tissue was transferred to EDTA decalcification solution (pH 7.2-7.4) and decalcified at room temperature, with fresh decalcification solution replaced every 3 days. The endpoint of decalcification was defined as the point at which a pin could easily penetrate the bone tissue without resistance (approximately 3-4 weeks). After decalcification, the tissue was rinsed with running water overnight, then dehydrated stepwise with 70%, 80%, 90%, 95%, and 100% graded ethanol (1-2 hours per grade), cleared with xylene (twice, 20-30 minutes each time), and finally impregnated with paraffin and embedded to form a tissue paraffin block.

[0137] Paraffin sections: Use a paraffin microtome to cut the paraffin block into continuous sections 4-5 μm thick. Place the sections in a 40℃ water bath to flatten them, remove them with a glass slide to prevent them from falling off, and bake them in a 60℃ oven for 2 hours or overnight at 37℃ for later use.

[0138] HE staining: Dewaxing to water: xylene I and II for 10 min each, anhydrous ethanol I and II for 5 min each, 95%, 85%, and 75% ethanol for 3 min each, followed by washing with distilled water for 2 min; Hematoxylin staining: hematoxylin staining solution for 5-8 min, followed by rinsing with running water for 5 min; Differentiation: 1% hydrochloric acid ethanol differentiation for 3-5 s, followed by rinsing with running water for 10 min; Eosin staining: 0.5% eosin staining solution for 2-3 min, followed by rinsing with running water for 1 min; Dehydration and clearing: 75%, 85%, 95%, and anhydrous ethanol I and II for 1-2 min each, followed by clearing with xylene I and II for 3 min each; Mounting: Mount with neutral resin, observe and photograph under an optical microscope. Results are as follows. Figure 13As shown in (a), observe the degree of synovial lining hyperplasia, the density of inflammatory cell infiltration (lymphocytes, neutrophils, macrophages, etc.) in the subsynovial tissue, joint cavity effusion, and cartilage / bone erosion. Figure 13 (a) The engineered cell group showed reduced synovial inflammation, inflammatory cell (neutrophil, macrophage) infiltration and tissue damage.

[0139] (3) Detection of TNF-α and Ly6G expression: Paraffin sections were placed in 0.01M citrate buffer (pH 6.0), microwaved on high (approximately 800W) for 10 min, then on medium-low (approximately 300W) for 10 min, and allowed to cool naturally to room temperature; Blocking endogenous peroxidase: Incubated with 3% H2O2-methanol solution at room temperature for 15 min, washed 3 times with PBS for 3 min each time; Blocking: Blocked with 5% goat serum at room temperature for 30 min, the blocking solution was discarded, and no washing was performed; Primary antibody incubation: Anti-mouse TNF-α primary antibody (dilution ratio 1:200) and anti-mouse Ly6G primary antibody were added dropwise respectively. Primary antibody (diluted 1:400), incubate overnight in a humidified chamber at 4°C; rewarming: remove the slides the next day, rewarm to room temperature for 30 min, wash 3 times with PBS for 5 min each time; secondary antibody incubation: add HRP-labeled goat anti-rabbit / anti-mouse universal secondary antibody, incubate at room temperature for 30-40 min, wash 3 times with PBS for 5 min each time; DAB staining: prepare fresh DAB staining solution, control the staining time under a microscope (about 1-5 min), stop the staining with distilled water immediately when a brownish-yellow color appears; counterstaining: hematoxylin counterstain for 1-2 min, rinse with running water for 10 min; dehydration, clearing, and mounting (same as HE staining process).

[0140] TNF-α and Ly6G inflammatory results as follows Figure 13 As shown in (b). Figure 13 (b) Immunofluorescence results showed that the number of Ly6G positive cells and TNF-α fluorescence signal in the synovial tissue of the engineered cell therapy group both showed a decreasing trend, indicating that neutrophil infiltration and local inflammatory response were relieved to a certain extent.

[0141] Example 6 This embodiment is used to verify the in vivo safety of the engineered macrophages prepared in Example 2.

[0142] 1. DiR labeling of engineered macrophages Engineered macrophages in the logarithmic growth phase were washed twice with PBS and then subjected to a 1×10⁻⁶ ppm solution. 6Cells were resuspended in serum-free medium at a density of 1 / mL. DiR fluorescent dye was added, and the cells were mixed with the dye and incubated at 37°C in the dark for 20 min, gently mixing every 5 min during incubation. After incubation, the cells were washed three times with complete medium containing 10% fetal bovine serum (centrifuged at 300 g for 5 min each time) to remove free DiR dye, resulting in DiR-labeled engineered macrophages (DiR labeling efficiency >95%, cell viability >90%).

[0143] 2. Animal grouping and administration 2×10 via tail vein injection 6 DiR-labeled engineered macrophages (resuspended in 200 μL PBS).

[0144] 3. In vivo fluorescence imaging Whole-body imaging of mice 72 hours after injection was performed using an IVIS Spectrum (PerkinElmer) small animal in vivo fluorescence imaging system. The excitation wavelength was 745 nm, the emission wavelength was 800 nm, and the exposure time was automatically adjusted according to the signal intensity. Quantitative analysis of fluorescence signals was performed after image acquisition, with fluorescence intensity expressed as radiance per unit area (photons / s / cm² / sr). The whole-body fluorescence imaging results are shown below. Figure 14 As shown. Semi-quantitative analysis of fluorescence signals from the liver, inflamed joints, and healthy joint areas was performed to obtain the total radiation efficiency (in μW / cm²) for each site. 2 The total radiation efficiency was used to characterize the relative enrichment and retention levels of engineered macrophages in different tissue regions. Results showed that strong fluorescence signals were detected in the liver region after intravenous injection of engineered macrophages, suggesting that some of the injected cells could be distributed or retained in the liver tissue. Simultaneously, fluorescence signals were detected in both inflamed and contralateral healthy joint regions, with the total radiation efficiency in the inflamed joint being slightly higher than that in the healthy joint, indicating that engineered macrophages could reach the inflamed joint region.

[0145] 4. Fluorescence imaging of ex vivo organs Mice in each group were sacrificed 108 hours after injection, and major organs such as the liver and kidneys, as well as joints, were dissected and harvested. The fresh organs were placed on the IVIS imaging system stage, and in vitro fluorescence imaging was performed using the same parameters as in vivo imaging. The fluorescence signal intensity of each organ was recorded. The fluorescence imaging results of the joints, liver, and kidney regions are shown below. Figure 15 As shown, where, Figure 15The images, from left to right, show the joint, liver, and kidney. In vitro imaging results show that, after tail vein injection, fluorescent signals related to engineered macrophages can be detected in the joint tissue, indicating that the engineered macrophages can reach the joint site via systemic circulation. Simultaneously, strong fluorescent signals were observed in the liver, suggesting that some cells may be taken up or retained by the hepatic reticuloendothelial system.

[0146] The above results demonstrate that the engineered macrophages of this invention have the ability to reach the joint lesion site after being administered via the tail vein, providing a basis for their in vivo intervention in gouty arthritis.

[0147] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. An engineered macrophage that regulates uric acid and inflammation, characterized in that, The genome of the engineered macrophages contains a gene encoding exogenous uricase. UOX Genes encoding exogenous interleukin-1 receptor antagonists IL- 1Ra .

2. The engineered macrophage according to claim 1, characterized in that, The gene encoding uricase UOX The nucleotide sequence is shown in SEQ ID NO:3; And / or, the gene encoding the interleukin-1 receptor antagonist IL-1Ra The nucleotide sequence is shown in SEQ ID NO:

4.

3. The engineered macrophage according to claim 1, characterized in that, The engineered macrophages were obtained from macrophage precursor cells through directed induction of differentiation and gene recombination. Preferably, the macrophage precursor cells are derived from at least one of bone marrow, peripheral blood, spleen, and stem cells.

4. A method for preparing engineered macrophages according to any one of claims 1-3, characterized in that, The method includes: introducing an exogenous gene encoding uricase into macrophages. UOX Genes encoding exogenous interleukin-1 receptor antagonists IL-1Ra .

5. The method according to claim 4, characterized in that, The method for preparing macrophages includes stimulating macrophage precursor cells with stimulating factors.

6. The method according to claim 5, characterized in that, The macrophage precursor cells are derived from at least one of the following: bone marrow, peripheral blood, spleen, and stem cells.

7. The method according to claim 5, characterized in that, The stimulating factor is selected from at least one of M-CSF, GM-CSF, PMA and interleukin, preferably M-CSF.

8. The method according to claim 4, characterized in that, Methods for introducing exogenous genes into macrophages include: viral vector-mediated transduction, transposon systems, CRISPR / Cas systems, and any one of base editing.

9. The use of the engineered macrophages according to any one of claims 1-3 in the preparation of medicaments for the prevention and / or treatment of gout.

10. The use of engineered macrophages according to any one of claims 1-3 in reducing uric acid levels and / or reducing inflammatory responses in vitro.

Images