Use of an engineered phage targeting Parasutterella secunda in the preparation of a medicament for treating osteoarthritis

Abstract

The application belongs to the technical field of microbial medicine, and particularly relates to application of an engineered bacteriophage, and particularly relates to application of a bacteriophage capable of specifically targeting and removing Parasutterella secunda, a conditional pathogenic bacterium in the intestinal tract, in preparation of a medicine for preventing, relieving or treating osteoarthritis, in particular, osteoarthritis driven by obesity or intestinal flora imbalance.

Description

Technical Field

[0001] This invention belongs to the field of microbial pharmaceutical technology, specifically relating to the application of an engineered bacteriophage, and more particularly to the application of a bacteriophage capable of specifically targeting and eliminating the intestinal conditional pathogen Parasutterella secunda in the preparation of drugs for the prevention, relief or treatment of osteoarthritis, especially obesity-related or gut microbiota-driven osteoarthritis. Background Technology

[0002] Osteoarthritis (OA) is one of the most common degenerative joint diseases, characterized by cartilage loss, subchondral bone remodeling, osteophyte formation, and synovitis, leading to chronic pain and functional impairment. Traditionally, OA has been considered primarily driven by mechanical wear and aging, with excessive joint load due to obesity being considered a core contributing factor.

[0003] However, clinical observations have revealed that obesity not only increases the risk of osteoarthritis (OA) in weight-bearing joints such as the knee and hip, but also in non-weight-bearing joints such as the hand, suggesting the existence of a systemic pathogenic pathway independent of mechanical stress. In recent years, the theories of "metabolic OA" and the "gut-joint axis" have received widespread attention. These theories suggest that obesity-induced chronic low-grade inflammation and gut microbiota dysbiosis may systematically exacerbate the OA process by releasing inflammatory factors and affecting immune cell function.

[0004] Although previous studies have revealed that gut microbiota metabolites such as GUDCA can alleviate osteoarthritis (OA) through the FXR-GLP-1 axis, the specific mechanisms by which specific bacterial species directly regulate immune cells and target and attack "immune-exempt" cartilage tissue remain unclear. Our previous research identified, for the first time, a type of gut-derived T-bet+ Th17 cells in the articular cartilage of obese OA patients, and further traced the key gut bacterium driving this pathological process—Parasutterella secunda. This bacterium is significantly enriched in the gut of obese OA patients and can directly promote Th17 cell differentiation and CXCR3-dependent migration to cartilage tissue, leading to chondrocyte senescence and MMP3-mediated matrix degradation.

[0005] Therefore, specifically clearing Parasutterella secunda from the gut and blocking the pathogenic axis of "bacteria-immune cells-joints" holds promise as a disruptive treatment strategy for osteoarthritis (OA). Phage therapy, due to its high host specificity, self-replication ability, and minimal impact on normal flora, has become an ideal tool for precisely targeting pathogenic bacteria in the gut. Currently, there are no reports on phages targeting Parasutterella secunda or their application in the treatment of osteoarthritis. Summary of the Invention

[0006] The first aspect of this invention provides an engineered phage capable of targeted removal of Parasutterella secunda. This engineered phage specifically recognizes, infects, and lyses Parasutterella secunda, thereby reducing its abundance in the gut. The engineered phage of this invention can be isolated and prepared by using Parasutterella secunda as the host bacterium, from environmental samples such as human feces and sewage, through conventional phage isolation techniques such as co-culture enrichment and purification using a double-layer agar plate method.

[0007] A second aspect of the present invention provides a probiotic agent or pharmaceutical composition comprising the aforementioned engineered bacteriophage. The probiotic agent or pharmaceutical composition is an oral formulation, wherein the bacteriophage titer per gram or milliliter of the formulation is not less than 1 × 10⁻⁶. 9 PFU.

[0008] A third aspect of the invention provides the use of the above-described engineered phage or the probiotic agent / pharmaceutical composition in the preparation of a medicament for the prevention, relief, or treatment of osteoarthritis. In this invention, osteoarthritis is associated with abnormal enrichment of Parasutterella secunda in the gut, specifically obesity-related osteoarthritis; the mechanism of action of this medicament is as follows: clearing Parasutterella secunda from the gut → reducing T-bet⁺ Th17 cells in the gut and joints → inhibiting Th17 cell migration to cartilage → alleviating chondrocyte senescence and MMP3-mediated matrix degradation → ultimately improving OA pathological scores, relieving pain, and improving joint function.

[0009] A fourth aspect of this invention provides a method for screening candidate drugs for osteoarthritis. The core of this method is to evaluate the inhibitory or scavenging ability of candidate drugs against Parasutterella secunda. Specifically, it includes using Parasutterella secunda as an indicator bacterium to detect the survival rate or growth activity of Parasutterella secunda after treatment with the candidate drug. If the candidate drug can significantly reduce the survival rate of Parasutterella secunda or inhibit its growth, then the candidate drug is a potential candidate drug for osteoarthritis. This screening method, based on the well-defined pathogenic role of Parasutterella secunda in the gut-joint axis, can rapidly and accurately target and screen for specific osteoarthritis treatment candidate molecules, significantly improving the screening efficiency of innovative osteoarthritis drugs.

[0010] Beneficial effects: The present invention achieves the following effects: a) Clears Parasutterella secunda from the intestines and restores the balance of the intestinal microecology; b) Reduce the number of T-bet+ Th17 cells in intestinal and / or articular cartilage tissue; c) Inhibit the migration of T-bet+ Th17 cells to articular cartilage tissue; d) Reduce chondrocyte senescence (reduce the positive rates of SA-β-Gal, p16, and p21). e) Inhibit the expression of matrix metalloproteinase MMP3 in cartilage tissue; f) Reduces articular cartilage degeneration, synovitis, and osteophyte formation; g) Improves mechanical hyperalgesia and gait abnormalities associated with osteoarthritis; H) It significantly accelerates the repair of cartilage tissue, thereby relieving and treating osteoarthritis. Attached Figure Description

[0011] Figure 1 Parasatella accelerates the progression of osteoarthritis. A: Linear discriminant analysis effect size (LEfSe) phylogenetic clade, showing taxa with significant differences in abundance between the OA group and the OA-obesity group; B: Linear discriminant analysis (LDA) scores of differentially abundant bacterial groups between the OA group and the OA-obesity group; C: Number of observed species, Chao1 index, and Shannon index in the OA group and the OA-obesity group; D: Relative abundance of the control group and the Parasatella treatment group.

[0012] Figure 2 Parasartella accelerates osteoarthritis progression by promoting Th17 cell migration to cartilage. AB: Representative images of the knee joints of mice treated with antibiotics by Parasartella spp., stained with Safranin O-Fix Green and using micro-CT. C: Comparison of serum IL-1β and IL-6 concentrations between the two groups of mice. D: Comparison of OARSI scores, synovitis grades, cartilage thickness (Ct.Th), number of osteophytes (Op.N), and total volume of osteophytes (Op.TV) between the two groups of mice. OARSI scores, Ct.Th, Op.N, and Op.TV were analyzed using one-way ANOVA + Tukey's multiple comparison test; synovitis grades were determined using the Kruskal-Wallis test + Dunn's post-hoc test. EF: Immunofluorescence and flow cytometry analysis of the proportion of K-red+CD4+ cells in the knee cartilage tissue of mice with IL17Acre-Rosa26-iDTR of Parasartella.

[0013] Figure 3Phage clearance of *Parasartella parasatella* can resist osteoarthritis. A: qPCR detection of *Parasartella parasatella* abundance in fecal samples from mice in the control and phage-treated groups; BC: Microscopic CT and Safranin O-Fix-Green staining representative images of the knee joints of mice in the control and phage-treated groups; D: CatWalk gait analysis of pain in mice in the control and phage-treated groups; EF: Flow cytometry analysis of the proportion of K-red+CD4+ cells and T-bet+IL-17+ cells in the knee cartilage tissue of mice in the control and phage-treated groups. Figure 4 Safranin O-Fixed Green staining of tissue sections (X100). Detailed Implementation

[0014] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0015] Example 1: Isolation, purification, and amplification of phages targeting Parasutterella secunda 1. Experimental Materials Host strain: Parasutterella secunda, isolated from fecal samples of obese OA patients and identified by 16S rDNA sequencing and whole genome sequencing.

[0016] Wastewater sample: collected from the inlet of a wastewater treatment plant in Nanjing, Jiangsu Province, China.

[0017] 2. Experimental Methods Phage enrichment: Take 200 mL of wastewater sample, centrifuge at 8000g for 20 minutes at 4℃ to remove precipitate, and filter the supernatant successively through 0.45 μm and 0.22 μm filter membranes. Take 10 mL of filtrate and mix it with 10 mL of 2× BHI medium and 1 mL of P. secunda bacterial culture in logarithmic growth phase, and incubate anaerobically at 37℃ overnight. After centrifugation and filtration, the culture yields the enriched phage solution.

[0018] Phage purification: The double-layer agar plate method was used. The enriched phage solution was serially diluted 10-fold. 100 μL of each dilution was mixed with 100 μL of *P. secunda* bacterial culture and added to 5 mL of 0.4% BHI agar top layer medium. After mixing, the mixture was poured onto 1.5% BHI bottom layer agar plates. The plates were incubated anaerobically at 37°C for 24–48 hours. Single, clear, well-defined phage plaques were picked. This "infection-plaque picking" process was repeated at least three times until uniform morphological plaques were obtained.

[0019] Preparation of high-titer phage stock solution: A purified single phage plaque was inoculated into 5 mL of logarithmic growth phase *P. secunda* bacterial culture and cultured overnight at 37°C. The next day, the culture was centrifuged and filtered, and the supernatant containing the phage was collected. The phage titer (PFU / mL) was determined using the double-layer agar plate method. The final engineered phage stock solution with a titer > 1 × 10^10 PFU / mL was obtained and named ΦPsec_1. The engineered phage ΦPsec_1 was used for subsequent animal experiments.

[0020] Example 2: Evaluation of the therapeutic effect of engineered bacteriophages on obesity-related OA model mice 1. Animal model construction and grouping Experimental animals: SPF-grade male C57BL / 6 mice, 8 weeks old.

[0021] Obesity-related OA model: After mice were induced to be obese by a high-fat diet (60% fat for energy) for 12 weeks, an OA model was constructed by medial meniscus instability (DMM) surgery.

[0022] Grouping and treatment: Mice that successfully modeled the mice were randomly divided into two groups (n=6 / group): Control group (PBS group): After DMM surgery, PBS buffer was administered orally by gavage once every 4 days.

[0023] Phage therapy group: After DMM surgery, the engineered phage ΦPsec_1 prepared in Example 1 was administered orally by gavage once every 4 days, with each dose being 8×109 PFU / phage, for a total of 7 doses.

[0024] A sham surgery group (Sham group) was set up as a healthy control group.

[0025] 2. Detection Indicators ① Abundance of P. secunda in the intestine: After the last administration, mouse feces were collected, DNA was extracted, and P. secunda-specific primers (F: 5'-GAGTAACGCGTAGGTAACAGCC-3'; R: 5'-TACCCCCAACACTAGCGATT-3') were detected by qPCR.

[0026] ② Joint pathological assessment: Micro-CT: After euthanizing the mice, the knee joints were harvested for Micro-CT scanning (SkyScan 1176).

[0027] Histological staining: Safranin O / Fix Green staining was performed on the knee joint after decalcification.

[0028] ③ Pain and behavioral assessment: CatWalk gait analysis: Footprints of mice during autonomous walking were detected using the CatWalk XT system.

[0029] Von Frey pain threshold measurement: Mechanical hyperalgesia is detected using an electronic Von Frey system.

[0030] ④ Molecular and cellular mechanism verification: Flow cytometry was used to detect cells in the digestive fluid of articular cartilage.

[0031] ⑤ Immunohistochemical staining of articular cartilage sections.

[0032] like Figure 2-3 As shown, phages targeting Parasutterella secunda can clear Parasutterella secunda from the gut, restoring gut microbiota balance; reduce the number of T-bet+Th17 cells in the gut and / or articular cartilage tissue; inhibit the migration of T-bet+Th17 cells to articular cartilage tissue; alleviate chondrocyte senescence (reduce the positive rates of SA-β-Gal, p16, and p21); inhibit the expression of matrix metalloproteinase MMP3 in cartilage tissue; alleviate articular cartilage degeneration, synovial inflammation, and osteophyte formation; and improve osteoarthritis-related mechanical hyperalgesia and gait abnormalities.

[0033] In other words, by specifically eliminating this pathogen, it is possible to effectively block the migration of T-bet+ Th17 cells driven by it to articular cartilage, thereby inhibiting chondrocyte senescence and matrix degradation, and ultimately significantly reducing the pathological process, pain and functional impairment of obesity-related osteoarthritis.

[0034] Example 3: Evaluation of the therapeutic effect of engineered bacteriophages on OA model mice Eight-week-old SPF-grade male C57BL / 6 mice, weighing 20-25g, were selected. The mice were housed in an SPF-grade animal facility at a temperature of 22±2℃ and humidity of 50-60%, with a 12-hour light / 12-hour dark cycle and free access to food and water. Experiments began after one week of acclimatization.

[0035] A knee osteoarthritis model was established using medial meniscus instability surgery. Grouping and treatment: Mice that successfully developed the model were randomly divided into two groups (n=6 / group). Control group (PBS group): After DMM surgery, PBS buffer was administered orally by gavage once every 3 days.

[0036] Phage therapy group (Phage group): After DMM surgery, the engineered phage ΦPsec_1 prepared in Example 1 was administered orally by gavage once every 3 days, with each dose being 8 × 10⁻⁶. 9 PFU / animal, administered 5 times in total.

[0037] A sham surgery group (Sham group) was set up as a healthy control group.

[0038] Joint pathological assessment: Micro-CT: After euthanizing the mice, the knee joints were harvested for Micro-CT scanning (SkyScan 1176).

[0039] Scoring will proceed according to the method outlined in Table 1: Table 1. Treatment Efficacy Scoring Criteria 0 The surface of articular cartilage is smooth and appears pale blue or colorless and translucent. 1 The surface of the articular cartilage is softened, but still smooth. 2 The articular cartilage begins to thin, showing changes resembling fine fibrous bundles. 3 Fiber bundle-like changes in articular cartilage 4 The articular cartilage shows wear-resistant, fibrous bundle-like changes, accompanied by exposure of subchondral bone and osteosclerosis. Histological staining: Safranin O / Fix Green staining was performed on the knee joint after decalcification. The results are shown in Table 2: Table 2. Gross observation and scoring results of articular cartilage Control group (PBS) 3.33±0.46** Treatment group (parasutterella) 0.83±0.21*## Healthy control group (n=6) 0.00 ±0.00## Compared with the healthy control group, *p<0.05, **p<0.01; compared with the PBS group, ##p<0.01.

[0040] The data in Table 2 show that there were significant differences in the gross cartilage scores between the PBS group, the healthy control group, and the treatment group, indicating that the model was successfully established. Compared with the PBS group, the degree of cartilage damage in the treatment group was significantly reduced, and the score decreased (p<0.01), suggesting that parasutterella has a strong repair effect on cartilage tissue.

[0041] like Figure 4As shown, in the normal articular cartilage group (healthy control group), the articular cartilage cells were arranged regularly, with clear structural layers, normal cell number, and intact tide lines; in the PBS group, the articular cartilage cells were arranged disorderly, with many fissures, reduced cell number, visible necrotic cells, blurred tide lines, and severe wear and degeneration of the cartilage surface; in the treatment group (parasutterella), the articular cartilage cells were arranged regularly, with no obvious fissures, mild safranin O staining on the cartilage surface, and relatively intact tide lines.

[0042] The above results indicate that phages targeting Parasutterella secunda can effectively repair cartilage tissue, thereby alleviating or treating osteoarthritis.

[0043] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. The application of an engineered bacteriophage in the preparation of drugs for the prevention, relief, or treatment of osteoarthritis, characterized in that, The engineered phage was obtained through phage enrichment, phage purification, and preparation of high-titer phage stock solution. The phage enrichment step involved co-culturing Parasutterella secunda with the phage for enrichment.

2. In the application according to claim 1, the phage purification step is as follows: the enriched phage solution is serially diluted 10-fold and cultured with Parasutterella secunda, and single, transparent phage plaques with clear edges are picked. The "infection-plaque picking" process is repeated at least three times until a single phage plaque with a consistent morphology is obtained.

3. According to the application described in claim 1, the high-titer phage stock solution preparation step involves inoculating a purified single phage plaque into Parasutterella secunda bacterial culture in the logarithmic growth phase to finally obtain an engineered phage stock solution with a titer > 1×10^10 PFU / mL.

4. According to claim 1, the bacteriophage can specifically infect and lyse Parasutterella secunda, has strong lytic activity against the host bacteria, and does not affect the normal growth of other symbiotic bacteria in the intestine, thus maintaining the homeostatic structure of the intestinal flora.

5. The application according to claim 1, wherein the medicament further comprises a pharmaceutically acceptable delivery carrier and / or excipient.

6. In the application according to claim 1, the drug may be an oral formulation, an injectable formulation, a suppository, a patch, an inhaler, or other clinically acceptable dosage form.

7. According to the application described in claim 1, the engineered phage specifically targets and clears Parasutterella secunda accumulated in the intestine, restores intestinal flora homeostasis, reduces the level of systemic inflammation in the body, reduces articular cartilage damage, and thus achieves the effects of delaying the progression of osteoarthritis and relieving joint pain and dysfunction caused by osteoarthritis.

8. In the application according to claim 1, the engineered phage accelerates the repair of cartilage tissue by specifically targeting and clearing Parasutterella secunda accumulated in the intestine, thereby treating osteoarthritis.

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