Bacterial outer membrane vesicle wrapping long-acting growth hormone and application of bacterial outer membrane vesicle

Abstract

The invention belongs to the technical field of biological medicine and nano-drug delivery, and particularly relates to a long-acting growth hormone (hGH) wrapped bacterial outer membrane vesicle and application, and the outer membrane vesicle GH-OMVs wraps a human growth hormone mutant with an amino acid sequence shown as SEQ ID NO: 1. The GH-OMVs provided by the invention can be used for preparing medicines or preparations for preventing and / or treating diseases related to growth hormone deficiency or growth hormone-related metabolic disorder, including but not limited to growth hormone deficiency, dwarf / low height, osteoporosis and the like, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and nanomedicine delivery technology, specifically relating to a bacterial outer membrane vesicle encapsulating long-acting growth hormone (hGH) and its application, wherein the growth hormone is a mutated human growth hormone, mainly used to improve growth hormone deficiency (GHD), short stature and related metabolic diseases. Background Technology

[0002] Growth hormone deficiency (GHD) is a clinical syndrome caused by insufficient or absent secretion of growth hormone (GH) from the pituitary gland. Its main manifestations include short stature, delayed skeletal development, insufficient muscle mass, and metabolic abnormalities. Recombinant human growth hormone (rhGH) has become the primary drug treatment for GHD since its clinical application. However, natural human growth hormone (hGH) has a short serum half-life (typically on the order of minutes), often requiring daily or every-other-day subcutaneous injections to maintain effective blood concentrations. This results in frequent dosing, a heavy long-term treatment burden, and poor patient compliance, potentially affecting the sustainability and stability of therapeutic efficacy. Therefore, the development of long-acting formulations and molecular modification of hGH has been a crucial research and industrialization direction in this field.

[0003] To prolong the in vivo action time of hGH, existing technologies have proposed a variety of long-acting strategies, such as: (1) chemical modification: such as polyethylene glycol (PEG) modification, which can increase the molecular hydration radius and prolong the in vivo circulation time, but may cause immune-related risks and may affect receptor binding activity or biological effects; (2) fusion protein: such as fusion with Fc fragment or human serum albumin (HSA) to improve in vivo stability, but often leads to a significant increase in molecular weight, complicated expression purification and quality control, and may bring about problems such as changes in immune safety and tissue distribution; (3) mutation modification: introducing specific mutations into the GH amino acid sequence to improve receptor affinity, reduce clearance rate or change in vivo dynamics is one of the more feasible molecular level solutions. However, relying solely on molecular modification may still be affected by factors such as in vivo protease degradation, non-targeted distribution and insufficient local retention, making it difficult to simultaneously achieve "high activity, long-acting effect and stable delivery".

[0004] Besides molecular long-term efficacy, the choice of drug delivery system also directly affects therapeutic effects. Traditional free GH may degrade or be rapidly eliminated in vivo, and its biodistribution lacks targeting and tissue retention advantages. In recent years, bacterial outer membrane vesicles (OMVs) have gradually attracted attention as a natural nanodelivery platform. OMVs are lipid bilayer vesicles spontaneously released by Gram-negative bacteria, with a particle size typically ranging from 50 to 200 nm, and can carry proteins, nucleic acids, and metabolites. Compared with artificial nanocarriers such as liposomes, OMVs have the advantages of structural stability, engineerability, and ease of loading biomolecules; at the same time, the natural membrane components on the surface of OMVs may provide a certain degree of protection for the loaded molecules and help improve in vivo stability and tissue retention. However, among the existing publicly available technologies, there is a lack of mature methods for effectively loading highly active mutant hGH onto OMVs for delivery, making it difficult to simultaneously solve the problems of short GH half-life and insufficient delivery efficiency / stability.

[0005] Therefore, there is an urgent need for a new delivery strategy that can combine highly active or mutation-optimized hGH with a vesicle delivery platform to improve in vivo stability and reduce dosing frequency while ensuring efficacy, thereby improving patient compliance and providing a new technical pathway for the treatment of GHD and related diseases. Summary of the Invention

[0006] This invention provides a formulation and its use for delivering mutant human growth hormone outer membrane vesicles (OMVs), in order to improve the problems of short duration of action, high frequency of administration and insufficient in vivo stability of existing rhGH.

[0007] This invention provides a human growth hormone mutant, the amino acid sequence of which is shown in SEQ ID NO:1.

[0008] In another preferred embodiment of the present invention, the present invention also provides outer membrane vesicles GH-OMVs that encapsulate the human growth hormone mutant described in the present invention.

[0009] Preferably, the human growth hormone mutant is further linked in tandem with a signal peptide, CTP, and a fluorescent tracer protein.

[0010] Preferably, the signal peptide is OmpA, and its amino acid sequence is shown in SEQ ID NO:2.

[0011] Preferably, the fluorescent tracer protein is mNeonGreen, and its amino acid sequence is shown in SEQ ID NO:3.

[0012] Preferably, the amino acid sequence of the CTP is shown in SEQ ID NO:5.

[0013] Preferably, the outer membrane vesicles are composed of genotypes of Nissle 1917 ; ΔNlpI Production of engineered bacteria.

[0014] In another preferred embodiment of the present invention, the present invention also provides a pharmaceutical composition comprising the human growth hormone mutant and / or outer membrane vesicle GH-OMVs described herein, and a pharmaceutically acceptable carrier or excipient.

[0015] In another preferred embodiment of the present invention, the present invention also provides a method for preparing outer membrane vesicles GH-OMVs, which includes the following steps: (1) Synthesize the encoding gene of OmpA-mNeonGreen-hGH-6×His and insert it into the expression vector; (2) The expression vector was introduced into the host bacterium ΔNlpI EcN; (3) After culturing the host and expressing it under induction conditions, the culture supernatant was collected; (4) The supernatant was separated and purified by tangential flow filtration (TFF) to obtain outer membrane vesicles GH-OMVs containing mutant human growth hormone.

[0016] Preferably, the amino acid sequence of the OmpA-mNeonGreen-hGH-6×His is as shown in SEQ ID NO:4.

[0017] In another preferred embodiment of the present invention, the present invention also provides the use of the human growth hormone mutants described in the present invention, the outer membrane vesicles GH-OMVs described in the present invention, and / or the pharmaceutical compositions described in the present invention in the preparation of medicaments for the prevention and / or treatment of diseases or symptoms related to growth hormone deficiency or growth hormone-related metabolic abnormalities.

[0018] Preferably, the disease or symptom includes, but is not limited to, growth hormone deficiency, dwarfism / short stature, and osteoporosis.

[0019] Compared with the prior art, the main advantages of this invention are: (1) The mutant human growth hormone (SEQ ID NO:1) of the present invention optimizes receptor interaction / biological activity through double mutation of amino acids; at the same time, it provides physical protection and delivery carrier for the protein by means of outer membrane vesicles (OMVs), thereby taking into account both activity and stability.

[0020] (2) Compared with wild-type hGH, the hGH mutants and GH-OMVs of the present invention have shown enhanced efficacy and extended duration of action in in vitro and in vivo drug evaluations, thus possessing the potential to reduce the frequency of administration and improve compliance.

[0021] (3) The lipid bilayer structure of OMVs can protect the loaded mutant GH, making it more stable in the environment of gastrointestinal proteases or plasma proteases, reducing the risk of rapid degradation and clearance in vivo; at the same time, it can improve storage stability and transport tolerance.

[0022] (4) The present invention uses engineered bacteria (ΔNlpI EcN) for expression and concentrates and purifies the supernatant by tangential flow filtration (TFF). The process route is clear and the operation unit is mature, which is convenient for scale-up production. The key quality attributes of the obtained GH-OMVs, such as particle size distribution, drug loading / drug loading rate, and protein content, can be quantitatively controlled.

[0023] (5) The GH-OMVs of the present invention can be used to prepare drugs or preparations for the prevention and / or treatment of diseases related to growth hormone deficiency or growth hormone-related metabolic abnormalities, including but not limited to growth hormone deficiency, short stature / low height, osteoporosis, etc., and have good clinical application prospects.

[0024] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0025] Figure 1 This is a flowchart of the production and enrichment process of engineered bacteria expressing hGH-OMVs.

[0026] Figure 2 The results show the binding ability of natural GH and mutant GH to GHR, as well as the cell proliferation ability.

[0027] Figure 3 These are the results of load detection and particle size potential characterization of the outer membrane vesicles of engineered bacteria.

[0028] Figure 4 These are the results of the efficacy and long-term effects test of GH-OMVs.

[0029] Figure 5 This is the result of the dose-response relationship between GH-OMVs and their muscle growth-promoting effect. Detailed Implementation

[0030] 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.

[0031] Example 1: Preparation of GH-loaded bacterial outer membrane vesicles The schematic diagram of the construction of engineered bacteria and the production and characterization of bacterial outer membrane vesicles is shown below. Figure 1 As shown, the specific operation is as follows: 1. Construct and assemble plasmid vectors for GH expression The gene encoding OmpA-mNeonGreen-hGH-6×His, as shown in SEQ ID NO:4, was synthesized. This fusion protein contains an OmpA signal peptide, a fluorescent tracer protein, CTP, and a mutant hGH, with a 6×His fused to the C-terminus for easy detection / purification. The sequence and function of each functional fragment are as follows: The OmpA signal peptide (SEQ ID NO:2) is used to guide the fusion protein to the outer membrane / peripheral space, promoting entry into the vesicle loading pathway; mNeonGreen (SEQ ID NO:3) is a tracer protein, facilitating visualization / quantitative detection of expression and vesicle enrichment; mutant hGH (SEQ ID NO:1) is the highly active human growth hormone of this invention; the N-terminus and C-terminus of mutant hGH are linked to 2X CTP (SEQ ID NO:5), and the fragments are linked by flexible linker peptides (Gly-Gly-Gly-Gly-Ser)n (n=2-4) to reduce steric hindrance between domains. The synthesized fragments were placed into the multiple cloning site (MCS) of the pET28 plasmid and expressed under the control of the T7 promoter and RBS; the insertion method was TA cloning, and the insertion direction and reading frame were confirmed by Sanger sequencing.

[0032] 2. Construction of engineered bacteria for high-yield GH-loaded vesicle production A small amount of plasmid vector solution was added to 100 μL of competent cells. E. coli In Nissle 1917; ΔNlpI, gently mix and place on ice for 30 min, followed by heat shock at 42 ℃ for 1.5 min, and then immediately on ice for 2 min. Add 900 μL of antibiotic-free LB medium to the mixture, and incubate at 37 ℃ and 220 rpm for 30 min to recover. Spread the bacterial culture on LB agar plates containing kanamycin and incubate at 37 ℃ for 12–14 h to obtain single-clone strains. Select morphologically regular colonies and amplify the insertion region using universal primers T7-R / F and insertion region primers. The amplified products were identified by Sanger sequencing, confirming the sequence was correct.

[0033] 3. Production and isolation of bacterial outer membrane vesicles encapsulating GH Select correctly identified monoclonal strains and inoculate them into 5 mL of LB liquid medium containing kanamycin, and culture at 37 ℃ and 220 rpm for 12 h. Inoculate 25 mL of TB medium containing antibiotics into a 500 mL Erlenmeyer flask and culture at 37 ℃ and 200 rpm (≥25 mm track distance) with shaking until the OD600 of the culture reaches 0.8–1.0. At this point, add IPTG to a final concentration of 20 μg / mL and induce at 37 ℃ for 12–14 h.

[0034] Collect the bacterial culture, centrifuge at 6000 g for 30 min to remove the bacterial cells, and obtain the supernatant containing OMVs.

[0035] 4. Tangential flow filtration (TFF) purification The supernatant was introduced into a tangential flow filtration (TFF) system for OMV concentration and purification. The TFF parameters were as follows: membrane material: PES; molecular weight cutoff (MWCO): 100 kDa; operating pressure: 6 Psi; flow rate: 30 mL / min; concentration factor: 10. Continuous circulation through the TFF system enabled impurity removal and OMV concentration. The OMVs were then washed and replaced with PBS to obtain high-purity GH-encapsulated bacterial outer membrane vesicles (GH-OMVs).

[0036] Example 2: Verification of the binding ability of mutant GH and wild-type GH to GHR Before the engineered long-acting mutant GH protein (SEQ ID NO:1) can be used for drug delivery, it needs to be verified whether it still retains its binding ability to the GH receptor (GHR). This embodiment compares the activity of mutant GH and wild-type GH using molecular docking simulation and cell proliferation experiments. The specific procedures are as follows: Modeling and Structure Preparation: The publicly available crystal structure of the GH-human GH receptor complex (PDB ID: 3HHR) was selected as a reference template. The amino acid sequence of the mutant GH (SEQ ID NO:1) was input into the AlphaFold2 platform for 3D structure prediction, and a pdb file was output.

[0037] Molecular docking simulation: The predicted GH protein structure and GHR receptor structure were imported into the molecular docking software ClusPro. The known GH binding region of GHR was set as the active site, and docking calculations were performed on wild-type GH and mutant GH. The binding conformations were output, and the binding free energy (ΔG), number of hydrogen bonds, hydrophobic interactions, and interfacial residue interactions were compared between the two.

[0038] Molecular docking simulation results ( Figure 2The results showed that interfacial residue analysis indicated that the mutant GH retained the key amino acids (Tyr103, Phe167, Asp171) required for binding with GHR, and the overall binding conformation was intact. The binding energy of the central region was -1044.7 kJ, and the lowest energy conformation was -1486.3 kJ, indicating that it possesses receptor activation function, providing theoretical support for its application.

[0039] The MTT assay was used to detect the effects of wild-type growth hormone and its mutants on cell proliferation activity, in order to evaluate their proliferative effects at the cellular level. The specific experimental procedures are as follows: Cell resuscitation and culture Remove the MC3T3 cell cryovials from the liquid nitrogen container and immediately place them in a 37°C water bath, shaking rapidly until the cryopreservation solution has just thawed. Transfer the cryopreservation solution to a centrifuge tube, add pre-warmed culture medium to dilute, and centrifuge (200-300×g, approximately 5 min). Discard the supernatant to remove the cryoprotectant. Resuspend the cells in approximately 1 mL of fresh culture medium, transfer to a culture dish, and add culture medium to approximately 5 mL. Incubate at 37°C in a 5% CO2 incubator. The next day, replace approximately half of the culture medium to remove non-adherent cells and metabolic waste, and continue culturing. When the cell confluence reaches approximately 80%, discard the culture medium and wash the cells twice with PBS. Add an appropriate amount of trypsin for digestion. Observe under a microscope as the cells gradually become rounded from a spread state and the intercellular spaces increase. Discard the trypsin and add fresh culture medium to stop digestion. Gently pipette to detach the cells and mix thoroughly. Passage the cells at a 1:4 ratio.

[0040] Cell counting and drug administration When cell confluence reached approximately 80%, the culture medium was discarded, and the cells were washed twice with PBS. A suitable amount of trypsin was added for digestion. Under a microscope, the cells were observed to gradually become rounder and the intercellular spaces increased. The trypsin was then discarded, and fresh culture medium was added to terminate the digestion. The decongested cell suspension was diluted with culture medium, and cell counting was performed under a microscope using a cell counting chamber. The cell suspension was then adjusted to 3 × 10⁻⁶ cells / mL with fresh culture medium. 4 Add 200 μL of cell suspension (approximately 6 × 10⁶ cells / mL) to each well of a 96-well plate. 3 (100 cells) were incubated at 37 ℃ and 5% CO2 for 24 h to allow cell adhesion and resume growth. After 24 h, when cell confluence was observed to be approximately 50%, drug administration was initiated. The concentration was set at 100 ng / mL, with 6 replicates. BSA was used as the negative control, and blank wells were not seeded with cells, only culture medium was added for background subtraction.

[0041] After drug administration, the cells were cultured for another 48 h. Then, 20 μL of MTT solution was added to each well, and the cells were incubated for another 4 h. When a blue-purple formazan precipitate appeared at the bottom of the well, the supernatant was discarded, and 100 μL of DMSO was added to each well to dissolve the precipitate. The plates were then shaken in a microplate reader for about 1 min to ensure complete dissolution, and the absorbance (OD) of each well was measured at a wavelength of 570 nm.

[0042] The effects of natural human growth hormone and its mutants on the proliferation of MC3T3-E1 cells, such as Figure 2 As shown, both wild-type growth hormone and the mutant had a certain promoting effect on the growth of MC3T3-E1 cells, and the effect was higher than that of the control group. At a concentration of 100 ng / mL, the mutant had a greater effect on the proliferation of MC3T3 cells than the wild-type growth hormone.

[0043] Example 3 Characterization and Load Detection of Outer Membrane Vesicles of Engineered Bacteria The experimental samples were the expression supernatant of the engineered bacteria constructed in Example 1 and the purified bacterial outer membrane vesicles. To confirm the potential and particle size distribution of the prepared GH-OMVs, the specific details are as follows: Zeta Potential Detection: GH-OMVs purified by TFF were diluted in PBS solution, and the sample concentration was adjusted to a suitable detection range (approximately 1×10^9-1×10^10 particles / mL). Electrophoretic mobility was measured using a dynamic light scattering / electrophoretic light scattering integrated instrument at 25 °C. The zeta potential value was calculated using the Smoluchowski equation.

[0044] Dynamic light scattering (DLS) detection: Dilute the GH-OMVs suspension to a suitable concentration, and add 1 mL of sample to a cuvette. Detect using a dynamic light scattering analyzer, with the temperature control set to 25 ℃. Repeat the test 3 times for each sample and take the average value.

[0045] Experimental results ( Figure 3 The results show that the surface zeta potential of GH-OMVs is stable at around -47 mV, indicating that the vesicle surface carries a negative charge and has good colloidal stability. The particle size of GH-OMVs is mainly distributed in the range of 100-400 nm, with Z-Average = 173.4 nm and PDI = 0.454, indicating good vesicle dispersion.

[0046] To confirm the prepared OMVs loading, Western blot (WB) analysis was performed. The specific method is as follows: Add 5×SDS loading buffer to the culture supernatant and the purified bacterial outer membrane vesicle samples, respectively. Heat at 95℃ for 5 min to fully denature. Separate proteins by SDS-PAGE electrophoresis. Take the protein gel separated by SDS-PAGE electrophoresis and gently rinse with pure water to remove residual electrophoresis solution. Cut a 0.22 μm PVDF membrane and completely immerse the PVDF membrane in methanol for about 1 min. Prepare transfer buffer and stack filter paper, gel, protein membrane, and filter paper in a transfer clamp. Assemble, add ice, and transfer the membrane in an ice bath. Transfer at a constant voltage of 300V for 20 min. Wash the membrane with TBST, then add 5% BSA blocking buffer and block overnight at 4℃. Wash the membrane with TBST, then place the membrane in His Tag primary antibody solution and incubate overnight. Remove the primary antibody incubation solution, wash the membrane with TBST, and repeat 3-5 times. Place the membrane in the appropriate secondary antibody solution and incubate at 37℃ and 100 rpm for 3 h. Add TBST and wash the PDVF membrane, repeating 3-5 times. Mix chromogenic agents A and B in a 1:1 ratio to completely impregnate the PDVF membrane, then expose it in an exposure unit. Determine protein expression and distribution based on band brightness and position.

[0047] Immunoblotting results showed that the target protein had a distinct band at a specific size in OMVs, indicating high abundance and specific expression of the target protein. Figure 3 ).

[0048] Example 4: Pharmacological study of GH-loaded engineered bacterial outer membrane vesicles in mice To verify the efficacy and long-term effects of GH-OMVs and evaluate their growth-promoting effect, efficacy testing was conducted in juvenile mice. The specific procedures are as follows: The experimental animals were 3-week-old male C57BL / 6J mice. They were housed in an SPF animal facility under constant temperature and humidity, with 12 hours of light / 12 hours of darkness, and free access to food and water. Animals were randomly assigned to three groups: a control group (PBS, 100 μL subcutaneously injected weekly); a positive control group (0.1 mg / kg natural growth hormone, subcutaneously injected daily); and a GH-OMVs group (0.1 mg / kg GH-loaded OMVs, subcutaneously injected weekly). Treatment continued for 4 weeks. Measured parameters included body weight, food intake, body length, and tail length.

[0049] Experimental results ( Figure 4 The results showed that, compared with the control group, GH-OMVs exhibited weight gain comparable to that of natural growth hormone, with both significantly higher than the control group. Meanwhile, the mutant growth hormone had a slightly better effect on food intake and body length in mice than natural growth hormone, while there was no difference in tail length among the three groups.

[0050] To verify the dose-dependent nature of GH-OMVs and determine the safe concentration for their growth-promoting effect, a dose-dependent efficacy assay was performed in juvenile mice. The specific procedures are as follows: Six-week-old male C57BL / 6J mice were used as experimental animals. They were housed in an SPF animal facility under constant temperature and humidity, with 12 hours of light / 12 hours of darkness, and free access to food and water. Animals were randomly assigned to three groups: control group (PBS, 100 μL subcutaneously weekly); vector group (empty OMVs, 100 μL subcutaneously weekly); low-dose GH-OMVs group (1 mg / kg, subcutaneously weekly); and high-dose GH-OMVs group (2 mg / kg, subcutaneously weekly). Administration lasted for 4 weeks. Measured parameters included body weight, tibia length, and muscle fiber diameter and density.

[0051] Experimental results ( Figure 5 The results showed that, compared with the control group, both the high- and low-dose GH-OMV groups exhibited weight gain and increased tibial length, suggesting that GH-OMVs promoted muscle and bone growth. No organ hypertrophy was observed in the weight of major organs (Table 1), indicating that GH-OMV has good safety at effective doses. Muscle H&E staining showed that muscle fibers were fuller and the average muscle fiber density increased after GH-OMV intervention.

[0052] Table 1 Weight of major organs

[0053] 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. A human growth hormone mutant, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. The human growth hormone mutant according to claim 1, characterized in that, The human growth hormone mutant is also linked to a signal peptide, CTP, and a fluorescent tracer protein.

3. The human growth hormone mutant according to claim 2, characterized in that, The signal peptide is OmpA, and its amino acid sequence is shown in SEQ ID NO:

2.

4. The human growth hormone mutant according to claim 2, characterized in that, The fluorescent tracer protein is mNeonGreen, and its amino acid sequence is shown in SEQ ID NO:

3.

5. The human growth hormone mutant according to claim 2, characterized in that, The outer membrane vesicles are composed of genotypes of Nissle 1917 ; ΔNlpI Production of engineered bacteria.

6. An outer membrane vesicle GH-OMVs encapsulating the human growth hormone mutant as described in any one of claims 1-5.

7. A pharmaceutical composition comprising the human growth hormone mutant of any one of claims 1-5 and / or the outer membrane vesicle GH-OMVs of claim 6, and a pharmaceutically acceptable carrier or excipient.

8. A method for preparing outer membrane vesicles GH-OMVs, comprising the following steps: (1) Synthesize the encoding gene of OmpA-mNeonGreen-hGH-6×His and insert it into the expression vector; (2) The expression vector was introduced into the host bacterium ΔNlpI EcN; (3) After culturing the host and expressing it under induction conditions, the culture supernatant was collected; (4) The supernatant was separated and purified by tangential flow filtration (TFF) to obtain outer membrane vesicles GH-OMVs containing mutant human growth hormone.

9. The method for preparing outer membrane vesicles GH-OMVs according to claim 8, wherein the amino acid sequence of OmpA-mNeonGreen-hGH-6×His is shown in SEQ ID NO:

4.

10. The use of the human growth hormone mutant according to any one of claims 1-5, the outer membrane vesicle GH-OMVs according to claim 6, and / or the pharmaceutical composition according to claim 7 in the preparation of a medicament for the prevention and / or treatment of diseases or symptoms related to growth hormone deficiency or growth hormone-related metabolic abnormalities, characterized in that, The diseases or symptoms mentioned include, but are not limited to, growth hormone deficiency, short stature / low height, and osteoporosis.

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