CN116159148A - Antisense oligonucleotide enema based on bacterial vector and preparation method and application thereof - Google Patents

Antisense oligonucleotide enema based on bacterial vector and preparation method and application thereof Download PDF

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CN116159148A
CN116159148A CN202310160524.6A CN202310160524A CN116159148A CN 116159148 A CN116159148 A CN 116159148A CN 202310160524 A CN202310160524 A CN 202310160524A CN 116159148 A CN116159148 A CN 116159148A
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antisense oligonucleotide
bacteria
enema
bacterial vector
adhesive
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张峻峰
吴锦慧
陈懿昀
许海恒
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Xishan Institute Of Applied Biotechnology Nanjing University Wuxi
Nanjing University
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Xishan Institute Of Applied Biotechnology Nanjing University Wuxi
Nanjing University
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Abstract

The invention relates to an antisense oligonucleotide enema based on a bacterial carrier, a preparation method and application thereof, which comprises bacteria and antisense oligonucleotides, wherein the surfaces of the bacteria are wrapped with positively charged adhesive, and the antisense oligonucleotides are adsorbed on the surfaces of the adhesive. The invention uses bacteria as a carrier, and coats the surface of the bacteria with the adhesive, and the adhesive has positive charges, so that antisense oligonucleotide can be adsorbed, thereby achieving the delivery effect, improving the bioavailability, avoiding systemic immunosuppression caused by systemic administration, and having better safety.

Description

Antisense oligonucleotide enema based on bacterial vector and preparation method and application thereof
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to an antisense oligonucleotide enema based on bacterial vector delivery, and a preparation method and application thereof.
Background
Inflammatory Bowel Disease (IBD) is a non-specific chronic recurrent inflammatory bowel disease, mainly including ulcerative colitis (Ulcerative colitis, UC) and Crohn's Disease (CD). Patients often experience symptoms such as abdominal pain, diarrhea, bloody stool, etc., and patients need intermittent treatment due to the recurrent nature of the disease, and serious patients need surgical treatment. Total colorectal resection, ileum Pouch Anal Anastomosis (IPAA), is the surgical intervention of choice for patients with ulcerative colitis. Although IPAA treatment results well, several complications can still occur, including the most common pouchitis (pouchitis).
The mechanism of occurrence of pouchitis is not known, and studies have shown that the structure of the pouch formed after IPAA surgery is gradually changed over time, which is indicated by loss or inactivation of villi and deepening of crypt, although it is composed of only the ileum, and this change is called "colonicmetaplasia". In addition, changes in total mucosal thickness, intrinsic layer chronic inflammatory cell infiltration, etc. all indicate that Chu Daiyan is histologically similar to ulcerative colitis, so it is speculated that the primary mechanism of occurrence of pouchitis is recurrence of ulcerative colitis in the ileal pouch of colitis.
For acute pouchitis, the antibiotic treatment effect is obvious, however, for recurrent pouchitis and chronic pouchitis, the antibiotic response rate is lower, and for the patients with the acute pouchitis and the patients with antibiotic resistance, the immunosuppressant medicine becomes an ideal treatment means.
Tumor necrosis factor TNF- α (Tumor necrosis factor- α) is a pro-inflammatory cytokine, produced primarily by activated macrophages. Intestinal macrophages produce a large amount of TNF- α, recruiting leukocyte infiltration to the site of inflammation, thereby promoting intestinal inflammation. Small molecule inhibitors of TNF- α block their pro-inflammatory effects, thereby alleviating the symptoms of the patient. However, since small molecule inhibitors have cell permeability, they can diffuse throughout the body along with the blood circulation, and blocking TNF- α action at non-inflammatory sites can lead to immunosuppression of the body, failing to provide normal immune resistance against foreign pathogens. Therefore, avoiding systemic exposure and immunosuppression at non-inflammatory sites is highly desirable.
Antisense oligonucleotides (Antisense oligonucleotides, ASO) are small, synthetic single-stranded nucleic acid polymers of about 18-30 nt capable of specifically binding RNA and modulating gene expression by both steric hindrance and cleavage of RNA by RNase H1. Currently, 9 ASO drugs are put on the market in batches, and tens of drugs are being researched. Because the half-life period of the nucleic acid medicine is short, the nucleic acid medicine is easy to be degraded by enzyme in blood, local administration can be effectively carried out without diffusing to the whole body, and the body immunosuppression is reduced. However, since nucleic acids are hydrophilic macromolecules and negatively charged, their membrane permeability is low, it is difficult to cross epithelial cell barriers, it is difficult to enter cells for gene regulation, and nucleic acids are easily degraded when exposed to the environment. Therefore, the development of a safe and effective delivery system is critical.
Studies have shown that gram-negative bacteria such as e.colinissle 1917, gram-positive bacteria such as Listeria, partial anaerobes and obligate anaerobes can cause up-regulation of TL1A protein expression in immune cells, which up-regulation of TL1A protein expression as a member of TNF superfamily (Tumor necrosis factor superfamily) causes immune cells to secrete pro-inflammatory factors such as IFN- γ, thereby causing immune responses and inflammation at the intestinal tract. In addition, lipopolysaccharide LPS expressed on the surface of bacteria, flagellin, and the like can activate Toll-like receptors (Toll-like receptors) on immune cells, thereby causing immune responses.
Disclosure of Invention
The applicant found in the study that although the above bacteria can cause inflammation at the intestinal tract, when the bacteria such as E.coli nissle1917 are used as a delivery carrier, the antisense oligonucleotide for inhibiting TNF-alpha can be adsorbed and delivered into immune cells under intestinal epithelial cells, so that the immune response of macrophages is not caused on the whole, and the secretion of pro-inflammatory factor TNF-alpha can be inhibited, and the effect of relieving the inflammation of the intestinal tract can be achieved.
Based on the above findings, the applicant constructs an antisense oligonucleotide enema based on a bacterial vector, which utilizes the characteristic that bacteria capable of entering an internal environment through epithelial cells are phagocytized by macrophages through immune cell surface pattern recognition receptors, and loads nucleic acid drugs on the surface of the bacterial vector, thereby completing in vivo delivery of the antisense oligonucleotide by means of the bacterial vector and achieving the purpose of gene silencing. The antisense oligonucleotide delivered by the bacterial vector can realize drug delivery more efficiently, improve the bioavailability, avoid systemic immunosuppression caused by systemic administration and have higher safety.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
an antisense oligonucleotide enema based on a bacterial vector comprising a bacterium and an antisense oligonucleotide, said bacterium surface being coated with a positively charged adhesive, said adhesive surface having said antisense oligonucleotide adsorbed thereon.
Preferably, the antisense oligonucleotide is an antisense oligonucleotide directed against TNF- α, JAK, TYK2, ICAM-1, IL-12, IL-23 or α4β7 integrin.
Preferably, the antisense oligonucleotide has a sequence of 5'-AACCCATCGGCTGGCACCAC-3' or 5'-GCCCAAGCTGGCATCCGTCA-3'.
Preferably, the adhesive is one or two of chitosan or protamine.
Preferably, the bacteria are attenuated salmonella, attenuated listeria or intestinal probiotics.
Preferably, the intestinal probiotics are one or more of L. rhamnosus GG, L. rhamnosus OLL2838, E. coli Nissle1917, S. thermophilus NCIMB41856 or bif. Longum CCM 7952.
Preferably, the ratio of the antisense oligonucleotide to the bacterium is (0.1-10 ug): 2×10 7 cfu)。
Preferably, the ratio of the adhesive to the bacteria is (1.5-2.5 mg): 1-2×10 7 cfu)。
The invention also provides a preparation method of the antisense oligonucleotide enema based on the bacterial vector, which comprises the following steps:
(1) Suspending the bacteria in a solvent and mixing with an adhesive to obtain a bacterial concentration of 1-2×10 7 cfuml −1 Incubating for 20-40 min to obtain wrapped bacteria;
(2) Mixing the encapsulated bacteria with the antisense oligonucleotide in a solvent, and incubating for 20-40 min to obtain the antisense oligonucleotide.
Preferably, the solvent is physiological saline, ringer's solution, phosphate buffer or water.
Preferably, the antisense oligonucleotide is prepared by a DNA synthesizer.
The invention also provides application of the antisense oligonucleotide enema based on the bacterial vector in preparing an enema medicine for relieving inflammation of a storage bag.
The invention has the beneficial effects that:
bacteria, especially probiotics, are used as carriers, and negative-charge antisense oligonucleotide drugs can be adsorbed after the carriers are coated with positive-charge adhesive, so that the antisense oligonucleotide drugs are not easy to degrade in vivo, the cycle period of the antisense oligonucleotide drugs is prolonged, and the bioavailability is improved. The bacterial delivery system can pass through intestinal epithelial cells, and after being ingested by intestinal macrophages, the surface-adsorbed antisense oligonucleotide is delivered into cells, is combined with intracellular mRNA and silences the gene of the intracellular mRNA, so that the intestinal macrophages stop or reduce the secretion of TNF-alpha, or prevent immune cells from migrating to an inflammation part, reduce the inflammatory reaction of a storage bag and relieve the symptoms of patients. Meanwhile, the probiotics can improve the deregulated intestinal flora, and the probiotic is low in cost, simple and convenient to prepare, high in safety and ideal in drug delivery carrier.
Drawings
FIG. 1 shows the results of Elisa in vitro experiments on macrophages in example 1.
Fig. 2 is a graph of the pathological scoring data for rats in example 1.
FIG. 3 is a graph of rat weight data in example 1.
FIG. 4 is a graph of colon length data for the rats of example 1.
Fig. 5 is a graph of the pathological scoring data for rats in example 7.
Description of the embodiments
Examples
1. Enema preparation based on e.coli Nissle 1917:
(1) The E.coli Nissle1917 used in this example was purchased from ATCC, streaked on plates, and single colonies were obtained and placed in liquid LB medium for shaking culture overnight (37 ℃,150rpm under aerobic conditions), and the strains were stored in a-80℃refrigerator using 25% glycerol.
(2) Single colonies were isolated (under aerobic conditions) prior to preparation using plate streaking, picked and shake-cultured overnight (37 ℃,150 rpm) in liquid medium to activate them.
(3) The bacterial solution was centrifuged at 3000rpm for 10min, and the supernatant was discarded, and the bacterial cells were resuspended in 0.5M NaCl solution.
(4) The above procedure was repeated and washed 3 times with 0.5M NaCl solution.
(5) Determination of OD with ultraviolet Spectrophotometer 600 And (5) counting the value.
(6) Chitosan (2 mg/mL) was dissolved in 0.5M NaCl solution to adjust the pH.
(7) Bacterial vectors were resuspended with a solution containing 2mg/mL chitosan and the bacterial concentration was adjusted to 2X 10 7 cfuml −1 。
(8) Incubating for 30min to enable chitosan to be entrapped on the surface of bacteria.
(9) The cells were collected by centrifugation at 3000rpm for 10min and washed 2 times with 0.5M NaCl solution to give a coated bacterial suspension.
(10) The lyophilized powder of TNF- α antisense oligonucleotide (SEQ ID NO: 5'-AACCCATCGGCTGGCACCAC-3') was centrifuged at 12000rpm for 1min.
(11) The lyophilized powder of TNF- α antisense oligonucleotide was dissolved using 0.5M NaCl solution.
(12) Adding TNF-alpha antisense oligonucleotide into the prepared encapsulated bacterial suspension, so that the final concentration is 1 mug/ml, 5 mug/ml and 10 mug/ml respectively, and incubating for 30min to obtain the final product of the chitosan-encapsulated escherichia coli Nissle1917 enema of the TNF-alpha antisense oligonucleotide.
2. In vitro drug efficacy verification:
the in vitro efficacy of the prepared enema is verified by utilizing RAW264.7 cell line and THP-1-derived macrophages. RAW264.7 cell line and THP-1-derived macrophages were purchased from ATCC and cultured in DMEM medium and RPMI 1640 medium containing 10% FBS, respectively (37 ℃,5% CO) 2 ）。
(1) And discarding the culture solution of RAW264.7 cell line and THP-1-derived macrophages, rinsing with PBS, and adding 25% pancreatin to digest for 1-2 min.
(2) Digestion was stopped by adding twice the volume of complete medium and cells were harvested by centrifugation at 1500rpm for 10 min.
(3) Counting cells with a blood cell counting plate, taking 2.5X10 5 Cell mass was plated in 24-well plates and cultured overnight.
(4) Cells were stimulated by addition of LPS at a final concentration of 2ug/mL, 2h later, the solution was changed, antisense oligonucleotide (ASO) was added in groups, the prepared enema (EcN 1917-ASO) was added, and the blank (blank) was not added with the drug, and after 4h, the solution was changed, and the supernatant and cells were collected, respectively.
(5) Cell lysate was added to the cell pellet to lyse it, and the supernatant was centrifuged at 1500rpm for 10 min.
(6) All collected supernatants were added to 100ul/well plates, respectively, and diluted Cytokinestandard was added to standard wells, and Diluionbuffer was added to blank wells, all at 100ul/well.
(7) Adding 50ul/well detection antibody, mixing, covering with sealing plate membrane, and incubating at 37deg.C for 90min.
(8) The well plate was discarded, 1 Xwash buffer,300ul/well was added, the well plate was discarded after 1min of residence, and washing was repeated 4 times, each time the well plate was required to be dried.
(9) 100ul/well Streptavidin-HRP was added, the plate membrane was covered and incubated at 37℃for 30min.
(10) The step 8 is repeated, and the plate is washed for 4 times.
(11) And adding 100ul/well TMB for color development, and incubating at 37 ℃ in a dark place for 5-30 min, and stopping the reaction according to an actual result. 100ul/well Stopsolution was added at the termination of the reaction.
(12) The data were analyzed using a microplate reader at a wavelength of 450nm within 10min after termination, combined with the label, and the results are shown in FIG. 1.
As can be seen from fig. 1, the macrophages secrete TNF- α under LPS induction, while the TNF- α values in the administered group are significantly reduced, indicating that the antisense oligonucleotide drug delivered by the bacterial vector is effective in cells.
3. Animal efficacy verification
And constructing a pouchitis model by adopting SD rats, verifying that the laboratory rats are purchased from the Ji-Cui-Xuankang, and are fed into a clean barrier of a laboratory animal center of Nanjing university, wherein the temperature of a feeding room is about 25 ℃, the humidity is 40% -70%, and the newly introduced laboratory animals are adapted for 1-3 days before the experiment. Is in line with the ethics of experimental animals at university of Nanjing.
(1) And (3) bag storage molding: SD rats are fasted and forbidden for more than 12 hours before operation, and are anesthetized by intraperitoneal injection of 10% chloral hydrate solution (3 ml/kg), the supine position of the rats is fixed, and after the abdominal hair of the rats is carefully removed by a blade, the rats are sterilized by 75% ethanol, and then complete colorectal resection and ileum storage bag anal anastomosis are carried out. The recovery was 30 days after surgery, and the survival rate of rats was observed and the status was recorded.
(2) Inflammation induction: the final concentration of 1-5% dextran sodium sulfate (dextran sulphate sodium, DSS) is added into the rat drinking water, and the inflammation induction progress of the rat is evaluated by scoring the disease activity index (disease activity index, DAI). DAI is a comprehensive score for various aspects such as weight loss, fecal matter, and bleeding.
(3) Drug treatment: the enema administration was performed on the inflammation-inducing pouch model rats, and the prepared enema was added for continuous administration 1 time per day.
(4) In the experimental process, the body temperature, the body weight, the food intake and the fecal condition of the rats are measured, and the scoring standard is as follows: the normal stool score is 5 minutes, the stool loose and unshaped score is 4 minutes, the stool is pasty and loose stool with 3 minutes, the diarrhea-like loose stool with 2 minutes and the stool with 1 minute is little loose stool.
(5) Rat pouch tissues were taken at the end of the experiment, HE stained after 4% paraformaldehyde fixation and subjected to histopathological analysis with the following scoring criteria:
erosion conditions: 0 minutes-no erosion, 1 minute-focal erosion, 2 minutes-multiple erosion, 3 minutes-extensive erosion;
ulcer range: 0 minutes-no ulcer, 1 minute-focal ulcer, 2 minutes-multiple ulcers, 3 minutes-extensive ulcer;
ulcer depth: 1 min-infiltrating into the upper 1/2,2 min-full mucosal layer of the mucosa, 3 min infiltrating into or beyond the muscular layer;
degree of villus atrophy: 0 min-no atrophy, 1 min-mild atrophy, 2 min-moderate atrophy, 3 min-severe atrophy;
intrinsic layer edema condition: 0 min-absent, 1 min-present.
(6) The results are shown in fig. 2 to 4: compared with a control group, the enema treatment group rats have the advantages that the inflammation of the storage bag is obviously relieved, the scores of histopathological sections are obviously reduced, and the Elisa detection result shows that the secretion of TNF-alpha is reduced, so that the bacteria vector prepared by the invention for delivering the antisense oligonucleotide drug enema has better treatment effect and higher safety.
Examples
Enema preparation based on l. rhamnosus GG:
(1) The Lactobacillus rhamnosus LGG used in this example was purchased from ATCC, streaked on plates and single colonies were taken and placed in MRS medium for shaking culture overnight (39 ℃ C., 150rpm under anaerobic conditions), and the strains were deposited in a-80 ℃ refrigerator using 25% glycerol.
(2) Single colonies were isolated (under anaerobic conditions) prior to preparation using plate streaking, picked and shake-cultured overnight (39 ℃,150 rpm) in liquid medium to activate them.
The chitosan-entrapped LGG enema, which adsorbs the TNF-alpha antisense oligonucleotide, was prepared by the method of steps (3) - (12) in example 1.
Examples
Enema preparation based on l. rhamnosus OLL 2838:
(1) Lactobacillus rhamnosus OLL2838 used in this example was purchased from ATCC, single colonies were obtained after streaking separation on a plate and placed in liquid MRS medium for shaking culture overnight (39 ℃ under anaerobic conditions at 150 rpm), and the strains were stored in a-80 ℃ refrigerator using 25% glycerol.
(2) Single colonies were isolated (under anaerobic conditions) prior to preparation using plate streaking, picked and shake-cultured overnight (39 ℃,150 rpm) in liquid medium to activate them.
The chitosan-entrapped rhamnose OLL2838 enema, which adsorbs the TNF-alpha antisense oligonucleotide, was prepared by the method of steps (3) - (12) in example 1.
Examples
Enema preparation based on s. thermophilus NCIMB 41856:
(1) Streptococcus thermophilus NCIMB41856 used in this example was purchased from ATCC, streaked on plates and single colonies were taken and placed in MRS broth for shaking culture overnight (39 ℃ under anaerobic conditions at 150 rpm) and the strains were stored in a-80 ℃ freezer using 25% glycerol.
(2) Single colonies were isolated (under anaerobic conditions) prior to preparation using plate streaking, picked and shake-cultured overnight (39 ℃,150 rpm) in liquid medium to activate them.
The preparation method of the steps (3) - (12) in the example 1 is used for preparing the chitosan-entrapped streptococcus thermophilus NCIMB41856 enema for adsorbing the TNF-alpha antisense oligonucleotide.
Examples
Preparation of an enema based on bif, longum CCM 7952:
(1) Bifidobacterium longum CCM7952 used in this example was purchased from ATCC, streaked on plates and single colonies were placed in MRS broth for shaking culture overnight (39 ℃, strict anaerobism, 150 rpm) and the strains were stored in-80 ℃ freezer using 25% glycerol.
(2) Single colonies (strictly anaerobic) were isolated before preparation by plate streaking, picked and shake-cultured overnight (39 ℃ C., 150 rpm) in liquid medium to activate them.
The method of steps (3) - (12) in example 1 was used to prepare a chitosan-entrapped bifidobacterium CCM7952 enema, which adsorbed TNF-alpha antisense oligonucleotides.
Examples
Preparation of an enema based on bif, longum CCM 7952:
(1) (4) in this example, the bif. Longum CCM7952 resuspended bacteria liquid was obtained by the method of (1) to (4) in example 1.
(5) Determination of OD with ultraviolet Spectrophotometer 600 And (5) counting the value.
(6) Protamine (2 mg/mL) was dissolved in 0.5M NaCl solution.
(7) Bacterial vectors were resuspended in a solution containing 2mg/mL protamine to adjust the bacterial concentration to 2X 10 7 cfuml −1 。
(8) Incubation is carried out for 30min, so that protamine is entrapped on the surface of bacteria.
(9) The cells were collected by centrifugation at 3000rpm for 10min and washed 2 times with 0.5M NaCl solution to obtain encapsulated bacteria.
(10) The antisense oligonucleotide (sequence: 5'-GCCCAAGCTGGCATCCGTCA-3') lyophilized powder was centrifuged at 12000rpm for 1min.
(11) The lyophilized antisense oligonucleotide powder was dissolved using a 0.5M NaCl solution.
(12) Mixing the encapsulated bacteria with antisense oligonucleotide 1:1, and incubating for 30min to obtain chitosan-encapsulated Escherichia coli bif, longum CCM7952 enema of final product ICAM-1 antisense oligonucleotide.
Examples
The encapsulated bacterial suspension prepared in example 1 (NaCl suspension with suspension of chitosan modified E.coli EcN 1917) was used as a comparative enema.
According to the animal efficacy verification experiment of example 1, enemas prepared in example 1 and the comparative enema are respectively adopted to perform enema administration on a pouchitis model rat, and the result of the obtained pathological section score map is shown in fig. 5.
As can be seen from fig. 5, the chitosan modified EcN1917 had a promoting effect on inflammation, consistent with the reported effect of the bacterial surface LPS on promoting the immune response of the organism. The enema of this invention has therapeutic effect on inflammation as a whole, because bacteria can promote endocytosis of macrophages, deliver a large amount of antisense oligonucleotide drugs into cells, down regulate gene expression after binding with mRNA, and prevent cells from secreting inflammatory factors.

Claims (10)

1. An antisense oligonucleotide enema based on bacterial vector, comprising bacteria and antisense oligonucleotide, wherein said bacteria are coated with positively charged adhesive, and said antisense oligonucleotide is adsorbed on the surface of said adhesive.
2. The bacterial vector-based antisense oligonucleotide enema according to claim 1, wherein said antisense oligonucleotide is an antisense oligonucleotide against TNF- α, JAK, TYK2, ICAM-1, IL-12, IL-23 or α4β7 integrin;
preferably, the antisense oligonucleotide has a sequence of 5'-AACCCATCGGCTGGCACCAC-3'
Or 5'-GCCCAAGCTGGCATCCGTCA-3'.
3. The bacterial vector-based antisense oligonucleotide enema according to claim 1, wherein said adhesive is one or both of chitosan or protamine.
4. The bacterial vector-based antisense oligonucleotide enema according to claim 1, wherein said bacterium is attenuated salmonella, attenuated listeria or intestinal probiotics.
5. The bacterial vector-based antisense oligonucleotide enema according to claim 4, wherein said intestinal probiotics are one or several of l. rhamnosus GG, l. rhamnosus OLL2838, e. coll Nissle1917, s. thermophilus NCIMB41856 or bif. Longum CCM 7952.
6. The bacterial vector-based antisense oligonucleotide enema according to claim 1, wherein the ratio of said antisense oligonucleotide to said bacterium is (0.1-10 ug): (2 x 10 7 cfu)。
7. The bacterial vector-based antisense oligonucleotide enema according to claim 1, wherein the ratio of said adhesive to said bacteria is (1.5-2.5 mg): (1-2 x 10) 7 cfu)。
8. A method for preparing a bacterial vector-based antisense oligonucleotide enema according to any one of claims 1 to 7, comprising the steps of:
(1) Suspending the bacteria in a solvent and mixing with an adhesive to obtain a bacterial concentration of 1-2×10 7 cfuml −1 Incubating for 20-40 min to obtain wrapped bacteria;
(2) Mixing the encapsulated bacteria with the antisense oligonucleotide in a solvent, and incubating for 20-40 min to obtain the antisense oligonucleotide.
9. The method according to claim 8, wherein the solvent is physiological saline, ringer's solution, phosphate buffer or water.
10. The method of claim 8, wherein the antisense oligonucleotide is produced by a DNA synthesizer.
CN202310160524.6A 2023-02-24 2023-02-24 Antisense oligonucleotide enema based on bacterial vector and preparation method and application thereof Pending CN116159148A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114317377A (en) * 2022-01-19 2022-04-12 南京大学 Modified bacterium and preparation method and application thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114317377A (en) * 2022-01-19 2022-04-12 南京大学 Modified bacterium and preparation method and application thereof

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