CN114606196A - A cell therapy for siRNA expression and in vivo delivery - Google Patents

Abstract

The invention provides a cell therapy for siRNA expression and in vivo delivery, in particular, the invention provides a cell in which an siRNA composition is expressed, the siRNA composition comprising: a first siRNA molecule that reduces expression of a first target gene; optionally, a coding sequence for a targeting peptide element; and optionally, a second siRNA molecule that reduces expression of a second target gene; wherein the first target gene is selected from the group consisting of: EGFR, KRAS, TNC, or a combination thereof, and the siRNA composition reduces the expression of one or more genes, and the invention also provides cell preparations comprising such cells, the cells and cell preparations of the invention being effective in treating diseases, such as cancer.

Description

A cell therapy for siRNA expression and in vivo delivery

technical field

The invention belongs to the field of biotechnology, and relates to a cell therapy for siRNA expression and in vivo delivery.

Background technique

siRNA can specifically bind to and degrade mRNA, interfering with gene expression at the post-transcriptional level. siRNA drugs have shown great potential in disease treatment, but how to safely, effectively and stably deliver siRNA to target cells or organs in vivo is one of the most critical issues in the development of siRNA drugs. At present, the in vivo delivery strategies of siRNA drugs can be mainly divided into several categories, such as direct delivery of naked siRNA, viral vectors, chemical modification, nanoparticles, and liposomes. Taking synthetic unmodified naked siRNA as an example, after intravenous injection, the siRNA needs to circulate with the blood until it reaches the target cells. During this period, a large part of the siRNA will be filtered out of the body by the kidneys, and some will be processed by phagocytes. However, the delivery methods of chemical modification, nanometer, liposome or viral vector all have their own safety problems. Therefore, an efficient and safe siRNA drug delivery system is urgently needed.

In recent years, siRNA delivery technology based on exosome delivery system has developed rapidly. barrier and reach recipient cells. This method has been widely reported to be successful in a variety of disease models, but these experiments are often cost-effective, and in practice, packaging siRNA into exosomes requires large-scale cell culture, which is time-consuming and time-consuming. In addition, the isolation and purification of exosomes also requires a lot of manpower and material resources. Therefore, it is not realistic to mass-produce siRNA-encapsulated exosomes in vitro, and it is difficult to meet the requirements of production quality control and industrialized production. .

Therefore, there is an urgent need in the field to develop a method that combines the in vivo delivery technology of cell therapy with miRNA secretion and circulation mechanisms, uses cells as siRNA drug delivery vehicles and natural siRNA drug synthesis machines, and utilizes genetic engineering technology in vitro in cells. A new method for stably expressing siRNA and then infusing cells back into the body for disease treatment.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method that combines the in vivo transmission technology of cell therapy with miRNA secretion and circulation mechanism, uses cells as siRNA drug delivery carrier and natural siRNA drug synthesis machine, and utilizes genetic engineering technology to stabilize in cells in vitro A new way to express siRNA and then infuse cells back into the body for disease treatment.

In a first aspect of the present invention, there is provided a cell in which a siRNA composition is expressed, the siRNA composition comprising:

a first siRNA molecule that reduces the expression of the first target gene;

Optionally, the coding sequence for the targeting peptide element; and

Optionally, a second siRNA molecule that reduces expression of a second target gene;

wherein the first target gene is selected from the group consisting of EGFR, KRAS, TNC, or a combination thereof, and the siRNA composition reduces the expression of one or more genes.

In another preferred embodiment, the second target gene is selected from the group consisting of EGFR, TNC, or a combination thereof.

In another preferred embodiment, the first target gene and the second target gene are the same or different.

In another preferred example, the first siRNA molecule has the sequence shown in SEQ ID NO.: 1 or 2.

In another preferred embodiment, the sequence of the first siRNA molecule is shown in SEQ ID NO.: 1 or 2.

In another preferred embodiment, the second siRNA molecule has the sequence shown in SEQ ID NO.:3.

In another preferred embodiment, the sequence of the second siRNA molecule is shown in SEQ ID NO.:3.

In another preferred embodiment, the targeting peptide element is one of all known targeting peptide segments.

In another preferred embodiment, the targeting peptide element is selected from the group consisting of RVG, LAMP2B, or a combination thereof.

In another preferred embodiment, the targeting peptide element is one of all known targeting peptides or a fusion protein with other proteins.

In another preferred embodiment, the targeting peptide element is a fusion protein composed of RVG and LAMP2B.

In another preferred embodiment, the sequence of the targeting peptide element is shown in SEQ ID NO.:4.

In another preferred embodiment, the cells include allogeneic or autologous cells.

In another preferred embodiment, the cells are selected from the group consisting of stem cells, precursor cells, differentiated cells, fibroblasts, or a combination thereof. In another preferred embodiment, the cells are selected from the group consisting of white adipocytes, brown adipocytes, mesenchymal stem cells, embryonic stem cells, immune cells, fibroblasts or a combination thereof.

In another preferred embodiment, the cells contain a vector for expressing the siRNA composition.

In another preferred embodiment, the carrier includes:

promoter element;

a first siRNA molecule that reduces the expression of the first target gene;

Optionally, the coding sequence for the targeting peptide element; and

Optionally, a second siRNA molecule that reduces the expression of a second target gene; wherein the first target gene is selected from the group consisting of EGFR, KRAS, TNC, or a combination thereof, and the siRNA molecule reduces one or more genes expression.

In another preferred embodiment, the carrier has the structure shown in formula I of 5'-3':

Z0-Z1-Z2-Z3(I)

Wherein, Z0 is a promoter element;

Z1 is the coding sequence for an optional targeting peptide element;

Z2 is the first siRNA molecule that reduces the expression of the first target gene;

Z3 is an optional second siRNA molecule that reduces the expression of the second target gene.

In another preferred embodiment, the promoter element comprises a constitutive promoter.

In another preferred embodiment, the promoter element is selected from the group consisting of CMV, U6, or a combination thereof.

In another preferred embodiment, the first target gene is selected from the group consisting of EGFR, KRAS, TNC, or a combination thereof.

In another preferred embodiment, the second target gene is selected from the group consisting of EGFR, TNC, or a combination thereof.

In another preferred embodiment, the first target gene and the second target gene are the same or different.

In another preferred example, the first siRNA molecule has the sequence shown in SEQ ID NO.: 1 or 2.

In another preferred embodiment, the second siRNA molecule has the sequence shown in SEQ ID NO.:3.

In another preferred embodiment, the sequence of the vector is shown in SEQ ID NO.:5.

In another preferred embodiment, the vector contains a promoter, an origin of replication and a marker gene.

In another preferred embodiment, the expression vector includes viral vectors and non-viral vectors.

In another preferred embodiment, the viral vector includes retrovirus, lentivirus, adenovirus, and adeno-associated virus vector.

In another preferred embodiment, the expression vector is a plasmid.

The second aspect of the present invention provides a cell preparation containing the cells of the first aspect of the present invention.

In another preferred example, in the cell preparation, the content of the cells is 1×10 ⁷ -2×10 ⁸ cells/person, preferably, 2×10 ⁷ -1.5×10 ⁸ cells/person, More preferably, 3×10 ⁷ -1×10 ⁸ per person.

In another preferred embodiment, the cell preparation further contains a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutically acceptable carrier is selected from the group consisting of phosphate buffer, physiological saline, trehalose solution, or a combination thereof.

In another preferred embodiment, the preparation is a liquid dosage form.

In another preferred embodiment, the preparation is an injection.

In another preferred embodiment, the cell preparation includes other drugs for treating cancer.

In another preferred embodiment, the other drugs for treating cancer include gefitinib, panitumumab, and sorafenib.

The third aspect of the present invention provides a use of the cells described in the first aspect of the present invention or the cell preparation described in the second aspect of the present invention for preparing a medicine or preparation for treating diseases.

In another preferred embodiment, the disease is selected from the group consisting of cancer, malignant tumor, digestive system disease, immune system disease, circulatory system disease, reproductive system disease, respiratory system disease, endocrine system disease, nervous system disease, motor system disease Disease, urological disease, cardiovascular disease, organ transplantation, inflammation, diabetes, blood disease, skin disease, infectious disease, psychiatric disease, infectious disease, organ damage, tissue trauma, or a combination thereof.

In another preferred embodiment, the cancer or malignant tumor is selected from the group consisting of lung cancer, glioblastoma, gastric cancer, colorectal cancer, liver cancer, breast cancer, bladder cancer, pancreatic cancer, prostate cancer, uterine cancer, ovary cancer cancer, or a combination thereof.

In another preferred embodiment, the preparation is a liquid preparation.

In another preferred embodiment, in the drug or preparation, 1×10 ⁷ -2×10 ⁸ cells/person, preferably 2×10 ⁷ -1.5×10 ⁸ cells/person, more preferably, 3 ×10 ⁷ -1 × 10 ⁸ per person.

A fourth aspect of the present invention provides a method for treating cancer, comprising:

The cells of the first aspect of the present invention or the cell preparation of the second aspect of the present invention are administered to a subject in need thereof.

In another preferred example, the administered dose is that the content of the cells is 1×10 ⁷ -2×10 ⁸ cells/person, preferably, 2×10 ⁷ -1.5×10 ⁸ cells/person, More preferably, 3×10 ⁷ -1×10 ⁸ per person.

In another preferred embodiment, the administration includes injection.

In another preferred example, the administration includes intravenous injection, intramuscular injection, subcutaneous injection, intracranial injection, and smearing.

In another preferred embodiment, the subject includes a human or a non-human mammal.

In another preferred embodiment, the subject includes rodents, such as mice and rats.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

Fig. 1 is a schematic diagram of a plasmid molecule composed of a gene element of the present invention.

Figure 2 is the detection of interference efficiency when siRNA is expressed in vitro.

293T cells expressing siRNA were constructed according to the method shown in Figure 2, and the interference efficiency was verified by cell experiments. AC: LLC cells were co-cultured with ^exosomes from 293T cells expressing CMV-siRNA, and the expression level of siRNA and its inhibition on the expression of EGFR gene mRNA (B) and protein (C) were detected. * means p<0.05, ** means p<0.01, *** means p<0.005.

Figure 3 is the content distribution of cell-transmitted siRNA in different tissues.

1, 3, 6, 9, 12, 24, and 48 hours after the cells were injected, the mice were sacrificed, and the mouse plasma and tissues were collected: A: The expression of siRNA in mouse plasma and its content in plasma exosomes were detected ; B: siRNA levels were detected in lung, kidney, spleen, brain, heart, pancreas, muscle, CD4 ⁺ T cells and other tissues at the above time points.

Figure 4 shows the therapeutic effect and survival statistics of siRNA delivered by cells on LLC orthotopic tumor-implanted lung cancer mouse model.

The LLC orthotopic tumor-planted lung cancer mouse models were equally divided into groups and injected with PBS, control 293T cells, gefitinib or siRNA expressing 293T cells every two days for 2 weeks. The tumor size of mice was detected by CT scan before and after treatment. and statistics of survival. A: Representative CT scan 3D imaging results; B: Survival statistics. Among them, * means p<0.05, ** means p<0.01, and *** means p<0.005.

Figure 5 is an in vivo safety assay of cell-delivered siRNA.

A-F: Effects of 293T cell injection on biochemical indexes such as alanine aminotransferase, aspartate aminotransferase, total bilirubin, urea, alkaline phosphatase and creatinine in serum of mice.

Detailed ways

After extensive and in-depth research, the inventors developed a cell expressing siRNA composition and a cell preparation containing the cell expressing the siRNA composition for the first time. The present invention unexpectedly found that the cell expressing the siRNA composition of the present invention or Cell preparations of cells expressing siRNA compositions can be used to treat diseases, such as cancer. On this basis, the present inventors have completed the present invention.

the term

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meaning given below. Additional definitions are set forth throughout the application.

The term "about" may refer to a value or composition within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the terms "containing" or "including (including)" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of," or "consisting of."

As used herein, the terms "host", "subject", "subject in need" refer to any mammal or non-mammalian. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates such as cattle, horses, dogs, cats, pigs, sheep, goats, camels, rats, mice, hares and rabbits.

first siRNA molecule

In the present invention, the first siRNA molecule refers to an siRNA molecule capable of reducing the expression of the first target gene (eg, EGFR, KRAS, TNC).

In a preferred embodiment, the sequence of the first siRNA is shown in SEQ ID NO.: 1 or 2.

SEQ ID NO. 1: AUACCUAUUCCGUUACACACU (EGFR siRNA);

SEQ ID NO. 2: 5'-GCAAAUACACAAAGAAAGCCC-3' (KRAS siRNA)

second siRNA molecule

In the present invention, the second siRNA molecule refers to an siRNA molecule capable of reducing the expression of the second target gene (eg, EGFR, TNC).

In a preferred embodiment, the sequence of the second siRNA is shown in SEQ ID NO.:3.

SEQ ID NO. 3: 5'-CACACAAGCCAUCUACACAUG-3' (TNC siRNA)

siRNA composition

In the present invention, a siRNA composition is provided, comprising:

a first siRNA molecule that reduces the expression of the first target gene;

Optionally, the coding sequence for the targeting peptide element; and

Optionally, a second siRNA molecule that reduces expression of a second target gene;

wherein the first target gene is selected from the group consisting of EGFR, KRAS, TNC, or a combination thereof.

In a preferred embodiment, the second target gene is selected from the group consisting of EGFR, TNC, or a combination thereof.

In a preferred embodiment, the first target gene and the second target gene are the same or different.

In the present invention, the siRNA compositions of the present invention can reduce the expression of one or more genes. In the present invention, the siRNA composition of the present invention is expressed in cells, and the cells are used to treat cancer.

carrier

The present invention also provides a carrier, which contains the siRNA composition of the present invention. The expression vector usually also contains a promoter, an origin of replication and/or a marker gene and the like. Methods well known to those skilled in the art can be used to construct the desired expression vectors of the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like. The expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as karamycin, gentamicin, hygromycin, ampicillin resistance.

In the present invention, representative promoters include (but are not limited to): CMV promoter, U6, T7 promoter.

targeting peptide element

In the present invention, the targeting peptide element is selected from the group including, but not limited to, RVG, LAMP2B. In a preferred embodiment, the targeting peptide element of the present invention comprises a rabies virus glycoprotein. Rabies virus glycoprotein (RVG) is a neurotropic protein that can bind to acetylcholine receptors expressed by nerve cells. Rabies virus belongs to the genus Rhabdoviridae, a single-stranded negative-stranded RNA virus with an envelope. The virus mainly encodes glycoprotein G, which is anchored on the surface of the viral envelope in the form of trimers, and can bind to receptors on the cell surface to mediate membrane fusion to allow the virus to invade cells. At the same time, G protein is the main antigenic protein of rabies virus, which stimulates the body to produce neutralizing antibodies. RVG peptides specifically bind to cholinergic bodies expressed by neuronal cells, and RVG targets are expressed outside the cell membrane, guiding exosomes through the blood-brain barrier and transporting them to neuronal cells.

In a preferred embodiment, the targeting peptide element of the present invention is RVG-LAMP2b, that is, a fusion protein composed of RVG and LAMP2B.

In a preferred embodiment, the targeting peptide element of the present invention may be absent.

cell preparation

The cell preparation of the present invention contains a safe and effective amount of cells expressing the siRNA composition of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, powders, and combinations thereof. The drug formulation should match the mode of administration.

The cell preparation of the present invention can be made into a liquid preparation, and its preparation can be carried out by conventional methods, and the liquid preparation is preferably manufactured under aseptic conditions. The active ingredient is administered in a therapeutically effective amount, eg, about 1 microgram/kg body weight to about 50 mg/kg body weight, about 5 microgram/kg body weight to about 10 mg/kg body weight, about 10 microgram/kg body weight to about 5 micrograms per day mg/kg body weight. In addition, the formulations of the present invention may also be used with other therapeutic agents.

When using the formulations of the present invention, a safe and effective amount of the drug is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, preferably Typically this dose is about 10 micrograms/kg body weight to about 20 mg/kg body weight. Of course, the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.

treatment method

The present invention also provides a method of treating diseases, such as cancer, by administering a safe and effective amount of cells expressing the siRNA compositions of the present invention or cell preparations of the present invention to a desired subject, thereby treating diseases, such as cancer.

The main advantages of the present invention include:

(1) The present invention finds for the first time that cells expressing the siRNA composition of the present invention or cell preparations containing cells expressing the siRNA composition of the present invention can effectively treat cancer.

(2) The present invention finds for the first time that the technical method for realizing the production and transportation of siRNA in vivo through cells of the present invention largely solves the current problems of high production cost and easy degradation of siRNA, and is a low-cost, high-efficiency interfering drug Manufacture delivery methods while demonstrating the safety of the novel cell-gene therapy modality based on the present invention.

(3) The genetically engineered cells designed in the present invention can be processed and expressed in vivo to generate siRNA, and then the siRNA molecules are secreted to other tissues and organs in the form of exosomes to play the function of regulating gene expression. The present invention utilizes the system to deliver the siRNA for inhibiting the EGFR gene, and obtains a good therapeutic effect in the in situ implantation of lung cancer.

Below in conjunction with specific embodiments, the present invention is described in further detail. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted detailed conditions in the following examples, usually according to normal conditions such as people such as Sambrook, molecular cloning: conditions described in laboratory manual (New York:Cold Spring Harbor LaboratoryPress, 1989), or according to manufacturer's instructions. recommended conditions. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

general method

I. Plasmid construction:

(1) Double enzyme digestion experiment

Take BamH1 (New England Biolab, article number: #R0136), XhoI (Biolabs, article number: #R0146) double enzyme digestion experiment as an example:

reaction system:

NEBuffer 3.1 5.0μL BamHI 1.0μL XhoI 1.0μL carrier DNA 1.0μg ddH₂0 to 50μL Total volume 50μL

Reaction program:

37 degrees, incubate for 60 minutes

Running gel (1% agarose)

The glue is recovered and the skeleton is temporarily stored in a -20 degree refrigerator.

(2) Annealing

Two pairs of synthesized oligomeric single-stranded DNA were dissolved into 100 μM with ddH2O, and 5 μl of each complementary single-stranded DNA was mixed in pairs, and annealed according to the system given in Table 2. Double-stranded DNA was formed by heating 2 parts of the oligo mixture at 95 degrees for 5 minutes and then at room temperature for 20 minutes.

oligo DNA annealing system

100μM top strand oligo 5μL 100μM bottom strand oligo 5μL 10×oligo annealing buffer 2μL ddH2O 8μL Total volUme 20μL

(3) Connection

The annealed double-stranded DNA was further diluted to a concentration of 10 nM, and ligated for 30 minutes at room temperature according to the system given in Table 3.

T4 enzyme ligation system:

5×ligation buffer 4μL carrier 2μL ds oligo (10nM) 4μL T4 DNA ligase (1U/μL) 1μL ddH2O 9μL Total volUme 20μL

(4) Conversion

Take 10 μL of the ligation product to transform 100 μL of competent cells DH5α, ice bath for 30 minutes, heat shock at 42 degrees for 90-120 seconds, and ice bath for 5 minutes.

After plating on LB plates (containing 50 μg/ml spectinomycin), incubate overnight at 37°C. Anti-resistant LB medium was added, and the bacteria were shaken at 37 degrees for 1 hour.

Take 500 μL, plate (spectacular), and incubate at 37 degrees for 16 hours.

(5) Sequencing verification

Three clones were picked from each transformation plate, and the plasmids were extracted by shaking bacteria and sequenced to verify whether the sequence of the inserted fragment in the recombinant clone was consistent with the designed oligomeric single-stranded DNA sequence.

II. Cell Culture Passaging

1. When the cell density reaches 80%-90% under a microscope, subculture it.

2. Discard old medium and rinse cells with PBS. Aspirate and discard the PBS.

3. Add mL of digestion solution (0.25% Trypsin) to the culture flask in a 10cm petri dish, and place it in a 37°C incubator to digest for 5 minutes.

4. Add 2 mL of new medium (10% FBS), gently pipette and mix, then aspirate, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 1-2 mL of culture medium, and blow evenly.

5. Divide the cell suspension 1/2 to 1/5 into new dishes containing 10 ml of medium.

III. Cell Cryopreservation and Recovery

1. When the cells are cryopreserved, digest the cells according to the same steps of passage, and count them with a cell counting plate. Add 1 ml of cryopreservation solution to each 1x10 ⁶ cells. The cryopreservation solution is a mixture of fetal bovine serum and DMSO, and the final concentration of DMSO is 10%. . Mix cells by pipetting and aliquot into cryovials.

2. Put the cryopreservation tube in the programmed cooling box, put it in a -80 degree refrigerator, and transfer it to liquid nitrogen for storage after overnight.

3. Quickly shake and thaw the cell cryopreservation tube in a 37°C water bath, aspirate the cryopreserved cell suspension, add 4 mL of medium and mix well. Centrifuge at 1000rpm for 4 minutes, discard the supernatant, resuspend the cells with new complete medium, add them to the culture flask and culture overnight, change the medium the next day and check the cell status.

IV. Cell Transfection

1. Inoculate the cells in culture plates (select the appropriate size according to the experimental purpose), and grow to about 50%-80% cell density.

2. According to the transfection instructions of Lipofectamine 2000, dilute Lipofectamine 2000 with OPTI-MEM, mix by pipetting, and let it stand for later use (Solution A).

3. Also refer to the transfection instructions, draw an appropriate amount of plasmid and dilute it with OPTI-MEM as solution B for use.

4. Mix liquid A and B, blow for 10-15 times, and let stand for 20 minutes. The cell culture medium to be transfected was changed to OPTI-MEM.

5. Add the mixed AB mixture dropwise to the cells evenly, and shake gently.

6. The medium was replaced with 2% fetal bovine serum medium after 6 hours of transfection, and the cells were collected after 36 hours for subsequent experimental analysis.

V. RNA extraction

1. Add 1 mL of Trizol to every 10 ⁷ cells or 10 mg of tissue (operated in a fume hood), shake vigorously and mix well, and let stand at room temperature for 10 minutes.

2. Add chloroform with 1/5 volume of Trizol (operated in a fume hood), shake vigorously and mix well, and then stand at room temperature for 5 minutes, then centrifuge at 12000g for 20 minutes.

3. Carefully aspirate the supernatant, avoid touching the protein layer, and add 2 times the volume of isopropanol (pre-cooling) to the supernatant, and let it stand at -20 degrees for at least 1 hour.

4. Centrifuge at 12000g for 20 minutes at 4 degrees, and wash with 75% ethanol prepared by adding an equal volume of Trizol DEPC water.

5. Centrifuge at 12000g for 15 minutes at 4 degrees, completely discard the supernatant, and dry at room temperature for no more than 10 minutes.

6. Dissolve with 25 μL of DEPC water.

VI. Removal of endotoxin plasmid

1. Inoculate the target strain into 10-15 mL of LB medium, and cultivate at 37°C for 14-16 hours.

2. Collect the bacteria: take 10-15 mL of bacterial liquid, centrifuge at 5000g at room temperature for 10 minutes.

3. Pour off the medium, dry the centrifuge tube to completely remove the bacterial liquid, add 500 μL of solution 1 mixture containing RNase A, and vortex vigorously to completely resuspend the cells.

4. Add 500 μL of solution 2 to the resuspended bacterial solution, and gently invert 7-10 times to achieve mixing. The lysis time in this step should not exceed 5 minutes.

5. Add 250 μL of solution 3 in the ice bath beforehand, invert the centrifuge tube gently 7-10 times to form a white flocculent precipitate, and centrifuge at ≥12000g for 10 minutes.

6. Transfer the supernatant to a 1.5mL centrifuge tube, add 0.1 times the volume of ETR solution, gently invert and mix, then ice bath for 10 minutes. The lysate turned from cloudy to clear.

7. Immediately transfer to 42°C, water bath for 5 minutes, and centrifuge at ≥12000g for 3 minutes at room temperature. ETR is layered at the bottom of the centrifuge tube.

8. Carefully transfer the supernatant to a centrifuge tube with a pipette, add 0.5 times the volume of absolute ethanol, and mix by gentle inversion. Transfer the mixture to the adsorption column and centrifuge at 10000g for 1 minute, and discard the filtrate.

9. Add 500 μL of HB Buffer to the spin column, centrifuge at 10,000 g for 1 minute, and discard the filtrate.

10. Add 700 μL DNA Wash Buffer, centrifuge at 10,000 g for 1 minute, and discard the filtrate.

11. Repeat step 12 washing process one more time.

12. Discard the filtrate and centrifuge at 12000g for 2 minutes.

13. Load the adsorption column into a new centrifuge tube, add 35-40uL ddH2O to the filter in the center of the column, stand at room temperature for 2-5 minutes, centrifuge at 12000g for 2 minutes, elute and collect DNA.

14. Detect the concentration, verify on agarose gel, and store at -20 degrees.

VII. Removal of endotoxin plasmid

Take 6L bacterial liquid as an example:

1. Shake bacteria: each 2L Erlenmeyer flask contains 1L LB, and 6 bottles of 6L of bacterial solution are shaken together. Shake the bacteria for no more than 16h

2. Put the bacterial liquid into 3 centrifuge bottles, balance the center symmetrically (no more than 1/2 of the domestic bottle, no more than 2/3 of the imported bottle), centrifuge at 5000 rpm for 10 min, and discard the supernatant to collect the bacteria.

3. Add 75mL of Solution 1 to each centrifuge bottle and shake vigorously until no lumps are visible.

4. Add 150mL of Solution 2 to each of the 3 centrifuge bottles. If a flocculent viscous substance appears, shake it gently, not too vigorously, and the lysis process does not exceed 10 minutes.

5. Add 112.5mL of pre-cooled Solution 3 to each of the 3 centrifuge bottles and shake it gently until the precipitate disperses, and a white precipitate can be seen at this time.

6. After balancing, centrifuge at 5000rpm, 20min, 4 degrees. Filter the supernatant into a domestic centrifuge bottle with the CSI filter in the kit.

7. Wash and dry the imported bottle, and transfer the filtrate from the domestic bottle to the imported bottle.

8. Add 210 mL of isopropanol to each of the 3 centrifuge bottles, invert for about 20 times and mix thoroughly. -20 degree precipitation for more than 1h.

9. Centrifuge the solution obtained above at 5000 rpm, 20 min, 4 degrees, and discard the supernatant.

10. Add 60 mL of P1 to one bottle, and after vigorous mixing, measure 30 mL and add them to the other two centrifuge bottles to obtain two centrifuge bottles each containing 30 mL of P1. Shake vigorously to dissolve the precipitate.

11. Let stand at 37 degrees for 10 min. Add 30 mL of P2 to each of the two centrifuge bottles, gently invert several times, and let stand for 7-9 min.

12. Add 30mL of P2 to each of the two centrifuge bottles, invert gently for several times until the solution appears white dispersed flocculent precipitate, and let it stand for 7-9min.

13. 5000rpm, 10min, 4 degree centrifugation.

14. Filter the supernatant into 1 centrifuge bottle using the CSI filter from the kit.

15. Add 19 mL of red endotoxin removal solution ER, invert and mix.

16. Add 60 mL of isopropanol, mix well, and precipitate at -20 degrees for more than 1 hour.

17. Column equilibration: take 6 adsorption columns, add 2.5 mL of BL to each, centrifuge at 8000 rpm for 2 min, and discard the waste liquid. (Angle rotor, round bottom, adsorption column treated with equilibration solution is best used immediately).

18. Passing the column: Pour 10 mL of liquid into each of the 6 adsorption columns, centrifuge at 8000 rpm for 2 min, and pour off the waste liquid until all the filtration is completed.

19. Pour 10 mL of buffer ED into each of the 6 adsorption columns, centrifuge at 8000 rpm for 2 min, and discard the waste liquid.

20.6 adsorption columns were poured into each adsorption column with 10 mL of rinsing solution PW (anhydrous ethanol was added in advance), centrifuged at 8000 rpm for 2 min, and the waste liquid was discarded.

21. Repeat 20.

22. Add 2 mL ddH2O to each adsorption column, let stand for 5 min, centrifuge at 7000 rpm for 2 min. Pour the liquid back into the adsorption column again.

23. Mix the liquid, measure the concentration, and store it at -20 degrees.

VIII. Fluorescence quantitative qRT-PCR detection

1. Quantitative PCR analysis of mRNA,

reverse transcription system

5×AMV buffer 2.0μL dNTP (10mM) 0.5μL RRI 0.20μL Oligo dT 1.0μL AMV RTase 0.5μL RNA 0.5-1μg DEPC water to 10μL total capacity 10μL

Reverse transcription steps:

16℃ for 15min,

42℃ for 60min,

85℃ for 5min,

12℃∞

2. mRNA real-time quantitative PCR (qPCR) system and steps are as follows:

PCR reaction conditions are:

95℃ for 10min,

(95℃15s, 60℃30s, 72℃30s)×40cycles

Dissolution curve: 95℃ for 10s, 60℃ for 10s, 72℃ for 30s

3. Reverse transcription PCR analysis of siRNA\miRNA using miScript RT Kit

5×Hispec buffer 2.0μL 10 x miScript Nucleics Mix 0.5μL miScript RTase 0.5μL RNA 0.5μg DEPC water to 10μL total capacity 10μL

Reverse transcription steps:

37℃ for 60min,

95℃ for 5min,

12℃∞

After 10-fold dilution

Reverse transcription PCR analysis of siRNA\miRNA using miScript SYBR Green PCR Kit:

Reverse primer 0.5μL 2×SYBR Green PCR Master Mix 1.0μL ddH₂O 13.1μL cDNA 1.0μL Total 20.0μL

PCR reaction conditions are:

95℃ for 15min,

(95℃15s, 55℃30s, 72℃30s)×40cycles

Dissolution curve: 95℃ for 10s, 60℃ for 10s, 72℃ for 30s

3. Unless otherwise specified, the internal reference for mRNA is GAPDH, and the internal reference for miRNA is U6. The relative expression level of the target gene can be expressed by equation 2 ^-ΔCT , where ΔCT=C _sample -C _{internal reference} .

Primer sequence:

EGFR (Forward): 5'-GCCATCTGGGCCAAAGATACC-3' (SEQ ID NO.: 6)

EGFR (Reverse): 5'-GTCTTCGCATGAATAGGCCAAT-3' (SEQ ID NO.: 7)

K-RAS (Forward): 5'-CAAGAGCGCCTTGACGATACA-3' (SEQ ID NO.: 8)

K-RAS (Reverse): 5'-CCAAGAGACAGGTTTCTCCATC-3' (SEQ ID NO.: 9)

RVG (Forward): 5'-CCAATAGCAGAGGGAAGAGAGC-3' (SEQ ID NO.: 10)

RVG (Reverse): 5'-TCCATCGTGTGTCGCCTTG-3' (SEQ ID NO.: 11)

GAPDH (Forward): 5'-GATATTGTTGCCATCAATGAC-3' (SEQ ID NO.: 12)

GAPDH (Reverse): 5'-TTGATTTTGGAGGGATCTCG-3' (SEQ ID NO.: 13)

EGFR-si precursor (Forward): 5'-GGCACAGACAGGCAGTCAGCA-3' (SEQ ID NO.: 14)

EGFR-si precursor (Reverse): 5'-CTGTCTGTGTGCTGTGTCAGTC-3' (SEQ ID NO.: 15)

EGFR-si (Forward): 5'-GGTGTTGCTTCTCTTAATTC-3' (SEQ ID NO.: 16)

EGFR-sh-ps (Forward): 5'-AGGAGTTAAGAGAAGCCAC-3' (SEQ ID NO.: 17)

EGFR-si-ps (Forward): 5'-GAGGAGTTAAGAAGCCACA-3' (SEQ ID NO.: 18)

U6 (Forward): 5'-ACACTCCAGCTGGGGTGCTCGCTTCGGCA-3' (SEQ ID NO.: 19)

miR-16 (Forward): 5'-TAGCTAGCAGCACGTAAAT-3' (SEQ ID NO.: 20)

IX.Western blot detection of protein expression level

(1) Protein sample preparation:

Add pre-chilled RIPA lysate to the cells (usually 100 microliters of lysate is added to each well of a 6-well plate, and PMSF is added to a concentration of 1 mM before use). After mixing by pipetting, the cells were lysed on ice for 30 minutes and centrifuged at 14,000g for 10 minutes at 4°C. If it is tissue, add an appropriate amount of RIPA lysis buffer according to the characteristics of the tissue (usually 100 microliters of lysis buffer is added per 20 mg of tissue). After adding steel balls, select appropriate conditions and grind with a tissue grinder.

After centrifugation, carefully aspirate the supernatant protein lysate.

Sampling, appropriate dilution (generally 10 times dilution), using BCA method to determine the protein concentration.

Add 1/4 of the supernatant volume to 5×SDS-PAGE buffer, mix by pipetting, take a metal bath at 95°C for 5 min, and store the samples in a -20°C refrigerator for later use.

(2) Preparation of polyacrylamide gel (SDS-PAGE gel):

Fix the cleaned and dried glass plate with the glue rack. Prepare a 10% subgel solution according to the table below. When preparing the separating gel solution, the 10% AP and TEMED solutions were added last, then mixed immediately, and the separating gel solution was immediately added between the two glass plates. After the added underlayer glue solution reaches a suitable height, add 1 mL of isopropanol on the surface of the underlayer glue solution. After the bottom glue has solidified (a clear interface can be seen under isopropanol), pour the isopropanol to prepare the top glue, add the top glue between the two glass plates, carefully suck out the air bubbles after filling, and insert the comb teeth. After the top layer glue has solidified, carefully pull out the comb and use it. For preservation, wrap the gel with plastic wrap and place it in a 4°C refrigerator.

5% top glue and 10% bottom glue formula

(3) Protein vertical gel electrophoresis:

Place the gel in the electrophoresis tank, add electrophoresis buffer to the tank, submerge the entire gel under the electrophoresis buffer, add protein samples to the sample wells, and add appropriate protein prestained markers. Electrophoresis is performed in a voltage-stabilized manner, and the voltage intensity is first adjusted to 80V. After the sample completely escaped from the upper layer of glue, adjust the voltage to 120V. Select the appropriate electrophoresis time according to the situation of Marker, and terminate the electrophoresis.

(4) Transfer film

Put the transfer buffer in the refrigerator to pre-cool at -20 degrees in advance. After the vertical gel electrophoresis is completed, disassemble the electrophoresis tank, carefully pry open the glass plate, cut off the excess part of the gel, soak it in the transfer buffer, and press Cut the PVDF membrane to the size of the gel, soak it in methanol for 1 minute to activate it, and arrange the membrane clips layer by layer in the order of negative electrode-sponge-filter paper-gel-PVDF film-filter paper-sponge-positive electrode. After soaking in transfer buffer, make sure that there are no air bubbles between the layers. Insert the transfer clip into the transfer film machine, then pour the transfer buffer, put it in a plastic ice box and bury the entire film transfer machine in an ice bath environment, so that the entire transfer film process is carried out at low temperature, and the transfer is carried out by the constant current method. membrane, set the current to 300mA, and determine the transfer time according to the size of the target protein.

(5) Closed

Block with 5% skim milk for 1 h.

(6) Western blot

According to the ratio of 1:2000 (the optimal concentration of different antibodies can be selected from the range of 1:200-2000), use 5% skim milk to prepare the primary antibody, put the PVDF membrane into the incubation box, and make the prepared primary antibody working solution completely Immerse the PVDF membrane, shake the incubation box slowly at room temperature, incubate for 1 hour, and let it stand at 4 degrees overnight.

The PVDF membrane was washed with TBST buffer on a shaker for 15 minutes each time, 4 times in total; then the secondary antibody was also diluted with 5% skim milk at a ratio of 1:5000, and incubated at room temperature for 1 hour with slow shaking. The strips were then washed 4 times with TBST for 15 minutes each.

(7) Exposure

Mix equal volumes of solution A and B in the Super Signal ECL kit to form a reaction solution, put the PVDF membrane into the exposure apparatus, add the reaction solution on the membrane, and perform exposure and development through the Tanon software.

X.LLC Orthotopic Lung Cancer Model:

To generate an orthotopic lung cancer model, we injected 5×10 ⁶ LLC cells into nude mice via the tail vein. After 30 days, the mice were monitored using a non-invasive Micro-CT scan to ensure successful tumor formation in the lungs. Then, tumor-bearing mice were randomly divided into 4 groups: intravenous injection of PBS or 5 mg/kg CMV-scr ^R or CMV-siR ^E gene circuit every 2 days, 1 group of 5 mg/kg gefitinib by gavage, and co-treatment 7 times. The course of treatment lasts 2 weeks.

Since mice need to be sacrificed at specific time points for tissue extraction for molecular biological analysis, mice with successful tumor engraftment were randomized and used to assess survival time and tumor progression. Mice used for survival analysis were monitored without any further treatment after treatment. For tumor progression analysis, only mice that survived the end of the 2-week treatment period were analyzed using Micro-CT. Following Micro-CT scanning, mice were sacrificed, and lung tissue was removed and analyzed using histopathological staining and immunohistochemical methods before adoption.

XI.KRAS ^LSL-G12D ; p53 ^fl/f1 transgenic lung cancer model

1. 6-week-old KRASLSL-G12D; p53fl/f1 mice were anesthetized with an appropriate amount of 5% chloral hydrate.

2. Aspirate Cre-expressing adenovirus Adeno-Cre at an amount of 5×10 6 PFU per mouse, and dilute with PBS in a volume of 50 μL per mouse for use.

3. Depilate the outer skin of the mouse neck, and make a longitudinal incision along the mid-axis of the ventral surface of the neck to expose the main trachea.

4. Use elbow forceps to fix the position of the airway, and guide the arterial monitoring needle to be inserted into the trachea through the mouth, and then push the syringe into the adenovirus diluent.

5. Suture the outer skin and treat the wound with erythromycin ointment to prevent infection.

Using this method, Adeno-Cre can be accurately and targetedly delivered to the lungs of mice without being trapped in the oral cavity and respiratory tract. Tumor formation was monitored by micro-CT at various times (30, 40 and 50 days) after inhalation. Fifty days after Adeno-Cre administration, mice were randomized into two groups and treated with 5 mg/kg CMV-scrR or CMV-siRK via tail vein for 2 weeks (7 injections). Then, the mice were monitored to determine survival time or to assess tumor growth.

XII. Small animal Micro-CT monitoring lung tumor progression

In this paper, small animal Micro-CT analysis was used to evaluate lung tumor growth, because even without any contrast agent, Micro-CT images clearly distinguish lung tumors from surrounding tissues, and the reconstructed 3-D lung images can more intuitively reflect The actual location of the tumor in the lung tissue. Micro-CT scans were performed using Bruker's SkyScan model 1176 Micro-CT analyzer, which scans a 180° area at a resolution of 35 μM with a rotation step size of 0.800. The system consists of two cermet tubes with fixed 0.5mm aluminium filters and two 1280 x 1024 pixel digital X-ray cameras. X-ray images were acquired at 50 kV and 500 μA. Mice were scanned in the supine position.

MicroCT data were batch sorted, processed and reconstructed using the N-Recon program according to the manufacturer's (Bruker Corporation) instructions. The reconstructed data were then imaged using DataViewer, and after identifying and identifying the tumor location, the CTan program was used to calculate the tumor volume, and the CTVol program was used to complete the whole lung reconstruction.

XIII. Isolation of exosomes:

Venous blood samples were collected from mice and placed in plasma separator tubes. Plasma was separated by centrifugation at 800 x g for 10 min at room temperature and cell debris was removed by centrifugation at 10,000 x g for 15 min at room temperature. The supernatant plasma was recovered and exosomes were isolated using the TotalExosome Isolation kit according to the manufacturer's instructions.

XIV. Co-immunoprecipitation

(1) Use RIPA lysis buffer (containing 1Mm PMSF, 1% PI) to lyse cells on ice for 30 minutes.

(2) Centrifuge at 12000g at 4 degrees for 10 minutes.

(3) Prepare at a concentration of 1:200, add 10ul of Flag antibody or IgG (primary antibody), and incubate at 4 degrees overnight on a shaker.

(4) Add 50ul Protein G agarose beads and incubate at room temperature for 2 hours.

(5) Centrifuge at 300 rpm for 5 min at room temperature.

(6) Western blot analysis was performed after the protein was eluted with the eluent.

XV. Statistical Analysis

All the results are expressed as means±SE. The comparative data of two groups were compared by student t-test, and the comparison of multiple groups was performed by one-way ANOVA, all of which were analyzed by Graphpad 7.0. A P value of <0.05 was considered statistically significant.

Example 1 Validation of different replaceable elements, and detection of interference efficiency of siRNA secreted by cells in vitro

The present invention first designs a plasmid molecule composed of genetic elements (Fig. 1). Based on this, a plasmid molecule targeting the EGFR gene was constructed, and the promoter element was connected in series with the siRNA expression element without targeting peptide element. The plasmid molecule was constructed, and the plasmid molecule was transfected into 293T cells. After 48 hours, the culture medium was collected and detected It was found that EGFR siRNA was significantly enriched in exosomes (Fig. 2A). The mouse lung cancer cell line LLC was treated after exosome isolation, and 36 hours later, the mRNA and protein expression levels of EGFR gene in the cells were detected by qRT-PCR and Western blotting experiments (Fig. 2A). Figure 2, B and C).

Example 2 Distribution of siRNA delivered by cells in various tissues after in vivo expression

The 293T cells expressing siRNA were injected into the tail vein of normal mice at a dose of 1*10^6/20g; after 1, 3, 6, 9, 12, 24, and 48 hours, the mice were sacrificed, and the mice were collected. siRNA levels were detected in plasma, liver, lung, kidney, kidney, spleen, brain, heart, pancreas, muscle, CD4 ⁺ T cells and other tissues. The results showed that siRNA could be detected in plasma, mainly in the form of encapsulated microvesicles (Fig. 3A). In contrast, substantial siRNA expression was detected in lung, kidney, spleen, pancreas, and CD4 ⁺ T cells, with low or no signal in other tissues (Fig. 3B).

Example 3 Therapeutic effect of cell-delivered siRNA on lung tumor model

In order to further confirm the therapeutic effect of cell-transmitted siRNA in vivo, we used the LLC lung cancer orthotopic tumor implantation mouse model as the experimental object to confirm the therapeutic effect on lung tumors. We randomly divided the mice with successful orthotopic tumor implantation into 4 groups, and injected control 293T cells, siRNA-expressing 293T cells, PBS and gefitinib respectively according to the dose of 1*10^6/20g. The drug was administered once a week for a total of two weeks of treatment. The changes of lung tumors were detected by CT imaging before and after treatment, and the survival of the mice was counted. The results showed that before and after treatment, the lung tumor volume of mice in the siRNA-expressing 293T cell group was significantly reduced, and completely disappeared in some mice, while the tumors in the other three groups were significantly enlarged (Figure 4A). The survival time of mice in the group injected with siRNA expressing 293T cells was significantly prolonged (Fig. 4B).

Example 4 In vivo safety detection of siRNA delivered by cells

In order to test the safety of this treatment, we also detected the serum alanine aminotransferase (Fig. 5A), aspartate aminotransferase (Fig. 5B), total bilirubin (Fig. 5C), urea (Fig. 5D), alkaline phosphatase (Fig. 5E) and creatinine (Fig. 5F) levels of biochemical indicators. The results showed that the above indicators of mice in the experimental group injected with siRNA expressing cells were not significantly different from those in the control group, which is a relatively safe way of administration.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

<110> Nanjing University

<120> A cell therapy for siRNA expression and in vivo delivery

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Claims (10)

1. a cell, is characterized in that, in described cell, expresses siRNA composition, and described siRNA composition comprises: a first siRNA molecule that reduces the expression of the first target gene; Optionally, the coding sequence for the targeting peptide element; and Optionally, a second siRNA molecule that reduces expression of a second target gene; wherein the first target gene is selected from the group consisting of EGFR, KRAS, TNC, or a combination thereof, and the siRNA composition reduces the expression of one or more genes. 2. The cell of claim 1, wherein the second target gene is selected from the group consisting of EGFR, TNC, or a combination thereof. 3. The cell of claim 1, wherein the first siRNA molecule has the sequence shown in SEQ ID NO.: 1 or 2. 4. The cell of claim 1, wherein the second siRNA molecule has the sequence shown in SEQ ID NO.:3. 5. The cell of claim 1, wherein the cell contains a vector expressing the siRNA composition. 6. The cell of claim 5, wherein the carrier comprises: promoter element; a first siRNA molecule that reduces the expression of the first target gene; Optionally, the coding sequence for the targeting peptide element; and Optionally, a second siRNA molecule that reduces expression of a second target gene; wherein the first target gene is selected from the group consisting of EGFR, KRAS, TNC, or a combination thereof, and the siRNA molecule reduces one or more genes expression. 7. The cell of claim 6, wherein the carrier has the structure shown in formula I of 5'-3': Z0-Z1-Z2-Z3(I) Wherein, Z0 is a promoter element; Z1 is the coding sequence for an optional targeting peptide element; Z2 is the first siRNA molecule that reduces the expression of the first target gene; Z3 is an optional second siRNA molecule that reduces the expression of the second target gene. 8 . A cell preparation comprising the cells of claim 1 . 9 . 9. Use of the cell according to claim 1 or the cell preparation according to claim 2, characterized in that it is used for preparing a medicine or preparation for treating diseases. 10. The use according to claim 9, wherein the disease is selected from the group consisting of cancer, malignant tumor, digestive system disease, immune system disease, circulatory system disease, reproductive system disease, respiratory system disease, endocrine system disease disease, neurological disease, motor system disease, urinary system disease, cardiovascular disease, organ transplantation, inflammation, diabetes, blood disease, skin disease, infectious disease, psychiatric disease, infectious disease, organ damage, tissue trauma, or its combination.

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