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

Cell therapy for siRNA expression and in vivo delivery Download PDF

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CN114606196A
CN114606196A CN202011416402.1A CN202011416402A CN114606196A CN 114606196 A CN114606196 A CN 114606196A CN 202011416402 A CN202011416402 A CN 202011416402A CN 114606196 A CN114606196 A CN 114606196A
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cell
disease
sirna
target gene
cells
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张辰宇
陈熹
王延博
付正
周祯
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Nanjing University
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Nanjing University
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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

Cell therapy for siRNA expression and in vivo delivery
Technical Field
The invention belongs to the technical field of biology, and relates to a cell therapy method for siRNA expression and in-vivo delivery.
Background
sirnas are capable of specifically binding to and degrading mRNA, interfering at the post-transcriptional level with gene expression. siRNA drugs have shown great potential in disease treatment, but how to deliver siRNA to target cells or target organs safely, efficiently and stably in vivo is one of the most critical issues for the development of siRNA drugs. At present, in vivo delivery strategies of siRNA drugs can be mainly divided into naked siRNA direct delivery, viral vectors, chemical modification, nanoparticles, liposomes and the like. Taking the synthetic unmodified naked siRNA as an example, after intravenous injection, the siRNA needs to flow with the blood circulation until reaching the target cell. During this period, a large fraction of the siRNA is cleared by renal filtration and a fraction is disposed of by phagocytes. However, the delivery methods using chemical modification, nano-, liposome or viral vectors all have their own safety problems, so that there is an urgent need for a highly efficient and safe siRNA drug delivery system.
In recent years, the development of siRNA transmission technology based on an exosome delivery system is rapid, and the advantages of the technology are that exosomes are natural vectors for transferring miRNA among cells and tissues, and can wrap and protect miRNA (siRNA analogues) to freely cross cell membranes and biological barriers and reach receptor cells. The method is widely reported to be successful on various disease models, but the cost and the cost are usually not counted in the experiments, in the actual operation process, the large-scale cell culture is needed for packaging siRNA into exosomes, the time and the labor are consumed, the cost is very expensive, and in addition, a large amount of manpower and material resources are needed for separating and purifying the exosomes, so that the in-vitro large-scale production of the exosomes wrapping siRNA is unrealistic, the requirement of production quality control is difficult to meet, and the industrial production cannot be realized.
Therefore, there is an urgent need in the art to develop a new method for combining the in vivo cell transmission technology in cell therapy with the miRNA secretion and circulation mechanism, using cells as the delivery vehicle of siRNA drugs and the natural siRNA drug synthesis machine, stably expressing siRNA in cells in vitro using genetic engineering technology, and then returning the cells to the body for disease treatment.
Disclosure of Invention
The invention aims to provide a novel method for combining a cell in vivo transmission technology in cell therapy with a miRNA secretion and circulation mechanism, taking cells as a delivery carrier of siRNA drugs and a natural siRNA drug synthesis machine, stably expressing siRNA in the cells by using a genetic engineering technology in vitro, and then infusing the cells back into the body for disease treatment.
In a first aspect of the invention, there is provided 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 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 embodiment, the first siRNA molecule has a sequence as 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 fragments.
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 peptide fragments or a fusion protein with other proteins.
In another preferred embodiment, the targeting peptide element is a fusion protein consisting of RVG and LAMP 2B.
In another preferred embodiment, the targeting peptide element has a sequence as shown in SEQ ID No. 4.
In another preferred embodiment, the cells comprise allogeneic or autologous cells.
In another preferred embodiment, the cell is selected from the group consisting of: stem cells, precursor cells, differentiated cells, fibroblasts, or a combination thereof. In another preferred embodiment, the cell is 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 cell contains a vector expressing the siRNA composition.
In another preferred embodiment, the carrier comprises:
a promoter element;
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 molecule reduces expression of one or more genes.
In another preferred embodiment, the vector has a structure of formula I from 5 'to 3':
Z0-Z1-Z2-Z3(I)
wherein Z0 is a promoter element;
z1 is the coding sequence of an optional targeting peptide element;
z2 is a first siRNA molecule that reduces expression of a first target gene;
z3 is an optional second siRNA molecule that reduces expression of a 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 embodiment, the first siRNA molecule has a sequence as 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 vector has the sequence 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 comprises a viral vector and a non-viral vector.
In another preferred embodiment, the viral vector comprises a retroviral, lentiviral, adenoviral, adeno-associated viral vector.
In another preferred embodiment, the expression vector is a plasmid.
In a second aspect, the invention provides a cell preparation comprising cells according to the first aspect of the invention.
In another preferred embodiment, the cell preparation contains 1X10 of the cells7－2×108Individual cell/human, preferably, 2X 107－1.5×108Per person, more preferably, 3X 107－1×108Person/person.
In another preferred embodiment, the cell preparation further comprises 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 formulation is a liquid dosage form.
In another preferred embodiment, the preparation is an injection.
In another preferred embodiment, the cell preparation comprises other drugs for the treatment of cancer.
In another preferred embodiment, the other drugs for treating cancer include gefitinib, panitumumab, sorafenib.
In a third aspect, the invention provides the use of a cell according to the first aspect of the invention or a cell preparation according to the second aspect of the invention for the manufacture of a medicament or formulation for the treatment of a disease.
In another preferred embodiment, the disease is selected from the group consisting of: cancer, malignancy, a disease of the digestive system, a disease of the immune system, a disease of the circulatory system, a disease of the reproductive system, a disease of the respiratory system, a disease of the endocrine system, a disease of the nervous system, a disease of the motor system, a disease of the urinary system, a cardiovascular disease, an organ transplant, inflammation, diabetes, a blood disease, a skin disease, an infectious disease, a psychiatric disease, an infectious disease, an organ injury, a tissue trauma, or a combination thereof.
In another preferred embodiment, the cancer or malignancy 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, ovarian cancer, or a combination thereof.
In another preferred embodiment, the formulation is a liquid formulation.
In another preferred embodiment, in the medicament or the preparation, 1 × 107－2×108Individual cell/human, preferably, 2X 107－1.5×108Per person, more preferably, 3X 107－1×108Person/person.
In a fourth aspect, the present invention provides a method of treating cancer, comprising:
administering to a subject in need thereof a cell according to the first aspect of the invention or a cell preparation according to the second aspect of the invention.
In another preferred embodiment, the dosage of said administration is such that the content of said cells is 1X107－2×108Individual cell/human, preferably, 2X 107－1.5×108Per person, more preferably, 3X 107－1×108Person/person.
In another preferred embodiment, the administration comprises injection.
In another preferred embodiment, said administering comprises intravenous injection, intramuscular injection, subcutaneous injection, intracranial injection, smearing.
In another preferred embodiment, the subject comprises a human or non-human mammal.
In another preferred embodiment, the subject comprises a rodent, such as a mouse, rat.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 is a schematic diagram of a plasmid molecule composed of the genetic elements of the present invention.
FIG. 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 FIG. 2, and the interference efficiency was verified by using cell experiments. A-C: expression of CMV-siRECo-culturing LLC cells with the 293T cell exosomes of (1), and detecting the expression level of the siRNA and the inhibition of the expression of mRNA (B) and protein (C) of the EGFR gene. Denotes p<0.05, represents p<0.01, represents p<0.005。
FIG. 3 shows the content distribution of siRNA delivered by cells in different tissues.
After 1, 3, 6, 9, 12, 24, 48 hours after cell injection, mice were sacrificed and mouse plasma and tissues were taken: a: detecting the expression level of siRNA in the plasma of the mouse and the content of the siRNA in plasma exosomes; b: lung, kidney, spleen, brain, heart, pancreas, muscle, CD4 at the time points mentioned above+And respectively detecting the siRNA level in tissues such as T cells and the like.
FIG. 4 shows the therapeutic effect of cell-delivered siRNA on LLC mouse model of in situ tumor lung cancer, and the statistics of survival.
The LLC tumor-in-situ lung cancer mouse models are averagely grouped, PBS, a control 293T cell, gefitinib or siRNA expression 293T cell is injected once every two days for 2 weeks, the tumor sizes of the mice are detected by CT scanning before and after treatment respectively, and the survival condition is counted. A: representative CT scan 3D imaging results; b: and (5) survival statistics. Wherein, represents p <0.05, represents p <0.01, and represents p < 0.005.
FIG. 5 is a test of in vivo safety of cell-delivered siRNA.
A to F: 293T cell injection has effects on glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, total bilirubin, urea, alkaline phosphatase, creatinine and other biochemical indexes in mouse serum.
Detailed Description
The present inventors have made extensive and intensive studies and, for the first time, have developed a cell expressing an siRNA composition and a cell preparation comprising the cell expressing the siRNA composition, and have unexpectedly found that the cell expressing the siRNA composition or the cell preparation comprising the cell expressing the siRNA composition of the present invention can be used for the treatment of diseases such as cancer. On this basis, the present inventors have completed the present invention.
Term(s)
In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.
The term "about" can refer to a value or composition that is within an acceptable error range for the 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 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the term "comprising" or "includes" 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," "desired subject" refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, e.g., cows, horses, dogs, cats, pigs, sheep, goats, camels, rats, mice, rabbits, and rabbits.
First siRNA molecule
In the present invention, the first siRNA molecule refers to an siRNA molecule capable of reducing the expression of a first target gene (e.g., EGFR, KRAS, TNC).
In a preferred embodiment, the sequence of the first siRNA is as 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 a second target gene (e.g., EGFR, TNC).
In a preferred embodiment, the second siRNA has the sequence shown in SEQ ID No. 3.
SEQ ID NO.3:5’-CACACAAGCCAUCUACACAUG-3’(TNC siRNA)
siRNA compositions
In the present invention, there is provided an 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.
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 a cell, and the cell is used to treat cancer.
Carrier
The invention also provides a carrier, which contains the siRNA composition. The expression vector usually further contains a promoter, an origin of replication, and/or a marker gene. Methods well known to those skilled in the art can be used to construct the expression vectors required by the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as kanamycin, gentamicin, hygromycin, ampicillin resistance.
In the present invention, representative promoters include (but are not limited to): CMV promoter, U6, T7 promoter.
Targeting peptide elements
In the present invention, the targeting peptide element is selected from the group including, but not limited to, RVG, LAMP 2B. In a preferred embodiment, the targeting peptide element of the present invention comprises a rabies glycoprotein. Rabies Virus Glycoprotein (RVG) is a neurotropic protein that binds to acetylcholine receptors expressed by nerve cells. The rabies virus is a single-strand negative-strand RNA virus of the genus rabies virus of the family Rhabdoviridae and having an envelope. The virus mainly encodes glycoprotein G, which is anchored on the surface of the viral envelope in the form of trimer and can be combined with a receptor on the cell surface to mediate membrane fusion so that the virus invades cells. Meanwhile, the G protein is the main antigen protein of rabies virus and stimulates the body to generate neutralizing antibodies. The RVG peptide is specifically combined with a choline body expressed by a neuron cell, and the RVG target point is expressed outside a cell membrane to guide an exosome to pass through a blood brain barrier and be transported to the neuron cell.
In a preferred embodiment, the targeting peptide element of the present invention is RVG-LAMP2b, i.e. a fusion protein consisting of RVG and LAMP 2B.
In a preferred embodiment, the targeting peptide element of the present invention may be absent.
Cell preparation
The cell preparation comprises a safe and effective amount of cells expressing the siRNA composition and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, dextrose, water, glycerol, ethanol, powders, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration.
The cell preparation of the present invention may be prepared as a liquid preparation, which can be prepared by a conventional method, and the liquid preparation is preferably prepared under aseptic conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 1 microgram/kg body weight to about 50 mg/kg body weight, from about 5 microgram/kg body weight to about 10 mg/kg body weight, from about 10 microgram/kg body weight to about 5mg/kg body weight per day. In addition, the formulations of the present invention may also be used with other therapeutic agents.
In 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 the dose is from about 10 micrograms/kg body weight to about 20 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
Method of treatment
The present invention also provides a method of treating a disease, such as cancer, by administering a safe and effective amount of a cell expressing an siRNA composition of the invention or a cell preparation of the invention to a subject in need thereof, thereby treating the disease, such as cancer.
The main advantages of the invention include:
(1) the present invention has for the first time found that cells expressing the siRNA composition of the invention or cell preparations containing cells expressing the siRNA composition of the invention are effective in the treatment of cancer.
(2) The invention discovers for the first time that the technical method for realizing siRNA in-vivo production and transportation through cells solves the problems of high production cost and easy degradation of the existing siRNA to a great extent, is a low-cost and high-efficiency interference drug production and delivery mode, and simultaneously proves the safety of the novel cell-gene therapy mode based on the invention.
(3) The gene engineering cell designed by the invention can be processed and expressed in vivo to generate siRNA, and then the siRNA molecule is secreted to other tissues and organs in an exosome form to play a role in regulating gene expression. The system is used for delivering siRNA for inhibiting EGFR gene, and a good treatment effect is obtained in lung cancer in-situ tumor implantation.
The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specifying the detailed conditions in the following examples, generally followed by conventional conditions such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.
General procedure
I. Plasmid construction:
(1) double restriction enzyme digestion experiment
A double digestion experiment with BamH I (New England Biolab, cat # R0136), Xho I (Biolabs, cat # R0146) was used as an example:
reaction system:
NEBuffer 3.1 5.0μL
BamHI 1.0μL
XhoI 1.0μL
vector DNA 1.0μg
ddH
20 to 50μL
Total volume 50μL
Reaction procedure:
incubation at 37 ℃ for 60 min
Running glue (1% agarose)
And (5) recovering the glue, and temporarily storing the framework in a refrigerator at the temperature of-20 ℃.
(2) Annealing
The 2 pairs of synthesized oligo single-stranded DNA were dissolved to 100. mu.M with ddH2O, 5. mu.l of each complementary single strand were mixed in pairs and annealed according to the system given in Table two. 2 parts of the oligo mixture was heated at 95 ℃ for 5 minutes and then left at room temperature for 20 minutes to form double-stranded DNA.
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 of
The annealed double stranded DNA was further diluted to 10nM concentration and ligated as given in Table III for 30 min at room temperature.
T4 enzymatic ligation system:
5×ligation buffer 4μL
carrier 2μL
ds oligo(10nM) 4μL
T4 DNA ligase(1U/μL) 1μL
ddH2O 9μL
Total volUme 20μL
(4) Transformation of
mu.L of the ligation product was transformed into 100. mu.L of competent cell DH 5. alpha. for 30 min in ice bath, 90-120 sec at 42 ℃ heat shock, 5min in ice bath.
After coating LB plates (containing 50. mu.g/ml spectinomycin), incubation was carried out overnight at 37 ℃. Adding a non-resistant LB culture medium, and culturing for 1 hour at 37 ℃ with a shake.
mu.L of the culture medium was plated (spectacular) and cultured at 37 ℃ for 16 hours.
(5) Sequencing validation
3 clones are picked from each conversion plate respectively, and sequencing is carried out after bacteria shaking and plasmid extraction so as to verify whether the sequence of the inserted fragment in the recombinant clone is consistent with the sequence of the designed oligomeric single-stranded DNA.
Cell culture passages
1. And (5) observing the cell density to 80-90% under a microscope, and then carrying out subculture.
2. Old medium was discarded and cells were rinsed with PBS. The PBS was removed by pipetting.
A3.10 cm dish was filled with mL of digest (0.25% Trypsin) in a flask and placed in an incubator at 37 ℃ for digestion for 5 minutes.
4. Adding 2mL of new culture medium (10% FBS), gently pumping, uniformly mixing, sucking, centrifuging at 1000rpm for 5min, discarding the supernatant, adding 1-2mL of culture solution, and uniformly blowing.
5. The cell suspension was dispensed in a ratio of 1/2 to 1/5 into new dishes containing 10ml of medium.
Cell cryopreservation and recovery
1. When the cells were frozen, after digesting the cells according to the same procedure as the passage, the cells were counted using a cell counting plate at 1X1061ml of frozen stock solution is added into the cells, the frozen stock solution is a mixed solution of fetal calf serum and DMSO, and the final concentration of the DMSO is 10%. And (4) blowing and beating the mixed cells, and subpackaging the cells into a freezing tube.
2. And (4) placing the freezing tube in a programmed cooling box, placing the freezing tube in a refrigerator at minus 80 ℃, and transferring the freezing tube to a liquid nitrogen tank for storage after overnight.
3. And (3) quickly shaking and unfreezing the cell cryopreservation tube in a water bath at 37 ℃, sucking the frozen cell suspension, adding 4mL of culture medium, and uniformly mixing. After centrifugation at 1000rpm for 4 minutes, the supernatant was discarded and the cells were resuspended in fresh complete medium, added to the flask and cultured overnight, and the next day the medium was changed and the cell status was checked.
Transfection of cells
1. Cells were seeded in culture plates (appropriate specifications were chosen for experimental purposes) and raised to approximately 50% -80% cell density.
2. Lipofectamine 2000 was diluted with OPTI-MEM according to the Lipofectamine 2000 transfection protocol, and the mixture was pipetted and left to stand (solution A).
3. Again with reference to the transfection instructions, appropriate amounts of plasmid were aspirated and diluted with OPTI-MEM as solution B for use.
4. Mixing the A, B solutions, beating for 10-15 times, and standing for 20 min. And the cell culture solution to be transfected was changed to OPTI-MEM.
5. The mixed AB mixed solution is evenly dripped into the cells and is shaken up gently.
6. The medium was replaced with 2% fetal bovine serum 6h after transfection, and cells were harvested 36h for subsequent experimental analysis.
V. RNA extraction
1. According to each 107To each cell or 10mg of tissue, 1mL of Trizol was added (fume hood operation), mixed well by vigorous shaking, and allowed to stand at room temperature for 10 minutes.
2. Chloroform was added in a volume of 1/5 Trizol (fume hood operation), mixed well by vigorous shaking, and after standing at room temperature for 5 minutes, centrifuged at 12000g for 20 minutes.
3. Carefully aspirate the supernatant, avoid touching the protein layer, and add 2 volumes of isopropanol to the supernatant (pre-cool), and leave it at-20 ℃ for at least 1 h.
4.12000g, centrifuged at 4 ℃ for 20 minutes, and washed with 75% ethanol in an equal volume of DEPC water.
5.12000g, centrifuging at 4 deg.C for 15 min, completely discarding supernatant, and air drying at room temperature for no more than 10min.
6. Dissolve with 25 μ L DEPC water.
VI, small extraction of endotoxin-removing plasmid
1. The target strain is inoculated into 10-15mL LB culture medium and cultured for 14-16 hours at 37 ℃.
2. And (3) collecting thalli: 10-15mL of bacterial solution is taken, and centrifuged for 10 minutes at room temperature of 5000 g.
3. The medium was decanted, the tube was allowed to drain and the bacterial solution was completely removed, 500. mu.L of solution 1 mixture containing RNase A was added and vortexed vigorously to resuspend the cells completely.
4. Add 500. mu.L of solution 2 to the resuspended suspension and gently invert 7-10 times to achieve mixing, which should not exceed 5 minutes lysis time.
5. Add 250. mu.L of solution 3 in an ice bath, gently invert the tube 7-10 times until a white flocculent precipitate forms, and centrifuge for 10 minutes at 12000g or more.
6. The supernatant was transferred to a 1.5mL centrifuge tube, 0.1 volume ETR solution was added, mixed by gentle inversion, and then ice-cooled for 10 minutes. The lysate turned clear from turbid.
7. Immediately transferred to 42 ℃ in a water bath for 5 minutes, centrifuged at room temperature of 12000g or more for 3 minutes. The ETR is layered on the bottom of the centrifuge tube.
8. Carefully transferring the supernatant to a centrifuge tube by using a pipette gun, adding 0.5 volume of absolute ethyl alcohol, and gently inverting and uniformly mixing. Transferring the mixed solution into an adsorption column at 10000g, centrifuging for 1 minute, and discarding the filtrate.
9. 500. mu.L of HB Buffer was added to the column, 10000g was centrifuged for 1 minute, and the filtrate was discarded.
10. Add 700. mu.L of DNA Wash Buffer, centrifuge at 10000g for 1 min, and discard the filtrate.
11. The washing process of step 12 is repeated once.
12. The filtrate was discarded and centrifuged at 12000g for 2 min.
13. The adsorption column was loaded into a new centrifuge tube, 35-40uL ddH2O was added to the central filter of the column, and the column was allowed to stand at room temperature for 2-5 minutes, centrifuged at 12000g for 2 minutes, eluted and the DNA was collected.
14. The concentration was checked, checked on agarose gel and stored at-20 ℃.
VII, extracting endotoxin from the plasmid
Taking 6L of bacterial liquid as an example:
1. shaking the bacteria: each 2L conical bottle contains 1L LB, 6 bottles of co-shake bacteria liquid 6L. The bacteria shaking time is not more than 16h
2. The bacterial liquid is filled into 3 centrifugal bottles, the center is symmetrically balanced (the domestic bottle is not more than 1/2, the imported bottle is not more than 2/3), centrifugation is carried out at 5000rpm for 10min, and the supernatant is poured off to collect thalli.
3. Add 75mL of Solution 1 to each centrifuge bottle and shake vigorously until no clumps are visible.
150mL of Solution 2 is added into each of 4.3 centrifuge bottles, flocculent viscous substances appear, the mixture is gently shaken and is not too violent, and the cracking process does not exceed 10min.
112.5mL of pre-cooled Solution 3 was added to each of 5.3 flasks and shaken thoroughly until the precipitate dispersed, at which time a white precipitate was visible.
6. After trimming, 5000rpm, 20min, 4 degrees centrifugation. The supernatant was filtered into a home-made centrifuge bottle using a CSI filter in the kit.
7. Cleaning and drying the imported bottle, and transferring the filtrate in the domestic bottle to the imported bottle.
8.3 centrifuge bottles 210mL of isopropanol were added to each bottle and mixed well by inverting about 20 cycles. Depositing at-20 deg.c for over 1 hr.
9. The solution obtained above was centrifuged at 5000rpm for 20min at 4 ℃ and the supernatant was decanted.
10. Adding 60mL of P1 into one bottle, violently mixing, respectively measuring 30mL of P1, adding into the other two centrifuge bottles to obtain 2 centrifuge bottles respectively filled with 30mL of P1, and violently shaking to dissolve the precipitate.
Standing at 11.37 ℃ for 10min.2 centrifuge bottles, 30mL of P2 was added to each bottle, gently inverted several times, and left to stand for 7-9 min.
30mL of P2 was added to each of 12.2 flasks, and the mixture was gently inverted several times until white flocculent precipitate appeared in the solution, and left to stand for 7-9 min.
13.5000 rpm, 10min, 4 degrees centrifugation.
14. The supernatant was filtered into 1 centrifuge bottle using the CSI filter in the kit.
15. 19mL of red de-endotoxin solution ER was added and mixed by inversion.
16. Adding 60mL of isopropanol, fully and uniformly mixing, and precipitating at-20 ℃ for more than 1 h.
17. Column balancing: 6 adsorption columns were added to each column 2.5mL BL at 8000rpm for 2min, and the waste solution was discarded. (Angle rotator, round bottom, adsorption column treated with equilibration fluid is preferably used immediately).
18. Column passing: 10mL of liquid is poured into each adsorption column of 6 adsorption columns respectively, the centrifugation is carried out at 8000rpm for 2min, and waste liquid is poured out until the filtration is finished.
10mL of the buffer ED was poured into each of the 19.6 adsorption columns, and the column was centrifuged at 8000rpm for 2min to remove the waste.
10mL of rinsing solution PW (added with absolute ethyl alcohol in advance) is poured into each adsorption column with 20.6 adsorption columns respectively, centrifugation is carried out at 8000rpm for 2min, and waste liquid is poured out.
21. Repeat 20.
22. 2ml ddH2O was added to each adsorption column, and the column was left to stand for 5min, 7000rpm, and centrifuged for 2 min. The liquid was poured back into the column and separated again.
23. Mixing the liquid, measuring the concentration, and storing at-20 deg.C.
Fluorescent quantitative qRT-PCR assay
Fluorescent 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 volume 10μL
A reverse transcription step:
16℃15min，
42℃60min，
85℃5min，
12℃∞
the mRNA real-time fluorescence quantitative PCR (qPCR) system comprises the following steps:
Figure BDA0002818961610000141
Figure BDA0002818961610000151
the PCR reaction conditions are as follows:
95℃10min，
(95℃15s，60℃30s，72℃30s)×40cycles
dissolution curve: 95 ℃ for 10s, 60 ℃ for 10s, 72 ℃ for 30s
Reverse transcription PCR analysis of siRNA \ miRNA using the MiScript RT Kit
5×Hispec buffer 2.0μL
10×miScript Nucleics Mix 0.5μL
miScript RTase 0.5μL
RNA 0.5μg
DEPC water to 10μL
Total volume 10μL
A reverse transcription step:
37℃60min，
95℃5min，
12℃∞
after diluting by 10 times
Reverse transcription PCR analysis of siRNA \ miRNA, adopting 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
the PCR reaction conditions are as follows:
95℃15min，
(95℃15s，55℃30s，72℃30s)×40cycles
dissolution curve: 95 ℃ for 10s, 60 ℃ for 10s, 72 ℃ for 30s
3. Unless otherwise specified, the mRNA internal reference is GAPDH and the miRNA internal reference is U6. The relative expression level of the target gene can be determined by equation 2-ΔCTWherein Δ CT ═ CSample (I)-CInternal reference。
The primer sequences are as follows:
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)
western blot detection of protein expression levels
(1) Protein sample preparation:
pre-cooled RIPA lysate was added to the cells (typically 100. mu.l of lysate per 6 well plate cell and PMSF was added to a concentration of 1mM before use). After being blown and mixed, the mixture was lysed on ice for 30 minutes and then centrifuged at 14000g for 10 minutes at 4 ℃. In the case of tissue, an appropriate amount of RIPA lysate is added according to the tissue characteristics (typically 100 μ l lysate per 20 mg tissue), and after adding the steel beads, the appropriate conditions are selected and ground with a tissue grinder.
After centrifugation, the supernatant protein lysate is carefully aspirated.
Samples were taken, diluted appropriately (typically 10-fold), and protein concentration was determined using the BCA method.
Adding 5 xSDS-PAGE buffer solution with the volume of the supernatant of 1/4, uniformly blowing, uniformly mixing, carrying out metal bath at 95 ℃ for 5min, and storing the sample in a refrigerator at-20 ℃ for later use.
(2) Preparation of Polyacrylamide gel (SDS-PAGE gel):
and fixing the cleaned and dried glass plate with the glue preparation frame. A 10% undergum solution was prepared as follows. When preparing the separating glue solution, 10% AP and TEMED solution are added at last, then mixed evenly immediately, and the separating glue solution is added between two glass plates immediately. After the addition of the lower gum solution reached the appropriate height, 1mL of isopropanol was added to the surface of the lower gum solution. After the lower layer glue is solidified (a clear interface can be seen under the isopropanol), the isopropanol is poured, the upper layer glue is prepared, the upper layer glue is added between the two glass plates, bubbles are carefully absorbed after the upper layer glue is filled, and the comb teeth are inserted. The comb teeth can be used after the upper layer glue is solidified and carefully pulled out. If the preservation is needed, the preservative film can be used for wrapping the glue and then the glue is placed in a refrigerator at 4 ℃.
5% of upper layer glue and 10% of lower layer glue formula
Figure BDA0002818961610000171
(3) Protein vertical gel electrophoresis:
and (3) placing the gel in an electrophoresis tank, adding electrophoresis buffer solution into the tank, immersing the whole gel below the electrophoresis buffer solution, adding a protein sample into the sample adding hole, and adding a proper protein for pre-staining a marker. Electrophoresis was performed in a voltage-stabilized manner, and the voltage intensity was first adjusted to 80V. After the sample completely runs out of the upper layer glue, the voltage is adjusted to 120V. And selecting proper electrophoresis time according to the condition of Marker, and terminating electrophoresis.
(4) Rotary film
Pre-cooling the membrane transferring buffer solution in a refrigerator at-20 ℃, after vertical gel electrophoresis is finished, disassembling an electrophoresis tank, carefully prying a glass plate, cutting off the redundant part of gel, soaking the gel in the membrane transferring buffer solution, shearing a PVDF membrane according to the size of the gel, soaking the PVDF membrane in methanol for 1 minute to activate the PVDF membrane, arranging membrane transferring clamps layer by layer according to the sequence of negative electrode-sponge-filter paper-gel-PVDF membrane-filter paper-sponge-positive electrode, finishing the whole process under the soaking of the membrane transferring buffer solution, and ensuring that no air bubbles exist among all layers. Inserting the film transferring clamp into a film transferring instrument, then pouring a transferring buffer solution, putting the film transferring instrument into a plastic ice box, burying the whole film transferring instrument in an ice bath environment, carrying out the whole film transferring process at a low temperature, adopting a constant current method to transfer the film, setting the current to be 300mA, and determining the film transferring time according to the size of target protein.
(5) Sealing of
Blocking with 5% skimmed milk for 1 h.
(6) Immunoblotting
According to the following steps of 1: 2000 (the optimal concentration of different antibodies can be selected from the range of 1: 200-2000), preparing a primary antibody by using 5% skimmed milk, putting the PVDF membrane into an incubation box, completely immersing the PVDF membrane in the prepared primary antibody working solution, slowly shaking the incubation box at room temperature, incubating for 1 hour, and standing at 4 ℃ overnight.
Washing the PVDF membrane with TBST buffer solution on a shaking table for 15 minutes each time for 4 times; secondary antibodies were then similarly 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 method
Uniformly mixing A \ B solution in a Super Signal ECL kit in equal volume to prepare reaction solution, putting the PVDF membrane into an exposure instrument, adding the reaction solution on the membrane, and carrying out exposure development through Tanon matched software.
Llc in situ lung cancer model:
to generate an orthotopic lung cancer model, we injected 5 × 10 via tail vein6LLC cells into nude mice. After 30 days, mice were monitored using non-invasive Micro-CT scanning to ensure successful tumor formation in the lungs. Tumor-bearing mice were then randomly divided into 4 groups: every 2 days, PBS or CMV-scr at 5mg/kg was injected intravenouslyROr CMV-siREGene loop, 1 group of 5mg/kg gefitinib gavage, total treatment 7 times. The course of treatment is 2 weeks.
Since mice need to be sacrificed at specific time points for tissue to be molecularly analyzed, mice that were successfully implanted with tumors were randomly grouped and used to assess survival time and tumor progression. Mice used for survival analysis, mice were monitored at all times after treatment without any further treatment. For tumor progression analysis, only mice that survived the end of the 2-week treatment period were analyzed using Micro-CT. After Micro-CT scanning, mice were sacrificed and lung tissue was taken and analyzed using histopathological staining and immunohistochemical methods for further adoption.
XI.KRASLSL-G12D；p53fl/f1Transgenic lung cancer model
1. KRASLSL-G12D, which is 6 weeks old; p53fl/f1 mice were anesthetized with an appropriate amount of 5% chloral hydrate.
2. Adenovirus Adeno-Cre expressing Cre was aspirated at a rate of 5X 106PFU per mouse, and 50. mu.L of each mouse was diluted with PBS for use.
3. The outer skin of the neck of the mouse is depilated, a small opening is longitudinally cut along the central axis of the ventral surface of the neck, and the main trachea is exposed.
4. The elbow forceps are used for fixing the position of the airway, and the injector is pushed into the adenovirus diluent after the artery monitoring needle is guided to be inserted into the trachea through the oral cavity.
5. The skin was sutured and the wound was treated with erythromycin ointment to prevent infection.
By using the method, the Adeno-Cre can be accurately and directionally delivered to the lung of the mouse without being retained in the oral cavity and the respiratory tract. Monitoring with micro-CT at different times (30,40 and 50 days) after inhalation ensured tumor formation. 50 days after Adeno-Cre administration, mice were randomized into two groups and treated with 5mg/kg CMV-scrR or CMV-sirK via the tail vein for 2 weeks (7 injections). The mice were then monitored to determine survival time or to assess tumor growth.
XII, monitoring lung tumor progression by using Micro-CT (Micro-computed tomography) of small animals
Lung tumor growth is assessed herein by means of small animal Micro-CT analysis, since Micro-CT images clearly distinguish lung tumors from surrounding tissue even without any contrast agent, and reconstructed 3-D lung images can more intuitively reflect the actual location of the tumor in lung tissue. Micro-CT scans were performed using a Bruker SkyScan 1176 model Micro-CT analyzer which scanned 180 region at 35 μ M resolution with a rotation step of 0.800. The system comprised two cermet tubes fitted with a fixed 0.5 mm aluminum filter and two 1280X 1024 pixel digital X-ray cameras. X-ray images were obtained at 50kV and 500 μ A. The mice were scanned in the supine position.
The micro-CT data were batch sorted, processed and reconstructed using the N-Recon program according to the manufacturer's (Bruker Corp.) instructions. The reconstructed data was then imaged using DataViewer, the tumor location identified and then further calculated using the CTan program and the whole lung reconstruction was done using the CTVol program.
Isolation of exosomes xiii:
venous blood samples were collected from mice and placed in plasma separation tubes. Plasma was separated using 800 Xg centrifugation for 10 minutes at room temperature and cell debris was removed by centrifugation at 10,000 Xg for 15 minutes at room temperature. Supernatant plasma was recovered and exosomes were isolated using the Total Exosome Isolation kit according to the manufacturer's instructions.
Co-immunoprecipitation of XIV
(1) RIPA lysate cells (containing 1Mm PMSF, 1% PI) were lysed on ice for 30 min.
(2) Centrifuge at 12000g 4 ℃ for 10min.
(3) The mixture was prepared at a concentration of 1:200, 10ul of Flag antibody or IgG (primary antibody) was added, and the mixture was incubated overnight at 4 ℃ in a shaker.
(4) 50ul of Protein G agar beads were added and incubated at room temperature for 2 hours.
(5) Centrifuge at 300rpm for 5min at room temperature.
(6) The immunoblot analysis was performed after eluting the proteins with an eluent.
XV. statistical analysis
All results are expressed as means ± SE, 2 sets of comparison data were compared using student t-test, multiple sets of comparisons were compared using one-way analysis of variance, and all were analyzed by Graphpad 7.0. P values <0.05 were considered statistically different.
Example 1 validation of different replaceable elements, and detection of the interference efficiency of siRNA secreted in vitro by cells
The present invention first designs a plasmid molecule composed of genetic elements (FIG. 1). Based on the method, a plasmid molecule aiming at the EGFR gene is constructed, a promoter element and a siRNA expression element are connected in series, a targeting peptide element is not used, the plasmid molecule is constructed, the plasmid molecule is transfected into 293T cells, a culture medium is collected after 48 hours, EGFR siRNA is obviously enriched in exosomes through detection (figure 2A), the mouse lung cancer cell line LLC is treated after the exosomes are separated, and mRNA and protein expression levels of the EGFR gene in the cells are detected by utilizing qRT-PCR and Western blotting experiments after 36 hours (figure 2, B and C).
Example 2 distribution of cellular delivered siRNA to various tissues following in vivo expression
Injecting 293T cells expressing siRNA into normal mice by tail vein according to the dosage of 1x10 ^6/20 g; the mice were sacrificed after 1, 3, 6, 9, 12, 24, 48 hours, respectively, and plasma, liver, lung, kidney, spleen, brain, heart, pancreas, muscle, CD4 of the mice were taken+T cells and other tissues are respectively used for detecting the siRNA level. The results indicated that siRNA could be detected in plasma and mainly in microvesicle-encapsulated form (fig. 3A). And in lung, kidney, spleen, pancreas and CD4+A high level of siRNA expression was detected in T cells, with low or no signal expression in other tissues (FIG. 3B).
Example 3 therapeutic Effect of cell-delivered siRNA on Lung tumor models
To further confirm the therapeutic effect of siRNA delivered by cells in vivo, we used LLC lung cancer in situ tumor implantation mouse model as an experimental subject to confirm the therapeutic effect on lung tumors. We randomly divided mice successfully implanted with in situ tumors into 4 groups, and injected with control 293T cells, siRNA expressing 293T cells, PBS and gavage gefitinib drugs at a dose of 1x10 ^6/20g, respectively. The drug is taken once a week for two weeks, lung tumor change conditions are detected by CT imaging before and after treatment, and the survival condition of the mice is counted. The results show that the lung tumor volume of mice in the group of 293T cells expressed by siRNA before and after treatment is significantly reduced, some mice completely disappear, and the tumors of other three groups of mice are significantly increased (fig. 4A). The survival time of mice in the group of 293T cells expressed by siRNA injection is remarkably prolonged (FIG. 4B).
Example 4 in vivo safety assay for cellular delivery of siRNA
In order to test the safety of the treatment, the levels of the biochemical indexes such as glutamic-pyruvic transaminase (fig. 5A), glutamic-oxaloacetic transaminase (fig. 5B), total bilirubin (fig. 5C), urea (fig. 5D), alkaline phosphatase (fig. 5E) and creatinine (fig. 5F) in the sera of the control group of mice and the experimental group of mice injected with siRNA expressing 293T cells were also tested. The results show that the above indexes of the mice in the experimental group injected with siRNA expressing cells have no obvious difference from those in the control group, and the administration mode is safer.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Nanjing university
<120> a cell therapy for siRNA expression and in vivo delivery
<130> P2020-1439
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> RNA
<213> Artificial sequence (artificial sequence)
<400> 1
auaccuauuc cguuacacac u 21
<210> 2
<211> 21
<212> RNA
<213> Artificial sequence (artificial sequence)
<400> 2
gcaaauacac aaagaaagcc c 21
<210> 3
<211> 21
<212> RNA
<213> Artificial sequence (artificial sequence)
<400> 3
cacacaagcc aucuacacau g 21
<210> 4
<211> 1356
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 4
atgtgcctct ctccggttaa aggcgcaaag ctcatcctga tctttctgtt cctaggagcc 60
gttcagtcca atgcattgat agttaatttg acagattcaa agggtacttg cctttatgct 120
cgatacacca tttggatgcc cgagaatccg agaccaggga caccttgtga catttttacc 180
aatagcagag ggaagagagc atccaacggg tccggaggtg cagaatggga gatgaatttc 240
acaataacat atgaaactac aaaccaaacc aataaaacta taaccattgc agtacctgac 300
aaggcgacac acgatggaag cagttgtggg gatgaccgga atagtgccaa aataatgata 360
caatttggat tcgctgtctc ttgggctgtg aattttacca aggaagcatc tcattattca 420
attcatgaca tcgtgctttc ctacaacact agtgatagca cagtatttcc tggtgctgta 480
gctaaaggag ttcatactgt taaaaatcct gagaatttca aagttccatt ggatgtcatc 540
tttaagtgca atagtgtttt aacttacaac ctgactcctg tcgttcagaa atattggggt 600
attcacctgc aagcttttgt ccaaaatggt acagtgagta aaaatgaaca agtgtgtgaa 660
gaagaccaaa ctcccaccac tgtggcaccc atcattcaca ccactgcccc gtcgactaca 720
actacactca ctccaacttc aacacccact ccaactccaa ctccaactcc aaccgttgga 780
aactacagca ttagaaatgg caatactacc tgtctgctgg ctaccatggg gctgcagctg 840
aacatcactg aggagaaggt gcctttcatt tttaacatca accctgccac aaccaacttc 900
accggcagct gtcaacctca aagtgctcaa cttaggctga acaacagcca aattaagtat 960
cttgacttta tctttgctgt gaaaaatgaa aaacggttct atctgaagga agtgaatgtc 1020
tacatgtatt tggctaatgg ctcagctttc aacatttcca acaagaacct tagcttctgg 1080
gatgcccctc tgggaagttc ttatatgtgc aacaaagagc aggtgctttc tgtgtctaga 1140
gcgtttcaga tcaacacctt taacctaaag gtgcaacctt ttaatgtgac aaaaggacag 1200
tattctacag cccaggagtg ttcgctggat gatgacacca ttctaatacc aattatagtt 1260
ggtgctggtc tttcaggctt gattatcgtt atagtgattg cttacctaat tggcagaaga 1320
aagacctatg ctggatatca gactctgtaa cactaa 1356
<210> 5
<211> 647
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 5
gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60
catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120
acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180
ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240
aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300
ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360
tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 420
ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480
ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 540
tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag 600
aacccactgc ttactggctt atcgaaatta atacgactca ctatagg 647
<210> 6
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 6
gccatctggg ccaaagatac c 21
<210> 7
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 7
gtcttcgcat gaataggcca at 22
<210> 8
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 8
caagagcgcc ttgacgatac a 21
<210> 9
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 9
ccaagagaca ggtttctcca tc 22
<210> 10
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 10
ccaatagcag agggaagaga gc 22
<210> 11
<211> 19
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 11
tccatcgtgt gtcgccttg 19
<210> 12
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 12
gatattgttg ccatcaatga c 21
<210> 13
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 13
ttgattttgg agggatctcg 20
<210> 14
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 14
ggcacagaca ggcagtcagc a 21
<210> 15
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 15
ctgtctgtgt gctgtgtcag tc 22
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 16
ggtgttgctt ctcttaattc 20
<210> 17
<211> 19
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 17
aggagttaag agaagccac 19
<210> 18
<211> 19
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 18
gaggagttaa gaagccaca 19
<210> 19
<211> 29
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 19
acactccagc tggggtgctc gcttcggca 29
<210> 20
<211> 19
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 20
tagctagcag cacgtaaat 19

Claims (10)

1. A cell that expresses an 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 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 a sequence as set forth 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 comprises a vector expressing the siRNA composition.
6. The cell of claim 5, wherein the vector comprises:
a promoter element;
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 molecule reduces expression of one or more genes.
7. The cell of claim 6, wherein the vector has a structure of formula I from 5 'to 3':
Z0-Z1-Z2-Z3(I)
wherein Z0 is a promoter element;
z1 is the coding sequence of an optional targeting peptide element;
z2 is a first siRNA molecule that reduces expression of a first target gene;
z3 is an optional second siRNA molecule that reduces expression of a second target gene.
8. A cell preparation comprising the cell of claim 1.
9. Use of a cell according to claim 1 or a cell preparation according to claim 2 for the preparation of a medicament or preparation for the treatment of a disease.
10. The use according to claim 9, wherein the disease is selected from the group consisting of: cancer, malignancy, a disease of the digestive system, a disease of the immune system, a disease of the circulatory system, a disease of the reproductive system, a disease of the respiratory system, a disease of the endocrine system, a disease of the nervous system, a disease of the motor system, a disease of the urinary system, a cardiovascular disease, an organ transplant, inflammation, diabetes, a blood disease, a skin disease, an infectious disease, a psychiatric disease, an infectious disease, an organ injury, a tissue trauma, or a combination thereof.
CN202011416402.1A 2020-12-04 2020-12-04 Cell therapy for siRNA expression and in vivo delivery Pending CN114606196A (en)

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