WO2019000151A1

WO2019000151A1 - Recombinant adeno-associated virus for simultaneously inhibiting expressions of three micrornas

Info

Publication number: WO2019000151A1
Application number: PCT/CN2017/089931
Authority: WO
Inventors: 毛吉炎
Original assignee: 深圳市博奥康生物科技有限公司
Priority date: 2017-06-26
Filing date: 2017-06-26
Publication date: 2019-01-03

Abstract

Provided is a recombinant adeno-associated virus for simultaneously inhibiting expressions of three microRNAs. The recombinant adeno-associated virus rAAV-Tud-140-185-424 can be stably expressed in vivo for a long time, thereby playing a role in knocking down the expressions of miR-140, miR-185 and miR-424.

Description

Recombinant adeno-associated disease for simultaneous inhibition of expression of three microRNAs

Technical field

[0001] The present invention belongs to the field of molecular biology and biomedical technology, and in particular relates to a recombinant adeno-associated virus for synchronously inhibiting the expression of three microRNAs.

Background technique

[0002] MicroRNAs (miRNAs) are a class of endogenous, non-coding RNAs found in eukaryotes, typically between 22 and 25 nt in size. miRNAs are widely distributed in plants, animals, and multicellular organisms, and can Play an important regulatory role, and in the study of human miRNAs, it is found that the expression of miRNA in normal tissues and tumor tissues is significantly different, some miRNAs are lowly expressed in tumor tissues, and some are highly expressed in tumor tissues. This suggests that miRNAs play a crucial role in tumorigenesis.

[0003] miR-140 is closely related to the development of various diseases, such as bone and joint diseases, liver diseases, pituitary adenomas, testicular development, head and neck tumors, ovarian and breast diseases. miR-140 can inhibit the proliferation and invasion and metastasis of hepatocellular carcinoma by targeting TG FBR1 and other gene expression. miR-140 is highly expressed in articular cartilage and plays a vital role in the pathogenesis of osteoarthritis. Under the control of multiple mechanisms, m iR-140 plays an oncogene role in some tumors, and plays a tumor suppressor role in other tumors, and is associated with multiple tumor chemotherapy resistance; miR-185 is a 22nt in length The miRNA, located at the human chromosome 22ql l.21, plays an important role as a tumor suppressor gene in the development and invasion of tumors such as colon cancer, gastric cancer, esophageal cancer, lung cancer, and liver cancer, and it is also associated with methylation. Anti-tumor miRNA can affect the methylation level of the whole genome by directly targeting the expression of DNMT1, thereby regulating the methylation status of some genes and affecting gene expression; miR-424 is a miRNA discovered in recent years. It affects the biological effects and development of tumor cells by acting on target genes and participating in the signal transduction pathway of target genes in a variety of tumors. , Tumor suppressor gene, or promoting, inhibition of tumor invasion and metastasis. Studies have shown that miR-424 is a multifunctional miRNA, which is associated with cell invasion and metastasis of cervical cancer, pancreatic cancer, etc.; it is associated with the expression of inflammatory factors such as IL-6 and TNF-ot; since the miR-424 promoter region has CpG islands It is also associated with methylation-induced gene silencing. By controlling the expression of miR-140, miR-185 and miR-424, peers work synergistically with other drugs to provide new treatments for cancer. Epigenetic ideas.

technical problem

[0004] Functional studies of MiRNAs are primarily accomplished through miRNA interference and overexpression techniques. Among the existing miRNA interference technologies, anti-miR and antagomiR are transient transfection techniques, and the interference effect cannot be stably maintained, while the mi RNA sponge effect is far from optimal. The prior art lacks a good interference effect and can achieve long-term effects. Stable interference technology.

[0005] Tough Decoy RNA (Tud RNA) is a novel miRNA-inhibiting miRNA that inhibits miRNA by introducing double-stranded RNA to target miRNAs. Because the inserted RNA is double-stranded and has a secondary structure of stem-loops, it is resistant to intracellular nuclease degradation and inhibits miRNAs in a long-term, stable, and efficient manner.

[0006] Adeno-associated virus (AAV) is a non-pathogenic human parvovirus that can infect a wide variety of cells, including mitotic and non-dividing cells, and is characterized by its safety, long-lasting, high-efficiency, and high specificity. Researchers are highly regarded and favored and are widely used in the field of biology. The use of AAV as a gene therapy vector has potential advantages.

Problem solution

Technical solution

[0007] It is an object of the present invention to provide a recombinant adeno-associated virus that inhibits the expression of miR-140, miR-185 and miR-424.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is: A recombinant adeno-associated virus for synchronously inhibiting the expression of three microRNAs is constructed by the following methods:

[0009] a. Based on the human miR-140, miR-185 and miR-424 sequences, the Tud RNA sequences specific for miR-140, miR-185 and miR-424 were designed, and the recombinant plasmid pAKD-Tud-140-185 was constructed. -424, and perform DNA sequencing analysis;

[0010] b recombinant plasmid pAKD-Tud-140-185-424, packaging plasmid pAAV-RC and helper plasmid pHelper were co-transfected into AAV-293 cells with calcium phosphate method, and recombinant adeno-associated virus rAAV-Tud-140- was harvested. 185-424, the titer of the virus was determined by quantitative PCR.

[0011] The specific Tud RNA sequence for miR-140, miR-185 and miR-424 in step a is: 5'- TGCCCAAGATGATCCTAGCGCCACCTTTTT -3,.

[0012] In step a, a Kpn I and Bg III endonuclease cleavage site sequence and a transcription termination sequence are added to the selected target sequence to synthesize a complete Tud DNA sequence:

[0013] Tud DNA-F: 5'-

TTGGTGCCCAAGATGATCCTAGCGCCACCTTTTTGTAC -3'

Tud DNA-R: 5,-

ATGAGTGCAAAACGTTGATGATCCTAGCGCC -3,.

Advantageous effects of the invention

Beneficial effect

The present invention constructs a recombinant adeno-associated virus rAAV-Tu d-140-185-424 for simultaneously inhibiting the expression of three microRNAs, and adopts a type 8 which can stably express a target protein for a long period of time and is highly biosafety. Adeno-associated virus (AAV8) is a transduction vector that stably expresses Tud RNA in vivo for a long time, thereby knocking down the expression of miR-140, miR-185 and miR-424.

Brief description of the drawing

DRAWINGS

1 is a schematic diagram showing the results of quantitative PCR detection of miRNA expression levels in each group of cells, wherein a. miR-140 expression, b. miR-185 expression, c. miR-424 expression. BEST MODE FOR CARRYING OUT THE INVENTION

BEST MODE FOR CARRYING OUT THE INVENTION

[0017] The present invention can be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions, and results described in the examples are merely illustrative of the invention and are not intended to limit the invention as described in the claims. . The methods used in the following examples are conventional methods unless otherwise specified; the reagents are commercially available products unless otherwise specified. Specific steps can be found in: "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor)

Example 1: Construction of a recombinant adeno-associated virus that inhibits the expression of miR-140, miR-185 and miR-424

[0019] 1. Construction of recombinant plasmid pAKD-Tud-140-185-424

[0020] Based on the sequence information of miR-140, miR-185 and miR-424 provided in the TuD RNA design sequence and miRBase, the Tud RNA sequences specific for miR-140, miR-185 and miR-424 were designed: 5' -

TGCCCAAGATGATCCTAGCGCCACCTTTTT -3,. Adding the restriction endonuclease sequence and transcription termination sequence of Kpn I and Bgl II endonuclease to the selected target sequence, and commissioning Shanghai Yingjun Biotechnology Co., Ltd. to synthesize the complete Tud DNA sequence ij ij.

[0021] The Tud DNA template was annealed, and after annealing, a double-stranded oligonucleotide fragment having a Kpn I and Bgl II restriction sites at both ends was formed, which was labeled Tud-140-185-424.

[0022] The pAKD-shRNA plasmid vector was obtained by linear digestion with Kpn I and Bgl II, and an appropriate amount of the product was detected by 1% agarose gel electrophoresis and recovered. The double-stranded DNA was ligated into the restriction vector according to the ligation reaction system, ligated and incubated at 4 ° C overnight, and transformed into competent E. coli DH5oc, which was plated and incubated overnight. Single colonies were picked for cultivation and sent to Shanghai Yingjun Biotechnology for sequencing. The bacteria with the correct sequencing results were cultured, and the pAKD-Tud-140-185-424 plasmid was extracted using the Promega plasmid extraction kit (endotoxin). [0023] 2. Virus packaging, purification and titer determination

[0024] AAV-293 cell culture is carried out by a conventional method, and when the cells reach 70-80% fusion, the calcium phosphate method is used for virus transfection, and rAAV8 expressing Tud-140-185-424 can be obtained, and virus collection is performed. Concentrate and purify.

[0025] The virus was titrated by a quantitative PCR method. The number of viral particles of rAAV is determined by detecting the genomic copy number of the rAAV vector in the viral genome, and the titer unit is expressed in vg/ml, that is, the number of viral genomes per ml (Vims Genome). The virus sample to be tested was digested with DNase and RNase at 37 ° C for 2~3 h, and the virus DNA was extracted and denatured in a boiling water bath for 5 min, immediately placed on ice for 2 min, and the virus sample and the standard product were diluted to different concentrations. For quantitative PCR detection, the primers used were: upstream: 5'-CCTTTCCGGGACTTTCGCTTT-3', downstream: 5,-GCAGAATCCAGGTGGCAAC A-3.

The reaction conditions were: denaturation at 94 ° C for 15 s, annealing at 52 ° C for 30 s, and extension at 72 ° C for 30 s, for a total of 40 cycles. After the end of the PCR, a standard curve was drawn based on the standard and the virus sample titer was calculated. The average virus titer was calculated to be 1.13x1012 vg/ml.

Example 2: AAV transfection T24 cells

[0028] T24 cells were inoculated into a six-well plate, 1000000 cells per well, and the cell density was about 50% after 12 hours. The virus solution was taken separately, and the virus was diluted 1000 times with DMEM complete medium to remove the medium in the 6-well plate. , add virus-containing DMEM complete medium (containing 10% fetal bovine serum), 24h

Discard the virus-containing DMEM complete medium and replace with fresh DMEM.

Complete medium and continue to culture for 96 h.

Example 3: Fluorescence quantitative PCR detection of changes in miRNA expression levels

[0030] The miRNAs of normal T24 cells and TuD-140-185-424 cells were extracted using the miRcute miRNA extraction and isolation kit, followed by S-Poly(T) hsa-miR-140 qPCR-assay primer set, S-Poly ( T) hsa-miR-185 qPCR-assay primer set and S-Poly(T) hsa-miR-424 qPCR-assay primer set kit for reverse transcription and tailing of miRNA to obtain the corresponding cDNA. The cDNA of each of the two cells was used as a template. The expression levels of miR-140, 11 11 -185 and 1^1 -424 were detected by real-time PCR. The experiment was repeated 3 times, and 3 parallel samples were set per well to snord 44. As an internal reference. As a result, as shown in Fig. 2, it can be seen that the expression level of miR-140 with TuD-140-185-424 cells is 49% lower than that of T24 cells, and the table of miR-185 The level of expression was 57% lower than that of T24 cells, and the expression level of miR-424 was 58% lower than that of T24 cells. The difference was statistically significant (/?<0.01), indicating that the TuD-140-185-424 cell line was successfully constructed.

Industrial applicability

The present invention constructs a recombinant adeno-associated virus rAAV-Tu d-140-185-424 for synchronously inhibiting the expression of three microRNAs, and adopts an adeno-associated virus type 8 which can stably express the target protein for a long period of time and is highly biosafety. (AAV8) is a transduction vector that stably expresses Tud RNA in vivo for a long time, thereby knocking down the expression of miR-140, miR-185 and miR-424.

Claims

Claim

[Claim 1] A recombinant adeno-associated virus for synchronously inhibiting expression of three microRNAs, characterized in that the recombinant adeno-associated virus is constructed by the following method:

a.

Tud specific for miR-140, miR-1 85 and miR-424 based on human miR-140, miR-185 and miR-424 sequences

The RNA sequence ij ij, the recombinant plasmid pAKD-Tud-140-185-424 was constructed and subjected to DNA sequencing analysis;

b. Recombinant plasmid pAKD-Tud-140-185-424, packaging plasmid pAAV-RC and helper plasmid pHelper were co-transfected into AAV-293 cells with calcium phosphate method, and recombinant adeno-associated virus rAA V-Tud-140-185- was harvested. 424, The titer of the virus was determined by a quantitative PCR method.

[Claim 2] The recombinant adeno-associated virus for synchronously inhibiting the expression of three microRNAs according to claim 1, wherein the serotype of the adeno-associated virus vector is type 8.

[Claim 3] The recombinant adeno-associated virus for synchronously inhibiting expression of three microRNAs according to claim 1, wherein step a is specific for miR-140, miR-185 and miR-424-specific Tud RNA The sequence is: 5'-

CTAGCGCCACCTTTTT -3,.

[Claim 4] The recombinant adeno-associated virus for synchronously inhibiting the expression of three microRNAs according to claim 1, wherein in step a, Kpnl and Bgl II incisions are added to the selected target sequence. Enzymatic cleavage site sequence and transcription termination sequence, synthesis of complete Tud DNA sequence 歹IJ

Tud DNA-F: 5'- ATCCTAGCGCCACCTTTTTGTAC -3' Tud DNA-R: 5,-

GATGATCCTAGCGCC -3' =