CN108359667B

CN108359667B - siRNA sequence expressed by silent programmed death receptor-ligand and application thereof

Info

Publication number: CN108359667B
Application number: CN201810012799.4A
Authority: CN
Inventors: 季爱民; 岑柏宏
Original assignee: Wuhan Zezhi Biological Pharmaceutical Co ltd
Current assignee: Wuhan Zezhi Biological Pharmaceutical Co ltd
Priority date: 2018-01-06
Filing date: 2018-01-06
Publication date: 2021-01-05
Anticipated expiration: 2038-01-06
Also published as: CN108359667A

Abstract

The invention discloses a siRNA sequence expressed by a silent programmed death receptor-ligand, which refers to any one of six nucleotide sequences capable of silencing human programmed death receptor-ligand 1, and particularly has the best silencing effect when the target sequence is GTAGTAGATGTTACAATTT or GGATAAGAACATTATTCAA. The invention can effectively reduce the expression level of PD-L1/CD274 protein on the surface of tumor cells, promote the killing of T lymphocytes to the tumor cells and further inhibit the growth of the tumor. The invention can be used for designing and preparing nucleic acid medicaments for preventing and treating various cancers.

Description

siRNA sequence expressed by silent programmed death receptor-ligand and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a siRNA sequence expressed by a silent gene or protein and application thereof.

Background

Small interfering RNA (siRNA) can specifically silence the expression level of target mRNA (Nobel biomedical prize 2006), so that the siRNA molecule can treat diseases caused by gene overexpression and gene mutation by specifically silencing genes caused or promoting disease occurrence or development (Science, 2016,352(6292): 1417-. Since 2004 the FDA has approved 20 or more siRNA molecule drug formulations into clinical trials, of which nearly 10 phase III clinical trial protocols were in progress. In 9 months 2017, the american company of alanam (www.alnylam.com) first announced that its siRNA drug, patrisiran, against Transthyretin (TTR) reaches primary and secondary endpoints in the third phase of a Familial Amyloid Polyneuropathy (FAP), which is the primary clinical endpoint of the siRNA drug in the third phase of the first time, meaning that the siRNA drug is about to come into the market for clinical application.

In a gene DNA double strand, a single strand that synthesizes a template as mRNA (messenger RNA, RNA carrying genetic information and serving as a template in protein synthesis) when transcribed is called a template strand or an antisense strand, and a single strand that cannot synthesize a template as mRNA when transcribed is called a sense strand or a sense strand. Among the siRNA duplexes, the single strand that forms an RNA Interference Silencing Complex (RISC) with the Argonaute protein (AGO protein) is called the Antisense strand (Antisense strand) or Guide strand (Guide strand), and the RISC complex formed by the strand binds to, cleaves and silences the target mRNA. While the other non-functional single strand is called the sense strand (sense strand) or the Passenger strand (Passenger strand). Using the characteristics of siRNA molecules, it is possible to design siRNA sequences for multiple silent target genes (Nature Reviews Genetics,2015,16(9): 543-552.).

PD-L1/CD274 is collectively called programmed cell death receptor-Ligand 1 (programmed cell death-Ligand 1), is a transmembrane protein of size 40kDa and is expressed in most tumors. The immune system normally responds to foreign antigens that accumulate in the lymph nodes or spleen, promoting proliferation of antigen-specific T cells. And the programmed death receptor-1 (PD-1) on the surface of the T cell is combined with the programmed death receptor-ligand 1(PD-L1) to transmit inhibitory signals and reduce the proliferation of the T cell. During tumorigenesis, PD-L1/CD274 expression on the surface of tumor cells is up-regulated, interacts with PD-1 receptors on the surface of T cells, and inhibits activation and proliferation of T cells, thereby generating tumor immune escape (Immunotherapy, 2016(8), 479-488). Therefore, designing small molecule inhibitors or antibodies to inhibit the PD-1 or PD-L1 pathway and thereby directly kill tumor cells using body T cells has become a hotspot of tumor immunotherapy. By the end of 10 months in 2017, the drugs aiming at the PD-1 target are Nivolumab (trade name Opdivo) of Behcet MeishiGuibao (BMS) company respectively; pembrolizumab (trade name Keytruda) by mshon. The drugs for the PD-1 target are Atezolizumab (trade name Tecnriq) by Roche, respectively; bavencio (trade name Avelumab) co-developed by merck and fevery, germany; durvalumab (trade name Imfinzi) from Aslikang.

The siRNA disclosed in International publication No. WO2017190695A1, which inhibits the expression of EGFR gene, can only inhibit the expression of EGFR gene, but cannot inhibit the expression of programmed death receptor-ligand.

Non-human animals having a humanized cluster of differentiation 274 gene, patent application No. 2015800670256, methods and compositions for making and using the non-human animals are provided, wherein the non-human animals comprise a humanization of the cluster of differentiation 274(CD274) gene.

Such non-human animals may be described in some embodiments as having a genetic modification to an endogenous CD274 gene such that the non-human animal expresses a programmed cell death ligand 1(PD-L1) polypeptide. The invention does not disclose an siRNA sequence capable of efficiently silencing the expression of the human PD-L1.

Disclosure of Invention

The purpose of the invention is as follows:

provides a siRNA sequence of expression of a silent programmed death receptor-ligand capable of effectively reducing the expression level of PD-L1/CD274 protein on the surface of a tumor cell and promoting the lethality of a T lymphocyte to the tumor cell and application thereof.

The technical scheme is as follows:

the siRNA sequence expressed by the silent programmed death receptor-ligand is human programmed death receptor-ligand 1 (PD-L1/CD 274 for short) existing on the surfaces of various tumor cells, and the siRNA sequence is any one of nucleotide sequences with the following target sequence, sense strand and antisense strand:

siPD-L1-1

target sequence: CAGAAAGATGAGGATATTT

Sense strand: 5 '-CAGAAAGAUGAGGAUAUUnn-3'

Antisense strand: 5 '-AAAUCCUCAUCUUUCUGn-3';

siPD-L1-2

target sequence: GTATGAGTTTTTCCTATTT

Sense strand: 5 '-GUAUGAGUUUUUCCCUAUUnn-3'

Antisense strand: 5 '-AAAUAGGAAAAACUCAUACn-3';

siPD-L1-3

target sequence: GTAGTAGATGTTACAATTT

Sense strand: 5 '-GUAGUAGAUGUUUACAAUUUNn-3'

Antisense strand: 5 '-AAAUUGUAACAUCUACUACnn-3';

siPD-L1-4

target sequence: GTAGATGTTACAATTTTGT

Sense strand: 5 '-GUAGAUGUUACAAUUUGUnn-3'

Antisense strand: 5' -ACAAAAUUGUAACAUCUACnn-3;

siPD-L1-5

target sequence: GTAGAGTATGGTAGCAATA

Sense strand: 5 '-GUAGUAUGGUAGCAAUAnn-3'

Antisense strand: 5 '-UAUUGCACCAUACUCACnN-3';

siPD-L1-8

target sequence: GGATAAGAACATTATTCAA

Sense strand: 5 '-GGAUAAGAACAUUUCAAnn-3'

Antisense strand: 5 '-UUGAAUGUUCUUCUCCn-3';

wherein n is one of A, G, C, U, dA, dC, dG and dT, and the two n can be the same or different; or one is absent from two n; or both n are missing.

Of the above sequences, the 19 nucleotide sequences after the 5 'end of the sense strand are complementary to the 19 nucleotide sequences before the 5' end of the antisense strand; wherein A is adenine ribonucleotide; g is guanine ribonucleotide; c is cytosine ribonucleotide; u is uracil ribonucleotide; dA is adenine deoxynucleotide; dC is cytosine deoxynucleotide; dG is guanine deoxynucleotide; dT is thymine deoxynucleotide.

In the present invention, preferably said nn is two free and consecutive deoxynucleotides dTdT.

In the invention, the siRNA is obtained by adopting an siRNA design rule, combining with computer aided design software and screening through a comparison experiment. Using a control sequence: SiPD-L1-PC is the siRNA sequence of the positive control, and siNC is the siRNA sequence of the negative control.

On the basis, through RT-qPCR and flow cytometry experimental screening, PD-L1/CD274 gene inhibition efficiency is selected to be more than 50% to kill the glioma cells by the T lymphocytes, and the designed sequence biological function is evaluated.

Among several siRNA sequences with different nucleotide sequences for silencing expression of programmed death receptor-ligand 1 (PD-L1/CD 274) gene, candidate siRNA target sequences are shown in the following table respectively, and nn in a sense strand and nn in an antisense strand are dtdt.

Sequence name	Target sequence
		siPD-L1-1	CAGAAAGATGAGGATATTT
siPD-L1-2	GTATGAGTTTTTCCTATTT
		siPD-L1-3	GTAGTAGATGTTACAATTT
siPD-L1-4	GTAGATGTTACAATTTTGT
		siPD-L1-5	GTAGAGTATGGTAGCAATA
siPD-L1-6	CAAATTCCCAGTAGAAAAA
		siPD-L1-7	CAGTAGAAAAACAATTAGA
siPD-L1-8	GGATAAGAACATTATTCAA
		siPD-L1-9	CAAAATCAACCAAAGAATT
siPD-L1-10	CAACAACTAATGAGATTTT
		siPD-L1-PC	GTATGGTAGCAATATGACA
siNC	No homology between human and mouse genes

In the above table, six of the siRNA duplexes for efficiently silencing PD-L1/CD274 expression are: sipD-L1-1, sipD-L1-2, sipD-L1-3, sipD-L1-4, sipD-L1-5 and sipD-L1-8.

Among the six siRNA double-stranded target sequences, particularly siPD-L1-3, siPD-L1-4, siPD-L1-5 and siPD-L1-8 have better silencing effect, and the sequences have basically the same CAAT or TCAA base short sequences, which is probably one of the important reasons that the sequences have better silencing effect.

When the siRNA sequence is siPD-L1-3 or siPD-L1-8, the effect of silent expression is the best.

The invention relates to the following three theoretical bases:

1) theory of RNA interference. siRNA is a double-stranded structure, after entering cells, double strands unwind and become single strands (sense strand and antisense strand, or sense strand-S, antisense strand-AS), wherein the AS strand is complementary to a target mRNA (mRNA of PD-L1/CD274 in the patent) sequence to form a new local double strand (different in length), and at this time, intracellular enzymes such AS AGO2 cut off the target mRNA. This is the RNA interference theory (the prize of nobel biomedical science in 2006), that is, as long as the double-stranded structure of RNA exists in the cell and can be cleaved by enzymes similar to AGO2, mRNA is not a complete structure, and thus the translation function is lost and cannot be translated into protein. The six siRNA segments protected by the patent can be respectively combined (with complementary sequences) at different positions of the mRNA of PD-L1/CD274, and can realize RNA interference and silence the translation function of the mRNA of PD-L1/CD 274.

2) Theory that tumor cells achieve immune escape through the PD-1/PD-L1 pathway: tumor cells and tumor microenvironment limit the host immune response by up-regulating PD-L1 expression (here, the tumor cells themselves or their microenvironment express PD-L1) and binding to PD-1 on the surface of cytotoxic CD8+ T cells. Or CD8+ T cells in humans, which can destroy tumors by cytotoxic effects; if a human body presents with tumor cells, the CD8+ T cells have anti-tumor effect under the general condition; however, PD-L1 expressed by the tumor cell itself binds to PD-1 receptor expressed by T cell membrane once the T cell is close to the tumor cell to kill the tumor cell, and after PD-L1 is combined with PD-1, the T cell can not perform the task of killing the tumor cell through a series of signal transduction pathways. If the tumor cells are not allowed to express PD-L1 (the siRNA in this patent silences the expression of PD-L1), or PD-L1 loses the ability to bind to the PD-1 receptor expressed on the surface of T cells with an anti-PD-L1 antibody drug, the function of T cells cannot be inhibited, and the T cells are not prevented from "performing the task of killing tumor cells". The siRNA sequence provided by the invention cuts mRNA of PD-L1 from a plurality of positions of RNA level, thereby terminating translation and expression of PD-L1 protein, and theoretically, can effectively reduce reduction or deletion of inhibition titer or effect caused by mutation of PD-L1 protein level. Therefore, the siRNA sequence of the invention has stronger function of inhibiting PD-L1 than antibody drugs which are applied clinically.

3) Obtaining siRNA sequences follows some known theories at present, a plurality of siRNA sequences capable of specifically silencing target mRNA can be obtained by inputting the target mRNA sequence into the software and setting some limited conditions through a large foreign research structure, after siRNA fragments are obtained through synthesis, the siRNA is transfected into cells through liposomes, and the silencing efficiency of the obtained siRNA can be judged by measuring the amount of the complete target mRNA in the cells through a PCR technology. However, the silencing efficiency is influenced by a plurality of factors, such as whether the mRNA fragment complementary to the siRNA has secondary structure (e.g., helix, etc.), the antisense strand in the siRNA cannot be bonded to the target mRNA in a "comfortable" manner, and cannot be completely and completely cut by AGO2, etc., so that the PCR result shows a difference in silencing efficiency. In practical application, the siRNA segment with the highest silencing efficiency is generally selected. The low silencing efficiency can be compensated by means of increasing the administration dosage.

Another object of the present invention is to provide a use of the siRNA sequence for silent programmed death receptor-ligand expression described above.

Is used for designing and preparing medicaments (especially antitumor medicaments aiming at tumor immune escape) for treating related diseases caused by the over-expression of PD-L1/CD274 in human pathological tissues and the abnormal immune system.

The related tumor diseases are any one or more of lung cancer, liver cancer, esophageal cancer, cervical cancer, colorectal cancer, pancreatic cancer, renal cancer, bladder cancer, breast cancer, prostatic cancer, gastric cancer, oral epithelial cancer, ovarian cancer, head and neck cancer, brain tumor and glioma.

Has the advantages that:

based on the existing biological medicine theory, the invention combines with experimental verification to obtain multiple siRNA with high expression level. The theory is combined with medical practice, and the conclusion is reliable. Has good popularization and application value.

The six siRNA sequences expressed by the efficient specific silent programmed death receptor-ligand 1 (PD-L1/CD 274) designed by the invention can effectively reduce the expression level of PD-L1/CD274 protein on the surface of tumor cells and promote the killing of T lymphocytes on the tumor cells, thereby generating the effect of inhibiting tumor growth similar to the effect of inhibiting a PD-1/PD-L1 pathway.

Moreover, from hundreds of possible sequences, 6 species with simple target sequences are preferred, and the species have good operability in synthesizing drugs and are not known to have small side effects.

Drawings

FIG. 1 is a graph showing the mRNA expression level analysis of PD-L1/CD274 in a human glioma cell line (U87 MG) following transfection of siRNA with lipofectamine 2000 in an RT-qPCR assay.

In fig. 1, the ordinate chinese meaning: relative expression level of PD-L1. This shows how much PD-L1 was expressed in the cells after siRNA transfection; higher expression levels indicate less efficient silencing of PD-L1 expression by the added siRNA. The siPD-l-8 in the figure is the most efficient in silencing.

FIG. 2 is a graph showing the effect of surface PD-L1/CD274 protein expression in a human glioma cell line (U87 MG) following transfection of siRNA with lipofectamine 2000 using flow cytometry.

In fig. 2, ordinate: count refers to the number of cells; the abscissa: PE refers to the intensity of red fluorescent PE and peak area refers to the percentage of the entire cell population.

FIG. 3 is a graph showing the effect of apoptosis in a human glioma cell line (U87 MG) after transfection of siRNA with lipofectamine 2000 and addition of human CD8 positive T cells by flow cytometry.

In fig. 3, ordinate: PI (propidium iodide) is a dye that stains the nucleus, with higher PI values, deeper staining of the nucleus, meaning that the entire cell membrane is destroyed, exposing the entire nucleus. AV (annexin V) is a dye for staining apoptotic antigens on cell membranes, and the higher the AV value, the more apoptotic antigens on cell membranes. Q4 is a normal cell population. Q1 is a mechanically damaged necrotic cell, meaning that the pancreatin is over-digested and the whole cell is mechanically damaged. Q2 refers to late apoptotic cells. Q3 is an early apoptotic cell.

Detailed Description

Example 1: RT-qPCR verified gene silencing efficiency of siPD-L1-1-10 at RNA level.

As shown in FIG. 1, according to the well-known cell biology method, a human glioma cell line (U87 MG) in a logarithmic growth phase is transfected in a cell culture plate, siPD-L1-1-10 with 100nM concentration forms lipid complex with liposome to transfect cells respectively under the assistance of lipofectamine 2000 transfection reagent, and the RT-qPCR experiment result shows that the expression of PD-L1/CD274 gene mRNA is effectively silenced at the RNA level by siPD-L1-1, siPD-L1-2 siPD-L1-3, siPD-L1-4, siPD-L1-5 and siPD-L1-8. Among them, siPD-L1-8 was most effective in silencing.

Example 2: the surface protein level gene silencing efficiency of siPD-L1-1-10 verified by flow cytometry.

As shown in FIG. 2, human glioma cell lines (U87 MG) in logarithmic growth phase were transfected in cell culture plates according to a known cell biology method, siPD-L1-1-10 at a concentration of 100nM was formed into lipid complexes with liposomes with the aid of lipofectamine 2000 transfection reagent to transfect cells, and after 48 hours, the cells were collected and subjected to flow cytometry to detect cell surface proteins. The results show that at the RNA level, siPD-L1-1, siPD-L1-2, siPD-L1-3, siPD-L1-4, siPD-L1-5 and siPD-L1-8 all silenced the expression of cell surface PD-L1/CD274 protein.

Example 3: the toxic effect of human CD8 positive T cells on glioma cells after silencing PD-L1/CD274 was verified by flow cytometry.

As shown in FIG. 3, according to the known methods of cell biology, the previous experiments showed that siPD-L1-3, siPD-L1-5 and siPD-L1-8 can inhibit cell surface PD-L1/CD274 protein by more than 50%. On the basis, a human glioma cell line (U87 MG) in a logarithmic growth phase is transfected in a cell culture plate, siPD-L1-3, siPD-L1-5 and siPD-L1-8 with the concentrations of 100nM respectively form lipid complex transfected cells with liposomes under the assistance of lipofectamine 2000 transfection reagent, and after 24 hours, human CD8 positive T cells are added for intervention. After the intervention is continued for 24 hours, the apoptosis of glioma cells is detected by flow cytometry. Experimental results show that siPD-L1-3, siPD-L1-5 and siPD-L1-8 can all cause apoptosis, wherein the effect of siPD-L1-3 is the most remarkable.

SEQUENCE LISTING

<110> Wuhan ze Zhi biopharmaceutical GmbH

<120> siRNA sequence expressed by silent programmed death receptor-ligand and application thereof

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<140> 2018100127994

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Claims

1. A siRNA sequence that silences programmed death receptor-ligand expression, comprising: the programmed death receptor-ligand is human programmed death receptor-ligand 1 (PD-L1/CD 274 for short) existing on the surfaces of various tumor cells, and the nucleotide sequences of a target sequence, a sense strand and an antisense strand of the siRNA sequence are shown as the following siPD-L1-1, siPD-L1-2 or siPD-L1-8:

siPD-L1-1

target sequence: CAGAAAGATGAGGATATTT

Sense strand: 5 '-CAGAAAGAUGAGGAUAUUnn-3'

Antisense strand: 5 '-AAAUCCUCAUCUUUCUGn-3';

siPD-L1-2

target sequence: GTATGAGTTTTTCCTATTT

Sense strand: 5 '-GUAUGAGUUUUUCCCUAUUnn-3'

Antisense strand: 5 '-AAAUAGGAAAAACUCAUACn-3';

siPD-L1-8

target sequence: GGATAAGAACATTATTCAA

Sense strand: 5 '-GGAUAAGAACAUUUCAAnn-3'

Antisense strand: 5 '-UUGAAUGUUCUUCUCCn-3';

wherein n is one of A, G, C, U, dA, dC, dG and dT, and the two n are the same or different; or one is absent from two n; or both n are missing.

2. A siRNA sequence of claim 1 that silences programmed death receptor-ligand expression, wherein: the nn is two free and continuous deoxynucleotides dTdT.

3. A siRNA sequence of claim 1 or 2 to silence programmed death receptor-ligand expression, wherein: the target sequence, the sense strand and the antisense strand of the siRNA sequence have the following sequences:

target sequence: GGATAAGAACATTATTCAA

Sense strand: 5 '-GGAUAAGAACAUUUAUCAAdTdT-3'

Antisense strand: 5 '-UUGAAUGUUCUUCCdTdT-3'.

4. Use of a sirtuin-ligand expressed siRNA sequence of claim 1, wherein: is used for designing or preparing a medicament capable of reducing the expression of programmed death receptor-ligand 1 on the surface of human tumor cells and inhibiting the growth of tumors.