CN116790580A

CN116790580A - Oncolytic virus-derived RNA and use thereof

Info

Publication number: CN116790580A
Application number: CN202310647014.1A
Authority: CN
Inventors: 姜孝明; 宋庆乐; 何裕华; 陈宇静; 谢天婕; 何凡翠; 夏鹏昌
Original assignee: Letu Youjia Shenzhen Pharmaceutical Technology Co ltd; Shenzhen Letu Biomedical Co ltd
Current assignee: Letu Youjia Shenzhen Pharmaceutical Technology Co ltd; Shenzhen Letu Biomedical Co ltd
Priority date: 2023-06-02
Filing date: 2023-06-02
Publication date: 2023-09-22

Abstract

An oncolytic virus-derived RNA and uses thereof, the source of the RNA comprising, or having homology to, an oncolytic virus. The RNA provided by the application can effectively inhibit cancer cell proliferation.

Description

Oncolytic virus-derived RNA and use thereof

Technical Field

The application relates to the field of biological medicine, in particular to an oncolytic virus-derived RNA and application thereof.

Background

Despite the great progress made in the treatment of cancer by various therapies, many cancers remain difficult to inhibit. In recent years, viral vectors have been developed for gene therapy against cancer, and the use of viruses as anticancer drugs has attracted considerable attention. Oncolytic viruses can be specifically replicated in tumor cells, have good effects in animal tumor models, and can effectively inhibit tumor growth. Various live viruses, such as mumps, newcastle disease, measles, reoviruses and vesicular stomatitis viruses, are injected in tumors to kill cancer cells using viral infection and viral replication. Based on this concept, there are a number of oncolytic viruses on the market for drugs to treat cancer, but there are fewer cases of success in more viruses in clinical trials. The main reasons are that oncolytic virus drugs show a certain toxic and side effect in human body, and the manufacturing process is complex, the quality control difficulty is high, and the cost and the technical barrier for manufacturing products are high.

Disclosure of Invention

According to a first aspect, in an embodiment, there is provided an RNA, the source of which comprises, or has at least 80% homology with, an oncolytic virus RNA.

According to a second aspect, in an embodiment, there is provided a DNA comprising a nucleotide sequence encoding the RNA of any one of the first aspects.

According to a third aspect, in an embodiment, a delivery formulation is provided loaded with the RNA of any of the first aspects.

According to a fourth aspect, in an embodiment there is provided the use of an RNA according to any one of the first aspect, or a DNA according to any one of the second aspect, in the manufacture of a medicament for the prevention and/or treatment of a disease.

In one embodiment, the RNA provided by the application is effective in inhibiting cancer cell proliferation.

Drawings

FIG. 1 shows the inhibitory effect of 6 RNAs on MFC cell viability in example 1;

FIG. 2 is a graph showing the effect of U5 on MFC in example 2 in inhibiting cell viability;

FIG. 3 is a graph showing the effect of U5 on inhibition of MB49 cell viability in example 3;

FIG. 4 shows the cell viability inhibition effect of U5 on 4T1 in example 4;

FIG. 5 shows the cell viability inhibition effect of U5 on B16F10 in example 5;

FIG. 6 shows the cell viability inhibition effect of U5 on CT-26 in example 6;

FIG. 7 is a graph showing the effect of U5 on inhibition of PC3 cell viability in example 7;

FIG. 8 is a graph showing the effect of U5 in example 8 on inhibition of GES-1 cell viability;

FIG. 9 is a graph showing the tumor volume growth of the human gastric cancer (NCL-N87) subcutaneous graft tumor model in example 9;

FIG. 10 is an in vitro diagram of tumor tissue of a human gastric cancer (NCL-N87) subcutaneous tumor graft model in example 9;

FIG. 11 is a graph showing the tumor volume growth of the mouse RENCA kidney cancer cell subcutaneous engrafting tumor model in example 10;

FIG. 12 is a graph showing the tumor weight results of the mouse RENCA kidney cancer cell subcutaneous tumor transplantation model in example 10.

Detailed Description

The application will be described in further detail below with reference to the drawings by means of specific embodiments. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted in various situations, or replaced by other materials, methods. In some instances, related operations of the present application have not been shown or described in the specification in order to avoid obscuring the core portions of the present application, and may be unnecessary to persons skilled in the art from a detailed description of the related operations, which may be presented in the description and general knowledge of one skilled in the art.

Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.

The numbering of the components itself, e.g. "first", "second", etc., is used herein merely to distinguish between the described objects and does not have any sequential or technical meaning.

In one embodiment, the application analyzes RNA of Sendai virus (HVJ, hemagglutinating virus of Japan), and discovers that short RNA fragments with certain structural characteristics can also inhibit the activity of tumor cells to a certain degree specifically, and the application has potential to be developed into novel antitumor drugs. The application provides RNA fragments, not viruses, but in the prior art, complete living viruses are used, and the RNA fragments can be prepared by a chemical synthesis method, and have the advantages of mature process flow, short period, low cost and high quality controllability.

According to a first aspect, in an embodiment, there is provided an RNA, the source of which comprises, or has homology to, an oncolytic virus.

In one embodiment, the RNA can inhibit cancer cell proliferation.

In one embodiment, the oncolytic virus includes, but is not limited to, sendai virus.

In one embodiment, the U-shaped sequence in the RNA and other nucleotides that do not affect self-pairing to form a hairpin structure can be altered.

In one embodiment, the RNA comprises the nucleotide sequence set forth in any one of SEQ ID NOS.1-6 or a nucleotide sequence having homology thereto.

In one embodiment, the RNA comprises a set of nucleotide sequences set forth in any one of SEQ ID NOS.1-6.

In one embodiment, the RNA comprises the set of nucleotide sequences set forth in SEQ ID NO. 1.

In the prior art, the preparation process of oncolytic viruses is complex, and the RNA fragment provided by the application can be prepared by a chemical synthesis method, and has the advantages of mature process flow, short period, low cost and high quality controllability. In vivo viruses bring about a large number of uncontrollable side effects, and by synthesizing RNA fragments, the toxic and side effects can be effectively avoided due to the fact that the sequences are designed and optimized.

In one embodiment, at least one nucleotide of the RNA is chemically modified from at least 3 nucleotides from the 5' end.

In one embodiment, at least one nucleotide of the RNA is chemically modified from at least 3 nucleotides from the 3' end.

In one embodiment, the nucleotides at both ends of the RNA may or may not have chemical modifications.

In one embodiment, the modification of both ends of the RNA helps to increase RNA stability and half-life.

In one embodiment, the chemical modification includes, but is not limited to, a 2' -O-ribose modification.

In one embodiment, the 2 '-O-ribose modification includes, but is not limited to, at least one of a 2' -O-ribomethyl modification, a 2 '-O-ribofluoro modification, a 2' -O-MOE ribose modification.

In one embodiment, the nucleotides with chemical modifications may be the same or different from one nucleotide to another.

In one embodiment, the nucleotide at the 3 'end of the RNA is 5' -methylcytidine.

In one embodiment, the RNA is single stranded.

In one embodiment, the RNA is double stranded.

In one embodiment, the RNA comprises a hairpin.

In one embodiment, the delivery formulation includes, but is not limited to, cationic polymers, lipid nanoparticles (Lipid Nanopa rticle, LNP).

In one embodiment, the cationic polymer includes, but is not limited to, PEI (Polyethylenimine).

In one embodiment, the disease includes, but is not limited to, cancer.

In one embodiment, the cancer includes, but is not limited to, at least one of gastric cancer, bladder cancer, breast cancer, melanoma, colon cancer, prostate cancer, renal cancer, and the like. The specific cancer species are not limited and are only exemplary lists herein.

In one embodiment, the drug is intact or nearly intact to normal cells.

In one embodiment, the normal cells include, but are not limited to, gastric mucosal cells. The gastric mucosa cells are only representative of normal cells, and the medicaments prepared from RNA provided by the application have almost no damage to all normal cells.

In one embodiment, by analyzing genomic RNA of Sendai virus (HVJ), RNA fragment sequences that inhibit tumor cell viability are designed and methylation modified at the 5 'and 3' segments to enhance RNA fragment stability, delivered to tumor cells by transfection techniques, and thereby inhibit tumor cell growth.

In one embodiment, the present application is directed to an oncolytic virus-derived RNA fragment and its anti-tumor application, which specifically inhibits tumor cell activity.

In one embodiment, an oncolytic virus-derived RNA fragment is provided, wherein the RNA is single stranded.

In one embodiment, the oncolytic virus-derived RNA fragments of the present application form a duplex.

In one embodiment, the oncolytic virus-derived RNA fragments of the present application, wherein the duplex comprises a hairpin.

In one embodiment, the oncolytic virus-derived RNA fragments of the present application, wherein the number of RNA bases is 30nt.

In one embodiment, the oncolytic virus-derived RNA fragments of the present application, wherein the sequence nucleotide is optionally subjected to a 2 '-O-ribose modification selected from the group consisting of 2' -O-ribomethyl modification, 2 '-O-ribofluoro modification, 2' -O-MOE ribose modification, and the like.

In a preferred embodiment, the oncolytic virus-derived RNA of the present application has a fragment of 5'-mG-s-mU-s-mG-s-CCCAUUCGGGGGGGGCCGAAUCAG-s-mC-s-mA-s-r5meC-3' (SEQ ID NO. 1), s represents a thio modification, m represents 2 '-O-methylation, and 5meC represents 5' -methylcytidine.

In one embodiment, the application also provides the use of the oncolytic virus-derived RNA fragments in the manufacture of a medicament for the prevention and/or treatment of a disease.

In one embodiment, the application also provides the use of the oncolytic virus-derived RNA fragments in the preparation of an antitumor drug.

In one embodiment, the oncolytic virus-derived RNA fragments of the present application can be loaded using a vector.

In one embodiment, the carrier includes, but is not limited to, at least one of a cationic polymer, a Lipid Nanoparticle (LNP).

In one embodiment, the oncolytic virus-derived RNA fragments provided by the present application can specifically inhibit tumor cell activity by inducing tumor cell apoptosis.

In one embodiment, the tumor cells include, but are not limited to, at least one of gastric cancer, prostate cancer, melanoma, bladder cancer, colon cancer, breast cancer, renal cancer, and the like.

In one embodiment, the oncolytic virus-derived RNA fragments of the present application can avoid damaging normal cells, such as human gastric mucosal cells.

In one embodiment, the anti-neoplastic agent may be administered by a convenient means, including but not limited to by intravenous, intratumoral, and the like routes of administration.

In one embodiment, the application adopts a short RNA sequence designed by referring to oncolytic viruses, and the short RNA sequence can be prepared by a chemical synthesis method, and has the advantages of mature process flow, short period, low cost and high quality controllability.

Example 1 nucleic acid sequence alignment experiments

The experimental steps are as follows:

1. cell plating:

mice gastric cancer cells (MFC) were inoculated at 4000 cells/well into 96-well plates and incubated at 37 ℃ for 24h for later use.

2. Dosing regimen:

5 centrifuge tubes were prepared, opti-MEM medium and U5 were added, and U5 stock solutions of different concentrations were prepared by a double dilution method with concentration gradients of 51.2. Mu.g/mL, 25.6. Mu.g/mL, 12.8. Mu.g/mL, 6.4. Mu.g/mL, 3.2. Mu.g/mL, 1.6. Mu.g/mL, 0.8. Mu.g/mL, 0.4. Mu.g/mL. PEI is taken and added into an opti-MEM culture medium to be diluted to obtain a PEI solution with the concentration of 0.4 mu g/mL, the PEI solution is added into 8 centrifuge tubes diluted in the above way in an equal volume, and the mixture is incubated for 10min to obtain U5 samples with the concentration gradient of 25.6 mu g/mL, 12.8 mu g/mL, 6.4 mu g/mL, 3.2 mu g/mL, 1.6 mu g/mL, 0.8 mu g/mL, 0.4 mu g/mL and 0.2 mu g/mL.

The original 100 mu L of culture medium in the well is reserved, 100 mu L of the diluted U5 sample is added into 96 wells, so that the concentration gradient of the drug in the wells is 12.8 mu g/mL, 6.4 mu g/mL, 3.2 mu g/mL and 1.6 mu g/mL, 3 compound wells are arranged for each concentration, and the culture is carried out at 37 ℃ for 48 hours.

3. Cell viability detection:

the cell supernatant was aspirated, 100. Mu.L of fresh medium and 100. Mu.L of chemiluminescent reagent were added to each well, incubated and mixed well, and transferred to a white plate at 120. Mu.L, and chemiluminescent detection was performed using a multifunctional microplate reader at a detection wavelength of 560nm.

Analysis of results:

FIG. 1 shows the following 5 RNAs and the inhibitory effect of U5 on MFC cell viability, U5 showed the best inhibitory effect on tumor cell viability, so U5 was selected for the subsequent study.

RNA sequence:

1：GUGCCCAUUCGGGGGGGGCCGAAUGGGCAC(SEQ ID NO.2)；

2：GUGCCCAUUCGGAAAAAACCGAAUCAGCAC(SEQ ID NO.3)；

3：GUGCUGAUUCGGGGGGGGCCGAAUCAGCAC(SEQ ID NO.4)；

4：GUGCCCAUUCGGAAAAAACCGAAUGGGCAC(SEQ ID NO.5)；

5：GUGCUGAUUCGGAAAAAACCGAAUCAGCAC(SEQ ID NO.6)。

by analyzing RNA fragments of Sendai virus, RNA sequences are designed, three bases at the 5 'and 3' ends are subjected to methylation modification, and the sequence is named U5,5'-mG-s-mU-s-mG-s-CCCAUUCGGGGGGGGCCGAAUCAG-s-mC-s-mA-s-r5meC-3' (SEQ ID NO. 1). s represents a thio modification, m represents 2 '-O-methylation, and 5meC represents 5' -methylcytidine.

EXAMPLE 2MFC cell viability assay

The experimental steps are as follows:

1. cell plating:

2. Dosing regimen:

8 centrifuge tubes were prepared, opti-MEM medium and U5 were added, and U5 stock solutions of different concentrations were prepared by a double dilution method with concentration gradients of 51.2. Mu.g/mL, 25.6. Mu.g/mL, 12.8. Mu.g/mL, 6.4. Mu.g/mL, 3.2. Mu.g/mL, 1.6. Mu.g/mL, 0.8. Mu.g/mL, 0.4. Mu.g/mL. PEI is taken and added into an opti-MEM culture medium to be diluted to obtain a PEI solution with the concentration of 0.4 mu g/mL, the PEI solution is added into 8 centrifuge tubes diluted in the above way in an equal volume, and the mixture is incubated for 10min to obtain U5 samples with the concentration gradient of 25.6 mu g/mL, 12.8 mu g/mL, 6.4 mu g/mL, 3.2 mu g/mL, 1.6 mu g/mL, 0.8 mu g/mL, 0.4 mu g/mL and 0.2 mu g/mL.

The original 100 mu L of culture medium in the well is reserved, 100 mu L of the diluted U5 sample is added into a 96 well, the concentration gradient of the drug in the well is 12.8 mu g/mL, 6.4 mu g/mL, 3.2 mu g/mL, 1.6 mu g/mL, 0.8 mu g/mL, 0.4 mu g/mL, 0.2 mu g/mL and 0.1 mu g/mL, 3 compound wells are arranged for each concentration, and the culture is carried out for 48 hours at 37 ℃.

3. Cell viability detection:

Analysis of results:

FIG. 2 is a graph showing the effect of U5 on inhibiting the cell viability of MFC. It can be seen that U5 can effectively inhibit cell viability of MFC and exhibit concentration dependence, with about 60% inhibition achieved at 12.8 μg/mL.

Example 3MB49 cell viability assay

The experimental steps are as follows:

1. cell plating:

mouse bladder cancer cells (MB 49) were seeded at 4000 cells/well in 96-well plates and incubated at 37℃for 24 h.

2. Dosing regimen:

5 centrifuge tubes were prepared, and the opti-MEM medium and U5 were added to prepare U5 stock solutions of different concentrations by a double dilution method, with concentration gradients of 51.2. Mu.g/mL, 25.6. Mu.g/mL, 12.8. Mu.g/mL, 6.4. Mu.g/mL, 3.2. Mu.g/mL. PEI is taken and added into an opti-MEM culture medium to be diluted, so that a PEI solution with the concentration of 0.4 mug/mL is obtained, the PEI solution is added into 5 centrifuge tubes diluted in the above way in an equal volume, and the mixture is incubated for 10min, so that U5 samples with the concentration gradient of 25.6 mug/mL, 12.8 mug/mL, 6.4 mug/mL, 3.2 mug/mL and 1.6 mug/mL are obtained.

The original 100 mu L of culture medium in the well is reserved, 100 mu L of the diluted U5 sample is added into 96 wells, the concentration gradient of the drug in the wells is 12.8 mu g/mL, 6.4 mu g/mL, 3.2 mu g/mL, 1.6 mu g/mL and 0.8 mu g/mL, 3 compound wells are arranged for each concentration, and the culture is carried out for 48 hours at 37 ℃.

3. Cell viability detection:

Analysis of results:

FIG. 3 is a graph showing the effect of U5 on inhibiting the cell viability of MB 49. It can be seen that U5 can effectively inhibit the cell viability of MB49 and exhibit a concentration dependence, with an inhibition effect of about 30% being achieved at 12.8. Mu.g/mL.

Example 4T1 cell viability assay

The experimental steps are as follows:

1. cell plating:

mouse breast cancer cells (4T 1) were inoculated into 96-well plates at 4000 cells/well and incubated at 37℃for 24 hours.

2. Dosing regimen:

3. Cell viability detection:

Analysis of results:

FIG. 4 is a graph showing the effect of U5 on inhibiting cell viability of 4T 1. It can be seen that U5 can effectively inhibit the cell viability of 4T1 and exhibit a concentration dependence, with an inhibition effect of about 20% being achieved at 12.8. Mu.g/mL.

Example 5B16F10 cell viability assay

The experimental steps are as follows:

1. cell plating:

mouse melanoma cells (B16F 10) were seeded at 4000 cells/well in 96-well plates and incubated at 37℃for 24 h.

2. Dosing regimen:

3. Cell viability detection:

Analysis of results:

FIG. 5 is a graph showing the effect of U5 on inhibiting the cell viability of B16F 10. It can be seen that U5 can effectively inhibit the cell viability of B16F10 and can achieve about 50% inhibition between 1.6 μg/mL and 6.4 μg/mL.

EXAMPLE 6CT-26 cell viability assay

The experimental steps are as follows:

1. cell plating:

the colon cancer cells (CT-26) of the mice are inoculated into a 96-well plate at a concentration of 4000 cells/well and are cultured for 24 hours at 37 ℃ for later use.

2. Dosing regimen:

4 centrifuge tubes were prepared and the culture medium opti-MEM was added by double dilution to give U5 stock solutions of different concentrations at a concentration gradient of 51.2. Mu.g/mL, 25.6. Mu.g/mL, 12.8. Mu.g/mL, 6.4. Mu.g/mL. PEI is taken and added into an opti-MEM culture medium to be diluted, so that a PEI solution with the concentration of 0.4 mug/mL is obtained, the PEI solution is added into 5 centrifuge tubes diluted in the above way in an equal volume, and the mixture is incubated for 10min, so that U5 samples with the concentration gradient of 25.6 mug/mL, 12.8 mug/mL, 6.4 mug/mL and 1.6 mug/mL are obtained.

The original 100 mu L of culture medium in the well is reserved, 100 mu L of the diluted U5 sample is added into 96 wells, so that the concentration gradient of the drug in the wells is 12.8 mu g/mL, 6.4 mu g/mL, 3.2 mu g/mL and 0.8 mu g/mL, 3 compound wells are arranged for each concentration, and the culture is carried out at 37 ℃ for 48 hours.

3. Cell viability detection:

Analysis of results:

FIG. 6 is a graph showing the cell viability inhibition effect of U5 on CT-26. It can be seen that U5 can effectively inhibit CT-26 cell viability and exhibit a concentration dependence, with about 40% inhibition achieved at 12.8 μg/mL.

Example 7PC3 cell viability assay

The experimental steps are as follows:

1. cell plating:

human prostate cancer cells (PC 3) were seeded at 4000 cells/well in 96-well plates and incubated at 37℃for 24 h.

2. Dosing regimen:

8 centrifuge tubes were prepared, opti-MEM medium and U5 were added, and U5 stock solutions of different concentrations were prepared by a double dilution method with concentration gradients of 180. Mu.g/mL, 140. Mu.g/mL, 102.4. Mu.g/mL, 51.2. Mu.g/mL, 25.6. Mu.g/mL, 12.8. Mu.g/mL, 6.4. Mu.g/mL, 3.2. Mu.g/mL. PEI is taken and added into an opti-MEM culture medium to be diluted to obtain a PEI solution with the concentration of 0.4 mu g/mL, the PEI solution is added into 5 centrifuge tubes diluted in the above way in an equal volume, and the mixture is incubated for 10 minutes to obtain U5 samples with the concentration gradient of 90 mu g/mL, 70 mu g/mL, 51.2 mu g/mL, 25.6 mu g/mL, 12.8 mu g/mL, 6.4 mu g/mL, 3.2 mu g/mL and 1.6 mu g/mL.

The original 100 mu L of culture medium is reserved in the holes, 100 mu L of the diluted U5 sample is added into 96 holes, the concentration gradient of the drug in the holes is 45 mu g/mL, 35 mu g/mL, 25.6 mu g/mL, 12.8 mu g/mL, 6.4 mu g/mL, 3.2 mu g/mL, 1.6 mu g/mL and 0.8 mu g/mL, 3 compound holes are formed for each concentration, and the culture is carried out for 48 hours at 37 ℃.

3. Cell viability detection:

Analysis of results:

FIG. 7 is a graph showing the effect of U5 on inhibiting the cell viability of PC 3. It can be seen that U5 can effectively inhibit PC3 cell viability and exhibit concentration dependence, with about 60% inhibition achieved at 25.6 μg/mL.

EXAMPLE 8GES-1 Normal cell viability assay

The experimental steps are as follows:

1. cell plating:

human gastric mucosal cells (GES-1) were seeded at 4000 cells/well in 96-well plates and incubated at 37℃for 24 h.

2. Dosing regimen:

5 centrifuge tubes were prepared, opti-MEM medium and U5 were added, and U5 stock solutions of different concentrations were prepared by a double dilution method with concentration gradients of 180. Mu.g/mL, 140. Mu.g/mL, 102.4. Mu.g/mL, 51.2. Mu.g/mL, 25.6. Mu.g/mL, 12.8. Mu.g/mL, 3.2. Mu.g/mL. PEI is taken and added into an opti-MEM culture medium to be diluted, so that a PEI solution with the concentration of 0.4 mug/mL is obtained, the PEI solution is added into 5 centrifuge tubes diluted in the above way in an equal volume, and the mixture is incubated for 10 minutes, so that U5 samples with the concentration gradient of 90 mug/mL, 70 mug/mL, 51.2 mug/mL, 25.6 mug/mL, 12.8 mug/mL, 6.4 mug/mL and 1.6 mug/mL are obtained.

The original 100 mu L of culture medium in the well is reserved, 100 mu L of the diluted U5 sample is added into a 96 well, the concentration gradient of the drug in the well is 45 mu g/mL, 35 mu g/mL, 25.6 mu g/mL, 12.8 mu g/mL, 6.4 mu g/mL, 3.2 mu g/mL and 0.8 mu g/mL, 3 compound wells are prepared for each concentration, and the mixture is incubated for 48 hours at 37 ℃.

3. Cell viability detection:

Analysis of results:

FIG. 8 is a graph showing the effect of U5 on inhibition of cell viability of GES-1. It can be seen that U5 does not significantly affect the cell viability of normal human gastric mucosal cells, and still exhibits less cytotoxicity in the concentration range of 0.8 μg/mL to 25.6 μg/mL.

Due to the limitation of PEI on the loading capacity of nucleic acid, the cell cannot be inhibited more effectively even if the concentration is increased continuously, and the cell viability is increased instead in some cases. If the PEI concentration is increased simultaneously, PEI will be toxic to cells and decrease cell viability (see FIGS. 6 and 7).

Example 9 efficacy experiment of human gastric cancer (NCL-N87) subcutaneous transplantation tumor model

The experimental steps are as follows:

1. animal model

40 male BABL/c nude mice of 5 weeks old were housed in an IVC system within an SPF-class barrier environment. N87 cells were first digested with pancreatin, resuspended in PBS and counted, and the cell suspension was prepared to 2.5X10 ⁷ 200 μl/mL was injected into the armpit of the right forelimb of 4 nude mice to obtain the 1 st generation tumor. The 1 st generation tumor is taken, after removing the attached connective tissue part, the tumor tissue is weighed and cut up by 4g, 4mL PBS is added for uniform mixing, and the tumor tissue suspension is injected into the right armpit of the mouse according to the dosage of 200 mu L/mouse. When the tumor grows to 75-100 mm ³ Afterwards, 32 mice which are most suitable for experimental conditions are selected from 40 mice, experimental animals are randomly divided into 4 groups, 8 mice in each group are respectively, and the average tumor volumes of the groups are ensured to be close when the mice are put into the groups.

2. Pharmaceutical formulation

2.1A 10% trehalose solution at 0.3mg/mL U5 was prepared.

2.2A 10% trehalose solution of 0.43mg/mL PEI was prepared and pH=3.60 was adjusted with 1M sodium hydroxide.

2.3, respectively dividing the two working fluids into two syringes, keeping the same amount of the liquid in the syringes, connecting the syringes in a mixer, fixing the syringes on an injection pump, starting the injection pump, and rapidly mixing to obtain the preparation stock solution.

2.4 lyophilizing overnight to obtain lyophilized powder.

2.5 re-dissolving the freeze-dried powder by adopting a proper amount of non-enzymatic sterile water to obtain a preparation solution containing 150 mug/mL U5.

2.6 dilution with 10% trehalose solution was performed at concentrations of 100. Mu.g/mL, 50. Mu.g/mL, 25. Mu.g/mL, respectively.

3. Administration of drugs

Followed by 1 tail vein injection every 3 days, each with a volume of 100. Mu.L/dose, with three doses, high (100. Mu.g/mL), medium (50. Mu.g/mL), and low (25. Mu.g/mL). The 1 st measurement administration time is marked as day 0, the tumor growth condition of the mice is observed and measured after the 16 th administration, and the mice are sacrificed for tumor sampling after 3 days, and the mice are photographed.

Analysis of results:

a tumor volume growth graph is shown in fig. 9. It can be seen that the tumor volumes of each experimental group showed a uniform growth trend. The low, medium and high dose groups of the test agent showed various degrees of tumor inhibition and were dose dependent.

As shown in fig. 10, which shows the tumor tissue in vitro, the tumor tissue is sampled and photographed on the 3 rd day after the last administration, and the volume of the tumor in vitro is observed, so that the low dose group and the control group have similar volumes, the medium dose group and the high dose group are slightly smaller than the control group, and the results suggest that the U5 has a certain inhibition effect on the growth of the N87 gastric cancer tumor and has dose dependency.

Example 10 efficacy experiment of mouse RENCA renal cancer cell subcutaneous transplantation tumor model

The experimental steps are as follows:

1. animal model

Quarantine-qualified BALB/c mice were collected, and the cell concentration was extracted to 2.5X10 s by using a 1mL syringe ⁷ 0.2mL of the cell suspension was inoculated subcutaneously in the right middle wing of the mice, i.e., 5X 10 cells were inoculated subcutaneously per mouse ⁶ Individual cells. Inoculation ofAfter 6 days, the average tumor volume was taken and grown to 100mm ³ The left and right animals were randomly divided into 2 groups of 8 animals each.

2. Pharmaceutical formulation

1) A10% trehalose solution was prepared at 0.3mg/mL U5.

2) A 10% trehalose solution of 0.43mg/mL PEI was formulated and ph=3.60 was adjusted with 1M sodium hydroxide.

3) The two working fluids are respectively and evenly distributed into two syringes, the liquid amounts in the syringes are kept the same, then the syringes are connected into a mixer, the syringes are fixed on an injection pump, the injection pump is started, and the preparation stock solution is obtained through rapid mixing.

4) Freeze-drying overnight to obtain freeze-dried powder.

5) And re-dissolving the freeze-dried powder by adopting a proper amount of non-enzymatic sterile water to prepare a preparation solution containing 300 mug/mL U5.

3. Administration of drugs

The first four doses were 0.3mg/mL (50. Mu.L/L) and the last four doses were 0.6mg/mL (50. Mu.L/L) for a total of 8 doses per 3 days of intratumoral administration 1 time. The experiment starts with the 1 st measurement administration time marked as 1 day, 1 time is measured every 3 days, the growth condition of the tumor of the mice is observed and measured, the mice are sacrificed for tumor sampling, and the tumor weight is measured.

Analysis of results:

the tumor volume growth curve is shown in fig. 11. It can be seen that the tumor volume of animals in the PBS group remained a rapidly increasing trend. There was a certain decrease in the average tumor volume in the U5 animals from the time of increasing the dose to day 28.

As shown in fig. 12, which shows the tumor weight results, the tumor weight of the U5 group was somewhat reduced compared to the PBS group on day 28.

The results show that U5 has a certain effect of inhibiting RENCA tumor cell growth.

The foregoing description of the application has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the application pertains, based on the idea of the application.

Claims

1. An RNA, wherein the source of the RNA comprises, or has homology to, an oncolytic virus.

2. The RNA of claim 1, wherein the RNA comprises or has homology to the nucleotide sequence set forth in any one of SEQ ID nos. 1 to 6.

3. The RNA of claim 1, wherein at least one nucleotide of the RNA is chemically modified from at least 3 nucleotides from the 5' end.

4. The RNA of claim 1, wherein at least one nucleotide of the RNA is chemically modified from at least 3 nucleotides from the 3' end.

5. The RNA of claim 3 or 4, wherein the chemical modification comprises a 2' -O-ribose modification;

or, the 2 '-O-ribose modification comprises at least one of 2' -O-ribomethyl modification, 2 '-O-ribofluoro modification, 2' -O-MOE ribose modification;

alternatively, the nucleotide at the 3 '-end of the RNA is 5' -methylcytidine.

6. The RNA of claim 1, wherein the RNA is single stranded;

or, the RNA is double-stranded;

alternatively, the RNA comprises a hairpin.

7. A DNA comprising a nucleotide sequence encoding the RNA of any one of claims 1 to 6.

8. A delivery formulation loaded with the RNA of any one of claims 1 to 6.

9. Use of an RNA according to any one of claims 1 to 6, or a DNA according to claim 7, for the preparation of a medicament for the prevention and/or treatment of a disease.

10. The use according to claim 9, wherein the disease comprises cancer;

or, the cancer comprises at least one of gastric cancer, bladder cancer, breast cancer, melanoma, colon cancer, prostate cancer and renal cancer.