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Modified nucleic acid and application thereof

A nucleic acid and chemical modification technology, applied in the modified nucleic acid and its application field, can solve the problems of RNA performance change and unclear application, and achieve the effect of reducing immune response, low immunogenicity and high immunogenicity

Pending Publication Date: 2022-07-29
SHENZHEN RHEGEN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, it is not clear about RNA modification and the performance change and application of modified RNA

Method used

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  • Modified nucleic acid and application thereof
  • Modified nucleic acid and application thereof
  • Modified nucleic acid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158] DNA template preparation

[0159] 1) PCR method to prepare luciferase (Luc) and erythropoietin (EPO) DNA templates, and carry out in a 96-well PCR instrument.

[0160] PCR products include at least

[0161] A) a promoter sequence (any one of T7 promoter, T3 promoter or SP6 promoter);

[0162] B) a 5'UTR comprising at least one Kozak sequence;

[0163] C) 3'UTR;

[0164] D) Luc or EPO coding sequence;

[0165] E) polyA tail.

[0166] The specific sequences involved in the present invention are shown in Table 1.

[0167] Table 1

[0168]

[0169]

[0170] Amplify the DNA template according to the following reaction system:

[0171] The reaction volume is 50 μL (the reaction volume of a single tube, and multiple tubes are simultaneously reacted at one time), and the specific reaction system is shown in Table 2.

[0172] Table 2 Reaction system

[0173] component volume PrimeSTAR Max Premix (2×) 25μl PrimeSTAR Max DNA Polymerase 1μl ...

Embodiment 2

[0314] According to the single-type modified or multiple-type modified mRNA encoding EPO of the present invention, its immunogenicity is detected, and the evaluation standard is the modified mRNA immunoprecipitation test (RIP analysis) to detect Toll-like receptors TRL3, TRL7, TRL8 and RIG-1 and mRNA The level of binding; the specific steps are as follows:

[0315] cell transfection

[0316] About 24 hours after inoculation of 293T cells (purchased from the cell bank of the Chinese Academy of Sciences), the state of the cells in the 6-well plate was observed, and the confluence was 88% to 92%. In a biological safety cabinet, prepare 90% (volume percent) DMEM+10% (volume percent) FBS medium. 30 min before transfection, the medium in the well plate was discarded, and 1 ml of fresh medium was added to each well, namely 90% (volume percentage) DMEM+10% (volume percentage) FBS medium.

[0317] Preparation of transfection system: take 200μl opti-MEM, add 10μg test substance (conce...

Embodiment 3

[0330] According to the single-modified or multi-modified mRNA encoding EPO of the present invention, its immunogenicity is detected, and the evaluation standard is the level of TNFα and IL-8 in serum after intramuscular injection of the modified mRNA into mice.

[0331] 6-8-week-old balbc mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were kept under SPF conditions and kept in ventilated cages under 12h light and 12h dark cycles. Various types of modified EPO mRNA were injected intramuscularly into balb / c mice, and the injection dose of each mouse was 100 μg. After 24 hours, the orbital blood was collected from the mice, and the serum was separated. Enzyme-linked immunosorbent assay (elisa) was performed with mouse IL-8 and TNFa kit (RayBio).

[0332] The result is as Figures 46 to 63 As shown, in mice, the immunogenicity of the mRNA synthesized by the modification strategy provided by the present invention is far less than that of the un...

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Abstract

The invention provides a modified nucleic acid and application thereof, and belongs to the technical field of nucleic acid modification, the modified nucleic acid comprises uridine, cytidine, adenine nucleoside, guanine nucleoside and chemically modified nucleoside; the chemically modified nucleoside comprises one or more of chemically modified uridine nucleoside, chemically modified cytidine nucleoside, chemically modified adenine nucleoside and chemically modified guanine nucleoside. The modified nucleic acid disclosed by the invention is high in stability, low in immunogenicity and long in in-vivo half-life period; the modified nucleic acid provided by the invention can be used as a diagnostic agent or a therapeutic agent, is applied to diagnosis and treatment of diseases, overcomes the defects of low stability, high immunogenicity, short in-vivo half-life period, need of repeated administration in a short time, high cost and the like compared with the existing nucleic acid in a natural state, and reduces the application cost while enhancing the curative effect of a nucleic acid drug.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid modification, and in particular relates to a modified nucleic acid and its application. Background technique [0002] As early as the 1970s, scientists discovered m6A modification in RNA, but its function has not been well revealed due to technical constraints. Until 2012, scientists' studies showed that m6A modification is related to mRNA stability, splicing processing, translation, and microRNA processing. In addition, m6A is also related to stem cell fate and biological rhythm, and can promote stem cells from a state of self-renewal to cell differentiation. Researchers found that methylation shortens the half-life of mRNA and reduces its abundance. It can be said that m6A modification affects almost every step of RNA metabolism. [0003] Although substantial progress has been made in the study of m6A modification in mRNA, there are still some technical challenges and basic scientific pro...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/50C12N15/53A61K39/215A61P31/14A61K48/00A61K38/18A61K49/00
CPCC07K14/505C07K14/005C07K14/495C12N9/0069C12Y113/12A61K39/12A61P31/14A61K48/005A61K49/0045C12N2770/20022C12N2770/20034A61K2039/53A61K38/00A61K39/00A61P43/00A61K47/65A61K47/54A61K31/7105A61P35/00
Inventor 胡勇张苗苗洪丹胡迅
Owner SHENZHEN RHEGEN BIOTECHNOLOGY CO LTD
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