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MRNA-fatty acid targeting compound as well as preparation method and application thereof

A compound and fatty acid technology, applied in the field of genetic engineering, can solve the problems of low delivery efficiency, inability to realize the coupling of fatty acid and mRNA, hindering the progress of muscle cell targeted therapy, etc., and achieve the effect of high specific expression

Pending Publication Date: 2022-07-01
WUHAN RHEGEN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although fatty acid-conjugated siRNA has been discovered for many years, its messenger RNA (Messenger RNA, mRNA) muscle delivery system has not been able to achieve a breakthrough. The existing technology cannot realize the coupling of fatty acid and mRNA. Inefficient delivery hampers progress in muscle cell-targeted therapies

Method used

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  • MRNA-fatty acid targeting compound as well as preparation method and application thereof
  • MRNA-fatty acid targeting compound as well as preparation method and application thereof
  • MRNA-fatty acid targeting compound as well as preparation method and application thereof

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preparation example Construction

[0047]The present invention also provides the preparation method of the mRNA-fatty acid targeting compound described in the above scheme, comprising the following steps:

[0048] Carry out RNA ligation reaction with the oligoadenylic acid-fatty acid compound with the structure shown in formula b and target mRNA under the catalysis of RNA ligase, obtain the mRNA-fatty acid targeting compound of the structure shown in formula I;

[0049]

[0050] In formula b: R is -C n H 2n+1 -or-C n H 2n-1 , n represents the number of carbon atoms, and A represents adenylate.

[0051] In the present invention, the preparation method of the oligoadenylic acid-fatty acid compound having the structure shown in formula b comprises the following steps:

[0052] The compound with the structure shown in formula a and the oligoadenylic acid are mixed and subjected to condensation reaction to obtain the oligoadenylic acid-fatty acid compound with the structure shown in formula b, and the structu...

Embodiment 1

[0079] The preparation of mRNA-palmitic acid targeting compound, the steps are as follows:

[0080] (1) 0.256g palmitic acid and 2.22mL triethylamine were dissolved in 1mL dichloromethane, 1.12g pentafluorophenyl trifluoroacetate (Pfp-TFA) was added, and the reaction was stirred at room temperature for 1 hour; Dilute with 6 mL of dichloromethane, then with 3 mL of 5 mmol / L NaHCO 3 solution and 3mL of 5mmol / L NaHSO 4 The solution was washed; the above solution was separated to obtain an organic layer, which was dried with anhydrous sodium sulfate solid, filtered and concentrated under negative pressure to obtain a crude product; the crude product was purified by silica gel column chromatography; the purification steps were as follows:

[0081] A crude product is dissolved in DMSO, acetonitrile and triethylamine are added to obtain crude product solution, wherein the concentration of crude product is 40mmol / L, the concentration of acetonitrile is 20mmol / L, and the concentration...

Embodiment 2

[0089] Validation of targeted delivery of mRNA-palmitic acid molecular compounds

[0090] The LUC mRNA-palmitic acid targeting compound prepared in Example 1 is compared with the targeted delivery characteristics of the nano lipid particle (LNP) delivery carrier, wherein the preparation method of LNP-LUC mRNA is as follows:

[0091] Prepare aqueous phase: Dilute LUC mRNA in citrate buffer at a final concentration of 2 μg / μl to obtain mRNA buffer;

[0092] Prepare ethanol phase solution according to Table 1;

[0093] Table 1 ethanol phase solution formula

[0094]

[0095] Prepare PBS solution as LNP dilution;

[0096] Syringe pump instrument operation steps:

[0097] (1) Load the A phase (mRNA buffer) into a 5mL syringe, and the B phase (ethanol phase solution) into a 5mL syringe, install it on a syringe pump, and clamp;

[0098] (2) Connect the chip to the syringe, and set the flow rate of the syringe pump;

[0099] (3) Click the start button of the syringe pump to in...

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Abstract

The invention relates to the technical field of gene engineering, and provides an mRNA-fatty acid targeting compound as well as a preparation method and application thereof. According to the invention, an oligonucleotide-fatty acid compound and a target mRNA are subjected to an RNA ligation reaction to obtain an mRNA-fatty acid targeting compound. The invention provides a brand new mRNA-fatty acid targeting compound, the problem that fatty acid cannot be efficiently coupled with mRNA is solved, and the coding target protein of the mRNA-fatty acid targeting compound is high in specific expression quantity in muscular tissues and can be directly delivered to muscle cells through intramuscular injection or systemic circulation injection.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an mRNA-fatty acid targeting compound and a preparation method and application thereof. Background technique [0002] Nucleic acid-based therapy is a unique approach to treatment, and the therapeutic effects of traditional therapies are often short-lived because they tend to target proteins rather than underlying genetic problems. In contrast, nucleic acid therapy can achieve long-term efficacy through gene suppression, addition, replacement or editing. Currently, the delivery of mRNA into specific tissue cells can be achieved by different methods, such as lipid nanoparticles, GalNac, etc., but these methods cannot specifically deliver to muscle cells. [0003] Bone and cardiomyocytes rely heavily on the oxidation of long-chain fatty acids for contraction. Fatty acids are transported to muscle tissue by blood complexed with albumin or covalently bound to triacylglyc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/02C07H1/00A61P3/00A61K31/7115A61K38/18A61K38/26C07K14/505C07K14/605C12N9/02C12N9/06
CPCC07H21/02C07H1/00A61P3/00A61K31/7115C07K14/505C07K14/605C12N9/0069C12N9/0048C12Y107/03003A61K38/26A61K38/1816
Inventor 胡勇
Owner WUHAN RHEGEN BIOTECHNOLOGY CO LTD
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