RNA vaccine for treating non-small cell lung cancer and construction method thereof

A non-small cell lung cancer and vaccine technology, applied in DNA/RNA vaccination, complete cell/virus/DNA/RNA components, vaccines, etc., to achieve good therapeutic effect, good stability, and easy delivery

Pending Publication Date: 2021-03-16
WUXI PEOPLES HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the efficacy of tumor neoantigen RNA vaccines is restricted by three important factors: 1. RNA sequence design

Method used

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  • RNA vaccine for treating non-small cell lung cancer and construction method thereof
  • RNA vaccine for treating non-small cell lung cancer and construction method thereof
  • RNA vaccine for treating non-small cell lung cancer and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Neoantigen Screening

[0038] 1. Construction and sequencing of animal models

[0039] Detection of tumor-specific non-synonymous mutations. The first step in identifying tumor neoantigens is to find non-synonymous mutations unique to tumor cells. With the advances in sequencing technology, it is not difficult to perform whole-genome sequencing (WGS) or whole-exome sequencing (WES) simultaneously on tumor tissue and normal tissue of the same individual. By comparing the results of the two, non-synonymous mutation sites unique to tumor tissues can be obtained, including point mutations, insertion-deletion mutations, and frameshift mutations. The latest research shows that, combined with RNA sequencing (RNAseq) technology, it can further prompt the expression of non-synonymous mutations from the transcription level and provide assistance for the screening of neoantigens.

[0040] The present invention conducts research on advanced NSCLC, selects C57BL6 mice a...

Embodiment 2

[0056] The construction of embodiment 2 neoantigen RNA vaccine (for the treatment of the RNA vaccine of non-small cell lung cancer)

[0057] In order to construct an RNA vaccine, in vitro transcription (IVT) of its full nucleotide sequence (DNA sequence) needs to be achieved.

[0058] Select the pCI-neo (pCI-neo mammalian expression vector) plasmid as the vector, first insert the DNA sequence of the designed RNA vaccine into the pCI-neo plasmid between the XhoI and NotI restriction sites, and amplify the plasmid loaded with the RNA vaccine sequence . Schematic diagram of the structure of pCI-neo, see figure 2 .

[0059] The plasmid carrying the RNA vaccine sequence was cut with Sap I endonuclease to make it linearized. After linearization, it was also verified by agarose gel electrophoresis. The results are as follows image 3 , indicating that the Sap I endonuclease has successfully linearized the plasmid and the in vitro transcription step can begin. Subsequent use of i...

Embodiment 3

[0062] Example 3 Construction of PEG--PLL--PLLeu self-assembled micelle carrier

[0063] Because of the structural specificity of RNA vaccines, its function is closely related to the delivery method. The RNA vaccine of the present invention is wrapped by nanoparticles and administered intravenously, and the effect is better: on the one hand, the nanoparticles can protect the loaded RNA from being degraded and destroyed; on the other hand, the physical and chemical properties of the nanoparticles can realize slow, Effects of sustained-release RNA vaccines. In addition, secondary lymphoid organs such as lymph nodes and spleen are important places for the identification and activation of T cells and DC cells, and intravenous delivery of nanoparticles is conducive to the distribution of RNA vaccines in secondary lymphoid organs throughout the body.

[0064] (1) Synthesis of PEG-PLL-PLLeu

[0065] Polyethylene glycol-polylysine-polyleucine (PEG-PLL-PLLeu) can self-assemble to for...

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Abstract

The invention provides an RNA vaccine for treating non-small cell lung cancer. The nucleotide sequence of the RNA vaccine is shown as SEQ ID NO. 1. The RNA vaccine comprises two parts, namely a basicskeleton sequence (backstone) and a tandem vaccine sequence. The invention further provides a construction method of the RNA vaccine for treating the non-small cell lung cancer. According to the RNA vaccine for treating the non-small cell lung cancer, provided by the invention, a nucleotide sequence of a new antigen is optimally designed by utilizing a codon, connection is carried out by utilizinglinker, insertion of sequences such as SEC and MITD and construction of poly A tails are perfected, plasmids are constructed after sequence design is finished, and a target RNA sequence, namely the new antigen RNA vaccine, is obtained through linearization and in-vitro transcription. The vaccine is good in stability, high in translation efficiency and good in treatment effect, and expression products are easy to transfer.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an RNA vaccine for treating non-small cell lung cancer and a construction method thereof. Background technique [0002] Lung cancer is the malignant tumor with the highest incidence rate in my country. According to statistics from the National Cancer Center, the number of new lung cancer cases and deaths from lung cancer in my country is increasing, and lung cancer ranks first among all malignant tumors in terms of morbidity and mortality , a serious threat to people's lives and health. [0003] Non-small cell lung cancer (NSCLC) is the main pathological type of lung cancer, accounting for about 80%. Among the diagnosed NSCLC, more than half of them are advanced inoperable patients, who are only suitable for drugs or best supportive care. The treatment of advanced NSCLC has always been a research hotspot. Since the last century, platinum-based combination chemotherapy ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N15/12A61K39/00A61P35/00A61P11/00
CPCC12N15/85C12N15/66C07K14/4748A61K39/0011A61P35/00A61P11/00C12N2800/107C12N2800/22A61K2039/53
Inventor 刘超英刘宝瑞张弢王惠宇
Owner WUXI PEOPLES HOSPITAL
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