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Tumor specific promoter and application thereof

A promoter and promoter element technology, applied in the field of tumor-specific promoters, can solve problems such as difficult to obtain good results and limited activity, and achieve the effect of inhibiting tumor growth and improving the function of tumor-specific promoters

Pending Publication Date: 2022-07-05
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the promoter of telomerase can be used as a tumor-specific promoter for oncolytic adenovirus therapy. Existing studies have shown that the activity of the hTERT promoter as a tumor-specific promoter is quite limited, and it is difficult to achieve good results in practical applications.

Method used

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  • Tumor specific promoter and application thereof
  • Tumor specific promoter and application thereof
  • Tumor specific promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Detection of mRNA expression of human telomerase reverse transcriptase in different human cells

[0074] The cells used in this example are human-derived normal cells, human embryonic lung fibroblasts MRC-5 (ATCC: CCL-171) and human primary skin fibroblasts PCS-201-010 (ATCC: PCS-201-010 TM), human tumor cells: malignant glioblastoma cells U87MG (ATCC: HTB-14), malignant melanoma cells A375 (ATCC: CRL-1619), lung cancer cells A549 (ATCC: CRM-CCL-185), breast cancer cells Cells MCF-7 (ATCC: HTB-22), cervical cancer cells Hela (ATCC: CRM-CCL-2) and ovarian cancer cells SKOV3 (ATCC: HTB-77). Cells were harvested, and total cellular RNA was extracted and reverse transcribed into cDNA. The expression of human telomerase reverse transcriptase was detected by RT-PCR, and GAPDH was used as an internal reference.

[0075] The result is shown ( figure 1 ), in normal cells MRC-5 and PCS-201-010, human telomerase reverse transcriptase was almost not expressed, while in ...

Embodiment 2

[0076] Example 2 Detection of hTERT promoter mutation in different human tumor cells

[0077] The tumor cells U87MG, A375, A549, MCF-7, Hela and SKOV3 were collected, the cell genome was extracted, and the hTERT promoter nucleic acid sequence was sequenced.

[0078] experiment result shows( figure 2 ): In U87MG cells, there are partial C69T and C47T double mutations in the hTERT promoter; in A375 cells, there is a C69T mutation; in other cells, there is no mutation in the hTERT promoter. Therefore, it was considered whether these mutations could be applied to the promoter optimization of oncolytic adenovirus and the improvement of oncolytic effect.

Embodiment 3

[0079] Example 3 Preliminary optimization of hTERT promoter

[0080] 1. Construction of hTERT promoter point mutation

[0081] This example uses the -378-+77 region sequence of the hTERT promoter (from the TERT 5' regulatory region of human chromosome 5, sequence ID: NG_055467.1), a total of 455 bp, marked as hTERT WT ( image 3 The winning bid is L-WT) sequence (SEQ ID No. 2) (see image 3 ). By studying its sequence, the segment containing 181 bp from -181 to +77 was selected as the core region with strong promoter activity, which was marked as 181-WT sequence (SEQ ID No. 1) (see image 3 ). image 3 Where +1 represents the first nucleotide in the mRNA sequence, and -1 represents the first 5' nucleotide to the transcription start site. Both C69T (nucleotide C at -69) and C47T (nucleotide C at -47) indicate that the hTERT promoter sequence has a single cytosine C to thymine T mutation. DT indicated that the sequence had the above-mentioned double mutation of C→T at posit...

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Abstract

The invention belongs to the field of tumor immunotherapy, and particularly relates to a tumor specific promoter and application thereof. The technical problem to be solved by the invention is that the tumor specific multiplication capacity of the oncolytic adenovirus is limited. According to the scheme for solving the problems, the core promoter is provided, has the function of independently promoting gene expression, can be independently used as a promoter and can also be used as a component construction promoter. The nucleotide sequence of the gene is as shown in SEQ ID No. 1; or a nucleic acid molecule which is obtained by inserting, deleting and / or replacing one or more basic groups in the nucleotide sequence as shown in the sequence SEQ ID No.1 and still has the promoter function, wherein the nucleotide sequence is obtained by inserting, deleting and / or replacing one or more basic groups in the nucleotide sequence as shown in the sequence SEQ ID No.1. The modified tumor specific promoter has a better tumor specific promoter function, and the oncolytic adenovirus constructed by the promoter has the characteristic of being capable of specifically proliferating in tumor cells but not proliferating in normal cells, and is an excellent immunotherapy drug aiming at various tumors.

Description

technical field [0001] The invention belongs to the field of tumor immunotherapy, and particularly relates to a tumor-specific promoter and its application. Background technique [0002] Surgery, radiotherapy and chemotherapy are the conventional methods for the treatment of tumors. In recent years, tumor immunotherapy has developed rapidly and has become a hot spot in clinical tumor treatment research. Oncolytic adenovirus is a recombinant replicating adenovirus. By inserting a tumor-specific promoter or deleting the relevant genes required for the virus to proliferate in normal cells, the virus that only proliferates in tumor cells can specifically kill tumor cells and kill normal cells. Cells have no cytotoxic activity. Oncolytic adenovirus has excellent characteristics such as easy production, high efficiency and clinical safety. In recent years, oncolytic adenovirus has become a hot spot in the field of tumor therapy because of its innovation and efficacy. [0003] ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/861C12N15/67C12N7/01A61K35/761A61P35/00C12R1/93
CPCC12N9/1276C12N15/86C12N7/00A61K35/761A61P35/00C12Y207/07049C12N2710/10343C12N2710/10321C12N2710/10352C12N2710/10332C12N2830/008Y02A50/30
Inventor 杨莉田要美
Owner SICHUAN UNIV
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