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Method for knockout of human papillomavirus E6E7 gene by zinc finger nucleases

A technology of human papillomavirus and zinc finger nuclease, which is applied in the fields of genetic engineering, plant genetic improvement, botany equipment and methods, etc., and can solve problems such as limitation of drug delivery methods and poor stability

Active Publication Date: 2015-03-11
WUHAN KDWS BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this type of technology can only target the transcribed RNA. Even if it can inhibit the expression of related genes into proteins to a certain extent, the oncogenes at the DNA level still exist and have not been fundamentally removed; in addition, the stability is poor, The defects of the way of administration etc. further limit the potential of its application in vivo (Shillitoe, E.J, 2006, Cancer gene therapy.13:445-450)

Method used

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  • Method for knockout of human papillomavirus E6E7 gene by zinc finger nucleases
  • Method for knockout of human papillomavirus E6E7 gene by zinc finger nucleases
  • Method for knockout of human papillomavirus E6E7 gene by zinc finger nucleases

Examples

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example 1

[0025] Example 1. Construction of ZFN16-603 expression vector

[0026] The E6E7 oncogene sequence information of HPV16 was obtained from the NCBI website, and the sequence was imported into the online design tool ZiFiT (http: / / zifit.partners.org / ZiFiT / ) to complete the design. The ZFNs pair targeting base 603 was selected and named ZFN16-603. The targeted DNA sequence is:

[0027] ZFN16-603: CAG AGG AGG AGG ATGAAA TAG ATG GTC CAG

[0028] The underlined part is the zinc finger protein binding sequence, and the middle part is the cleavage site of FokI endonuclease. The corresponding ZFNs expression vectors were named ZFN16-603F and ZFN16-603R, respectively.

[0029] For the construction method of zinc finger ribozyme expression vector, refer to Wright, D A et al., Nature Protocol, (2006) 1: 1637-1652, and the vector used for construction is pcDNA3.1+ (Invitrogen, USA, catalog number: V790-20).

[0030] After the construction is completed, it is confirmed th...

example 2

[0031] Example 2. ZFN16-603 induces apoptosis

[0032] ZFN16-603 targeting HPV16E6E7 is transfected into cells and expressed zinc finger ribozyme can quickly recognize and bind to the target sequence, and then play a cutting role, making it generate DSB at the target site, DSB activates the cell to use easily The wrong NHEJ repair mechanism is repaired; the base insertion or deletion caused by the repair causes the frame shift mutation of the codon, and finally the expression of the HPV16E6E7 protein is inhibited. The E6E7 protein can enhance the proliferation ability of cervical cancer cells and escape apoptosis through a series of mechanisms. After the expression of E6E7 protein is inhibited, the proliferation ability of the cells decreases and the apoptosis increases. On the other hand, ZFN16-603 can only specifically recognize and bind to the HPV16E6E7 sequence, but cannot recognize the E6E7 sequence of other subtypes such as HPV18. Therefore, ZFN16-603 can only induce ap...

example 3

[0041] Example 3. Verification of cutting efficiency of ZFN16-603

[0042] Zinc finger ribozymes cut and form DSBs after functioning in cells, inducing cells to use the error-prone NHEJ repair method to repair. After the repair, the insertion or deletion of bases is caused at the target site, thereby causing frame-shift mutations and achieving the purpose of gene knockout. At this time, if the wrongly repaired base sequence is annealed and extended with the normal (wild-type) base sequence, a hybrid double strand can be formed, and the base sequence of this hybrid double strand will occur at the original site. Mismatch (corresponding to the base insertion or deletion after repair), and then use T7Endonuclease I (New England Biolabs company, product number: M0302S) that can recognize and cut the mismatch base in the sequence to process, that is, indirect Reflect the cleavage efficiency of ZFN. Due to the limitation of transfection efficiency, the successfully transfected cell...

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Abstract

Relating to the field of gene therapy, the invention provides a method for knockout of a high-risk human papillomavirus (HPV) E6E7 gene by zinc finger nucleases (ZFNs). According to a high-risk type HPV E6E7 gene sequence, the method designs ZFNs expression vectors of a targeted specific site to targetedly cut the HPVE6E7 gene sequence in cells, thereby knocking out a target gene. In corresponding subtype HPV infected cells, the ZFNs can significantly induce cell apoptosis and proliferation inhibition, and has no effect on other subtype HPV positive cells and HPV negative cells. Transfection of the ZFNs expression vectors in a subcutaneous tumor model of HPV positive cervical cancer cells can obviously inhibit the growth rate of subcutaneous tumors and alleviate tumors. The method for targeted knockout of the high-risk HPV E6E7 gene by ZFNs can efficiently destroy virus oncogenes at the DNA level so as to make HPV infected cells undergo apoptosis and proliferation inhibition, and has enormous treatment value to HPV infection related diseases, especially cervical intraepithelial neoplasias and other precancerous lesions.

Description

technical field [0001] The present invention relates to the field of gene therapy, and the disclosure includes constructing a targeted zinc finger nuclease for the high-risk human papillomavirus E6E7 oncogene sequence, and using the zinc finger nuclease to knock out the high-risk human papillomavirus E6E7 oncogene Methods. Background technique [0002] Cervical cancer is one of the most common gynecological malignancies in women worldwide after breast cancer, and its incidence rate ranks second among female malignancies. Its incidence rate ranks first in some developing countries. According to the International Cancer Research Center, there are approximately 3,710,200 new cases of cervical cancer each year, accounting for 9.8% of all tumors and 15% of new cases in women, of which 78% occur in developing countries. my country has a vast territory and a large population. According to the large-scale national population death survey organized by the National Cancer Prevention...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N15/55C12N9/22
Inventor 马丁汪辉胡争丁文成于兰
Owner WUHAN KDWS BIOLOGICAL TECH CO LTD
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