TOP > 外国特許検索 > METHOD FOR INTEGRATING LONG FOREIGN GENE INTO SAFE REGION OF HUMAN PLURIPOTENT STEM CELL AND ALLOWING SAME TO NORMALLY FUNCTION THEREIN

METHOD FOR INTEGRATING LONG FOREIGN GENE INTO SAFE REGION OF HUMAN PLURIPOTENT STEM CELL AND ALLOWING SAME TO NORMALLY FUNCTION THEREIN NEW

外国特許コード F200010235
整理番号 (S2019-0189-N0)
掲載日 2020年10月29日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2020JP007164
国際公開番号 WO2020171222
国際出願日 令和2年2月21日(2020.2.21)
国際公開日 令和2年8月27日(2020.8.27)
優先権データ
  • 特願2019-030699 (2019.2.22) JP
発明の名称 (英語) METHOD FOR INTEGRATING LONG FOREIGN GENE INTO SAFE REGION OF HUMAN PLURIPOTENT STEM CELL AND ALLOWING SAME TO NORMALLY FUNCTION THEREIN NEW
発明の概要(英語) The purpose of the present invention is to safely and efficiently introduce a cytotoxic gene into human pluripotent stem cell genome and then allow the gene to exert the function thereof to such an extent that undifferentiated cells, which remain after the differentiation into target cells, can be efficiently and exhaustively removed thereby. By homologous recombination using genome editing technology, a foreign (target) gene sequence was successfully and efficiently inserted into a safe harbor region of hPSC genome for the first time. Moreover, the following findings were obtained, namely: AAVS1 region that is defined as a safe harbor region is liable to show a lower gene expression level compared to random gene transfer using, for example, a lentiviral vector; in the homologous recombinant cells thus obtained, a promoter is required to have a strong expression ability; and a toxicity selective for undifferentiated cells can be achieved by employing, as a functional gene, a suicide gene correlating to cell growth activity and controlling the concentration of a prodrug corresponding thereto. As a result, the present invention successfully provides a mechanism whereby a gene is safely introduced into a safe harbor region of a genome by homologous recombination and, after inducing differentiation, undifferentiated cells alone can be exhaustively and selectively exterminated from the differentiated cells.
従来技術、競合技術の概要(英語) BACKGROUND ART
Establishment of human pluripotent stem cells (hPSC), such as human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC), has attracted a high expectation to create medical and medicaments such as cell transplantation therapy instead of organ transplantation, and research of regenerative medicine has been rapidly progressed. In Japan, clinical studies and clinical trials mainly including hiPSC have been carried out, and in U.S. and the like, clinical trials mainly including hESC have been carried out. Some of them are expected to lead to future practical use. However, intermingling of undifferentiated cells remaining in differentiated cells leads to the risk of formation) of teratoma or carcinogenesis (malignancies after cell transplantation, which is concerned with safety. Therefore, in order to establish regenerative medicine such as cell transplantation therapy using hPSC as a widely used safe medicine, the development of techniques for directly and specifically killing undifferentiated cells and carcinogenic cells which become "tumorigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigen@@
(Non-Patent Document 3) is a case in which leukemia has occurred at a high rate in clinical studies of ex vivo gene therapy using hematopoietic stem cells, which have been performed above after scientific consensus that safety has been established in a sufficient non-clinical trial was obtained. That is, in many basic studies and non-clinical studies using animals throughout the world for many years, data of "no tumorigenesis" are accumulated and consensus is obtained in scientific world, In clinical trials in which gene treated hematopoietic stem cells were transplanted into patients, it was found that leukemia, which is a serious side effect, has been developed for 2 years after the treatment. It should be noted that cancer cells that caused leukemia were clones derived from 1 cells. In this case, it has been concluded that the tumor can be generated from only 1 cells because of the proliferation dominant of stem cells, but it has been concluded that the probability of tumor generation is low in clinical applications in subsequent non-clinical studies. However, in actual clinical trials, it has been demonstrated by human being that the technique of directly eradicating the tumorigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigen hPSC is highly plasticity to form teratoma, and it is suggested that genetic mutant cells leading to carcinogenesis may occur during culture because of unstable chromosomes, and there is a possibility that results similar to clinical tests using hematopoietic stem cells may occur in regenerative medicine using hPSC. Therefore, in addition to conventional strategies of "reducing" the mixture of remaining undifferentiated cells (tumorigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigen
For gene transfer to hPSC, a non-viral DNA delivery method in which a plasmid vector is transferred by electroporation or lipofection, an infection transfer method using a viral vector, and the like are mainly used. The efficiency of gene transfer to hPSC is higher in a method using a viral vector than in a non-viral DNA delivery method. In the case of gene transfer, only transient gene expression is possible, and long-term stable expression is possible, depending on the vector used. Lentiviral vectors and retroviral vectors are very suitable for establishing long-term stable expression cells since the foreign (target) gene is integrated into the genome of hPSC. Therefore, the present inventors have developed a tumorigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigeni@@ By using the platform, various TC-LV s expressing reporter and suicide genes in different types of promoters can be efficiently and simultaneously produced, and by infecting hPSC with the produced TC-LV s and analyzing it, a promoter which most effectively kills tumorigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigenigeni@@ However, when lentiviral vectors are used, they are introduced at random positions into the genome, so that some introduction sites may lead to the risk of oncogenic transgenes. Therefore, in order to utilize this system more safely in clinical applications, it is known that it is called a safe harbor region on the genome, that is, it is known that the phenotype change does not occur in the cell by gene insertion, but more likely that the chromosome part to which the gene is introduced is identical, The expression of the introduced gene is stably and highly expressed, resulting in a stable and difficult to be suppressed (because the influence of the structure of the chromosome and the beat is the same, It has been necessary to develop a technique for inserting a reporter and a suicide gene which are foreign genes, that is, knock-in of a foreign target gene into a specific chromosome site called a safe harbor region.
Genome editing techniques performed using zinc finger nucleases or TALEN have recently developed dramatically due to the appearance of CRISPR / Cas9. However, even when CRISPR / Cas9 is used, the gene editing efficiency is lower in hPSC than in differentiated cells such as 293 T cells and K562 cells. Most studies on gene modification using genome editing techniques have been performed by gene knockout by a heterologous terminal binding (NHEJ) mechanism, and there are very few reports on knockin of a foreign (target) gene into a specific chromosome site by homologous recombination using genome editing techniques. Ruan , J et al. report that 9 kb foreign (target) gene containing neomycin resistance gene was homologous recombination inserted into a safe harbor region called Hipp11 (H11) region of the genome using porcine fibroblasts using CRISPR / Cas9 technology. In primate pluripotent stem cells, CRISPR / Cas9-mediated genome editing using iPS cells of macaqua monkey has been reported, but only expression of reporter genes was confirmed. Therefore, an example in which a foreign (target) gene having a long base sequence is integrated into hPSC and a gene functioning in a cell such as a suicide gene as well as a marker gene has not been reported so far.
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • KAGOSHIMA UNIVERSITY
  • 発明者(英語)
  • KOSAI, Ken-ichiro
  • MITSUI, Kaoru
  • IDE, Kanako
国際特許分類(IPC)
指定国 National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JO JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN WS ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
特許の内容に興味を持たれた方、ライセンスをご希望の方は、下記「問合せ先」までお問い合わせください。

PAGE TOP

close
close
close
close
close
close