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

Foreign code F200010235
File No. (S2019-0189-N0),S2020-0442-N0
Posted date Oct 29, 2020
Country WIPO
International application number 2020JP007164
International publication number WO2020171222
Date of international filing Feb 21, 2020
Date of international publication Aug 27, 2020
Priority data
  • P2019-030699 (Feb 22, 2019) JP
Title METHOD FOR INTEGRATING LONG FOREIGN GENE INTO SAFE REGION OF HUMAN PLURIPOTENT STEM CELL AND ALLOWING SAME TO NORMALLY FUNCTION THEREIN
Abstract 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.
Outline of related art and contending technology 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.
Scope of claims (In Japanese)[請求項1]
 ヒト細胞内において強い活性を有するプロモーター、及び前記プロモーターに機能的に連結した外来遺伝子をゲノム中のセーフハーバー領域に含むヒト多能性幹細胞。

[請求項2]
 前記セーフハーバー領域が、ヒト第3染色体上のRosa26遺伝子座又はヒト第19染色体上のAAVS1領域又はヒト第22番染色体上のH11領域である、請求項1に記載のヒト多能性幹細胞。

[請求項3]
 前記ヒト細胞内において強い活性を有するプロモーターが、CAプロモーター、survivinプロモーター、PGKプロモーター、TERTプロモーター、又は、Nanogプロモーターである、請求項1又は請求項2に記載のヒト多能性幹細胞。

[請求項4]
 前記外来遺伝子が自殺遺伝子である、請求項1~請求項3のいずれか1項に記載のhPSC。

[請求項5]
 前記自殺遺伝子が、ヘルペスウイルス由来チミジンキナーゼ遺伝子、ヒト由来チミジンキナーゼ遺伝子、シトシンデアミナーゼ遺伝子、水痘ウイルスチミジンキナーゼ、又はCaspaseである、請求項4に記載のヒト多能性幹細胞。

[請求項6]
 請求項1に記載の細胞を樹立する方法であって、
 相同組換え法により、ヒト多能性幹細胞のゲノム中のセーフハーバー領域に、ヒト細胞内において強い活性を有するプロモーター、及び前記プロモーターに機能的に連結した外来遺伝子を導入することを含む方法。

[請求項7]
 請求項1~請求項5のいずれか1項に記載の細胞を分化誘導処理することにより得られた細胞。

[請求項8]
 分化誘導処理後のヒト多能性幹細胞に含まれる、残存する未分化細胞及び存在する腫瘍化原因細胞に選択的に傷害を与える方法であって、
 請求項4又は請求項5に記載の細胞を分化誘導処理することにより得られた細胞からなる細胞群を、該自殺遺伝子に対応する薬剤と接触させることにより、前記細胞群に含まれる未分化細胞及び/又は腫瘍化原因細胞に選択的に傷害を与えることを含む方法。

[請求項9]
 請求項8に記載の方法であって、
 更に、請求項4又は請求項5に記載の細胞を分化誘導処理することにより、分化誘導処理後のヒト多能性幹細胞からなる細胞群を得ることを含む方法。

[請求項10]
 請求項7に記載の方法であって、
 更に、相同組換え法により、ヒト多能性幹細胞のゲノム中のセーフハーバー領域に、前記ヒト細胞内において強い活性を有するプロモーター、及び前記プロモーターに機能的に連結した外来遺伝子を導入して、請求項1に記載の細胞を得ること含む方法。

[請求項11]
 接触させる前記薬剤のレベルが、未分化細胞及び/又は腫瘍化原因細胞への毒性が分化細胞への毒性よりも強いレベルである、請求項8又は請求項9に記載の方法。

[請求項12]
 接触させる前記薬剤のレベルが、未分化細胞及び/又は腫瘍化原因細胞の生存率が分化細胞の生存率よりも3倍以上高いレベルである、請求項11に記載の方法。

[請求項13]
 分化誘導処理後の請求項4又は請求項5に記載の細胞のうち、未分化細胞及び/又は腫瘍化原因細胞に傷害を与える可能性が高く、かつ、分化細胞には障害を与えない可能性が高い、前記薬剤のレベルを決定する方法であって、
 請求項4又は請求項5に記載の細胞を分化誘導処理することにより得られた細胞と分化誘導処理されていない請求項4又は請求項5に記載の細胞を、複数のレベルの前記薬剤と接触させること、及び、
 前記分化誘導処理された細胞と比較して、前記分化誘導処理されていない細胞において細胞傷害効果が高い前記薬剤のレベルを未分化細胞及び/又は腫瘍化原因細胞に傷害を与える可能性が高く、かつ、分化細胞には障害を与えない可能性が高いレベルとして決定することを含む方法。

[請求項14]
 請求項13に記載の方法であって、
 相同組換え法により、ヒト多能性幹細胞のゲノム中のセーフハーバー領域に、前記強い発現をもたらすプロモーターと機能的に連結した自殺遺伝子を導入して、該自殺遺伝子が導入されたヒト多能性幹細胞を得ること、
 得られた自殺遺伝子が導入されたヒト多能性幹細胞を分化誘導処理すること、
 得られた分化誘導処理された細胞と、分化誘導処理していない自殺遺伝子が導入されたヒト多能性幹細胞のそれぞれを、複数のレベルの前記薬剤と接触させること、
 前記分化誘導処理された細胞と比較して、前記分化誘導処理されていない細胞において細胞傷害効果が高い前記薬剤のレベルを、未分化細胞及び/又は腫瘍化原因細胞に傷害を与える可能性が高く、かつ、分化細胞には障害を与えない可能性が高いレベルとして決定することを含む方法。

[請求項15]
 分化誘導処理後のヒト多能性幹細胞に含まれる、残存する未分化細胞及び/又は存在する腫瘍化原因細胞に選択的に傷害を与える方法であって、
 請求項4又は請求項5に記載の細胞を分化誘導処理することにより得られた細胞からなる細胞群を、前記薬剤と接触させることにより、前記細胞群に含まれる未分化細胞及び/又は腫瘍化原因細胞に選択的に傷害を与えることを含み、
 ここで、接触させる前記薬剤のレベルが、請求項13又は請求項14において決定されたレベルであることを特徴とする方法。

[請求項16]
 分化誘導処理後の、ヘルペスウイルス由来チミジンキナーゼ遺伝子をゲノム中のセーフハーバー領域に含むヒト多能性幹細胞の細胞群に残存する未分化細胞及び/又は存在する腫瘍化原因細胞に選択的に傷害を与える方法であって、
 請求項4又は請求項5に記載の細胞を分化誘導処理することにより得られた細胞からなる細胞群を、0.01~1μg/mLのガンシクロビルと接触させることにより、前記細胞群に含まれる未分化細胞及び/又は腫瘍化原因細胞に選択的に傷害を与えることを含む方法。

[請求項17]
 分化誘導処理後のヒト多能性幹細胞に含まれる、残存する未分化細胞及び/又は存在する腫瘍化原因細胞に選択的に傷害を与える方法であって、
 請求項13又は請求項14に記載の方法により、分化誘導処理後の請求項4又は請求項5に記載の細胞のうち、未分化細胞及び/又は腫瘍化原因細胞に傷害を与える可能性が高く、かつ、分化細胞には障害を与えない可能性が高い、前記薬剤のレベルを決定すること、
 請求項4又は請求項5に記載の細胞を分化誘導処理すること、
 分化誘導処理した細胞を、前記レベルの前記薬剤と接触させることにより、残存する未分化細胞及び存在する未分化細胞及び/又は腫瘍化原因細胞に選択的に傷害を与えることを含む方法。
  • Applicant
  • ※All designated countries except for US in the data before July 2012
  • KAGOSHIMA UNIVERSITY
  • Inventor
  • KOSAI, Ken-ichiro
  • MITSUI, Kaoru
  • IDE, Kanako
IPC(International Patent Classification)
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