Generation of HIV-1 resistant T cells from induced pluripotent stem cell using CRISPR/Cas9 system

使用 CRISPR/Cas9 系统从诱导多能干细胞生成 HIV-1 抗性 T 细胞

• 批准号: 19K17925
• 负责人: オフィンニ ユディル
• 金额: $ 2.75万
• 依托单位: Osaka University (2021)Kobe University (2019)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Early-Career Scientists
• 财政年份: 2021
• 资助国家: 日本
• 起止时间: 2021-03-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-19K17925/
• 关键词: HIV-1; iPSC; CRISPR/Cas9; T cell; Feeder-free; Lentivirus

The Step 1 of the study, "to establish anti-HIV-1 CRISPR-Cas9", has been completed successfully with its results published in the journal Viruses in 2020, as well as selected as the cover story of the journal's #11 issue. Here, we showed that our CRISPR construct blocked infectious HIV-1 NL4-3 replication in T cell culture for up to 45 days. In Step 2, "to transduce iPSC with CRISPR-Cas9", even though we were successful in introducing the transgene, we now aim to increase the transduction efficiency to ensure constitutive CRISPR-Cas9 and gRNA expression even after iPSC differentiation, Here, we made a new lentivector with Cocal virus (COCV) pseudotyped-envelope to replace VSV-G, which has been reported to increase transduction efficiency in hematopoietic stem cells. For Step 3, "to differentiate iPSC into T cells", we will adopt the "feeder-free" protocol which was recently published. However, the current lab of the grantee is not an iPSC lab, so setbacks are likely and differentiation into HSC and T cells may be more difficult and take longer time than first expected.

该研究的第一步“建立抗HIV-1 CRISPR-Cas9”已成功完成，其结果于2020年发表在《病毒》杂志上，并被选为该杂志第11期的封面故事。在这里，我们证明了我们的CRISPR构建体在T细胞培养物中阻断了感染性HIV-1 NL 4 -3复制长达45天。在步骤2“用CRISPR-Cas9转染iPSC”中，尽管我们成功地引入了转基因，但我们现在的目标是提高转导效率，以确保即使在iPSC分化后也能进行组成型CRISPR-Cas9和gRNA表达。在此，我们制备了具有Cocal病毒（COCV）假型包膜的新慢病毒载体，以取代VSV-G，据报道其提高了造血干细胞中的转导效率。对于步骤3，“将iPSC分化为T细胞”，我们将采用最近发表的“无饲养层”方案。然而，受赠者目前的实验室不是iPSC实验室，因此可能会遇到挫折，分化成HSC和T细胞可能会更加困难，需要的时间比最初预期的更长。

项目成果

CRISPR-Cas9 as a curative drug for HIV-1 infection

CRISPR-Cas9作为HIV-1感染的治疗药物

• 发表时间: 2021
• 作者: 石島花穂;石綱遥;服部真一朗;満屋裕明;六本木誠;紺野奇重;Youdiil Ophinni
• 通讯作者: Youdiil Ophinni

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• 发表时间: 2021-07-09
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• 发表时间: 2021
• 作者: 1.Tsukada K;Shinki S;Kaneko A;Murakami K;Irie K;Murai M;Miyoshi H;Dan S;Kawaji K;Hayashi H;Kodama E;Hori A;Salim E;Kuraishi T;Hirata N;Kanda Y;Asai T.;Youdiil Ophinni
• 通讯作者: Youdiil Ophinni