| 研究課題/領域番号 |
19K17925
|
|---|
| 研究機関 | 大阪大学 |
|---|
研究代表者 |
オフィンニ ユディル 大阪大学, 免疫学フロンティア研究センター, 特任研究員(常勤) (30837901)
|
|---|
| 研究期間 (年度) |
2021-03-01 – 2023-03-31
|
| キーワード | HIV-1 / iPSC / CRISPR/Cas9 / T cell / Feeder-free |
|---|
| 研究実績の概要 |
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.
|
| 現在までの達成度 (区分) |
現在までの達成度 (区分)
4: 遅れている
理由
Even if the results of Step 1 has been successfully published in several papers, the overall study is delayed. The COVID-19 pandemic starts exactly in the middle of the project in Spring 2020, and any iPSC culture was halted at the time, reversing all progress. And then, the grantee moved for postdoctoral work for 2 years in Harvard, United States, and thus the KAKEN funding was completely stopped at that time. Currently, the grantee has come back to do research in Osaka University and so the grant can be continued. However, the current lab of the grantee does not bears expertise in iPSC, and thus necessary instruments and reagents are not readily available. This would likely put severe obstacles in finishing Step 3, which is successful differentiation of iPSC into HSC and T cells.
|
| 今後の研究の推進方策 |
The plan from now on is to complete Step 3 of the project. This includes:
(a) Maintenance of iPSC feeder-free culture; (b) Pluripotency check of iPSC in vivo in mouse, (c) Cytokine differentiation of iPSC towards HSC
and T cells, (d) Phenotype check for produced T cells with flowcytometry, (e) Confirmation of CRISPR/Cas9 insertion in differentiated cells, (f)
Off-target screening to ensure safety, (g) HIV-1 challenge to CRISPR/Cas9-equipped, differentiated T cells, and (h) Confirmation of long-term
immunity of T cells under replicative HIV-1 infection.
|
| 次年度使用額が生じた理由 |
Due to reasons stated above, the research was paused temporarily and delayed. Subsequently, budget use to purchase necessary reagent for ongoing experiments was also halted. This cause an incurring amount for use for the next continuation of the study. This amount will be fully use for Step 3 of the project, as well as scientific publication fee, and participation as well as travel fee to international academic conferences.
|
