Rc3h1 gene and/or Zc3h12a gene knockout recombinant T cells and application thereof
A cell and gene technology, applied in genetically modified cells, blood/immune system cells, cells modified by the introduction of foreign genetic material, etc., to achieve the effect of increasing the effectiveness, wide application range, and reducing the number of cells
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Embodiment 1
[0039] Example 1 Knockout of Rc3h1 and / or Zc3h12a can promote high-efficiency expansion of reinfused T cells without pretreatment
[0040] 1. Using CRISPR-Cas9 technology to knock out Rc3h1 and / or Zc3h12a in CD8 T cells activated in vitro, the specific steps are as follows:
[0041] 1. Gene knockout vector construction: In this embodiment, a retrovirus-based sgRNA expression vector, namely pMSCV-hU6-sgRNA-EF1a-Thy1.1, was constructed, wherein pMSCV-hU6-sgControl-EF1a-Thy1.1 (SEQ ID NO.1, the 1686-1705th position of SEQ ID NO.1 is a random sequence that does not target any gene) as a control without knocking out any gene, pMSCV-hU6-sgRc3h1-EF1a-Thy1.1 (the vector sequence is the The 1686-1705 positions of SEQ ID NO.1 are replaced by SEQ ID NO.2, and the sequence obtained by keeping other sequences unchanged, said SEQ ID NO.2 is the target sequence targeting Rc3h1 gene) is used to knock out Rc3h1, pMSCV -hU6-sgZc3h12a-EF 1a-Thy1.1 (the carrier sequence is the sequence obtained ...
Embodiment 2
[0056] Example 2 T cells knocking out Rc3h1 and / or Zc3h12a can inhibit the growth of solid tumors without pretreatment
[0057] Divide 6-8 week-old C57b1 / 6 mice weighing 20-25g into four groups, namely PBS group (8 mice), sgControl group (10 mice), sgRc3h1 group (11 mice), and sgZc3h12a group (10 mice) and sgRc3h1 / Zc3h12a group (10 rats), each rat in each group was subcutaneously inoculated with EL4 tumor cells, and after 7 days of inoculation, the CD8 T cells without gene knockout prepared in Example 1 were prepared into a cell suspension with PBS, and the tail of the rats was Transfuse to each mouse in the sgControl group, and reinfuse each mouse tail to 4×10 5 CD8 T cells that do not knock out the gene; CD8 T cells that knock out Rc3h1 were made into a cell suspension with PBS, and the mouse tail was reinfused into each mouse in the sgRc3h1 group, and the tail of each mouse was reinfused to 4×10 5 CD8 T cells knocked out of the sgRc3h1 gene; CD8 T cells knocked out of Zc3h...
Embodiment 3
[0059] Example 3 CD19-CAR-T cells knocked out of Rc3h1 and / or Zc3h12a can effectively eliminate CD19-positive target cells without any pretreatment
[0060] In order to verify whether the above experimental results can be directly used to improve the existing CAR-T cell therapy, we prepared CD19-CAR-T cells knocked out of Rc3h1 and / or Zc3h12a, and used them to eliminate CD19-positive targets in vivo. cell. Specific steps are as follows:
[0061] 1. Construction of gene knockout vector: A retroviral vector expressing both CD19-CAR and sgRNA was constructed, i.e. hU6-sgRNA-EF1a-Thy1.1-P2A-CD19-CAR (structural schematic diagram as shown in image 3 Middle a), wherein pMSCV-hU6-sgControl-EF1a-Thy1.1-P2A-CD19-CAR (SEQ ID NO.5, the 1686-1705th position of SEQ ID NO.5 is a random sequence that does not target any gene) as Control CAR-T cells without knockout of any gene, pMSCV-hU6-sgRc3h1-EF1a-Thy1.1-P2A-CD19-CAR (the carrier sequence is to replace the 1686-1705th positions of SEQ ...
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