Universal car-t cell and its preparation method and application
A general-purpose, cellular technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, and applications, which can solve problems such as missing, low probability of successful HLA matching, and long time consumption.
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Embodiment 1
[0070] Example 1, Chinese population HLA-A typing frequency detection
[0071] 1. SBT (Sequenced Based Typing) detection:
[0072] This method belongs to HLA high-resolution typing technology.
[0073] 4ml of venous blood was extracted from EDTA anticoagulant tubes (blood routine tubes), stored at 4°C for temporary storage, and long-term frozen at -20°C. Then send to BGI for HLA typing test.
[0074] 2. Bioinformatics methods: refer to Boegel, Sebastian, Valesca et al. (2013)
[0075] Using the seq2HLA software, the HLA high-resolution typing (MHC-I and MHC-II, 4 bits) of the sample and the corresponding expression value and p-value were calculated directly from the raw data (fastq format) of RNA-Seq sequencing.
[0076] Experimental results such as figure 1 The 8 HLA-A types with the highest frequency in 8 Chinese are shown, namely A*02, A*11A*24, A*30, A*33, A*03, A*01 and A*26, The gRNA molecules were designed respectively for these 8 subtypes, and the gRNA sequences a...
Embodiment 2
[0077] Example 2, Knockout of TCR and MHC Class I and Class II Molecule Related Gene Vector Construction
[0078] 1. sgRNA molecular design:
[0079] Confirm the genes related to TCR and MHC class 1 and class 2 molecules: B2M, CIITA and TRAC genes are closely related to the functions of genes related to TCR and MHC class 1 and class 2 molecules Confirm the knockout of B2M, CIITA and TRAC genes. B2M, CIITA and TRAC gene sequence numbers were obtained from NCBI to obtain CDS sequence, B2M sequence number AH002619.2, CIITA in NCBI gene ID: 4261, TRAC in NCBI gene ID: 28755, as shown in SEQ ID NO: 1-3. The gRNAs were designed for the identified targeted SEQID NO: 1-3 regions of the CDS region, and the gRNA sequences are shown in Table 1.
[0080] Table 1: gRNA sequences
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[0082]
[0083] 2. Construction of expression vector for CRISPR / Cas9 gene knockout
[0084] The following primers were designed and synthesized by Nanjing GenScript Biotechnology Co., Ltd. The s...
Embodiment 3
[0099] Example 3. Universal CAR-T cell construction and phenotypic detection
[0100] 1. Confirmation of electroporation conditions
[0101] Under different conditions, 293T cells were used to transfect GFP to optimize the electroporation conditions. After confirming better electroporation conditions, PBMC transfection was carried out. The experimental results are shown in Table 2 and Table 3. The preferred electroporation conditions are PBMC or T cell activation. After 48 hours, the voltage was 1000V, the pulse width was 35ms, and the number of electric shocks was twice for electroporation, and the total number of cells was 2×10 5 , cultured with fresh medium containing IL-2 after electroporation.
[0102] Table 2: Comparison of transfection efficiency and cell viability of transfected 293T cells under different electroporation conditions
[0103] serial number efficiency% Survival % Voltage / V Pulse width / ms Number of shocks 1 0 100 0 1 1 2 ...
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