Genetically engineered t cells with improved persistence in culture
A technique of genetic engineering, cell, applied in the field of genetically engineered T cells with improved persistence in culture
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example 1
[0271] Example 1: Effects of TET2 knockout (KO) or mutation on primary human T cells
[0272] (i) Efficient Cas9 knockout or mutation of TET2:sgRNA RNPs in T cells
[0273] This example describes the efficient ex vivo editing of the TET2 gene in T cells derived from primary human PBMC cells using CRISPR / Cas9 gene editing. The genomic fragment of the TET2 gene containing the fourth, fifth and sixth protein-coding exons was used as input to the gRNA design software. Desired gRNAs are those that will create deletions in the coding sequence, disrupt the amino acid sequence of TET2, and result in out-of-frame / loss of functional alleles (referred to as "TET2 knockout" alleles or truncations of the TET2 protein). Four (4) in silico-identified gRNA spacer sequences targeting the TET2 gene were synthesized and the gRNAs were specifically modified, as shown in Table 1. Although the gRNAs in Table 1 have been modified with 2'-O-methyl phosphorothioate modifications, unmodified gRNAs ...
example 2
[0288] Example 2: FAS knockout (KO) increases proliferation and reduces apoptosis
[0289] (i) Cas9 knockout FAS:sgRNA RNP in T cells
[0290] This example describes the efficient ex vivo editing of the FAS gene in primary human T cells using the CRISPR / Cas9 gene editing approach. Desired gRNAs are those that will create insertions or deletions in the coding sequence, disrupt the amino acid sequence of FAS, resulting in out-of-frame / loss of functional alleles (referred to as "FAS knockout" alleles). All five (5) in silico identified gRNA spacer sequences targeting the FAS gene were synthesized.
[0291] Ribonucleoprotein particles (RNPs) or controls ( Primary human T cells from two healthy donors were transfected (electroporated with no Cas9, no gRNA). Six (6) days after transfection, cells were treated by flow cytometry (primary antibody: PEDazzle 594 anti-human FAS antibody, clone DX2, Biogen Biotech) to assess the level of FAS expression on the cell surface.
[0292] ...
example 3
[0305] Example 3: Triple knockout of CD70, TET2 and FAS enhances cytolytic activity of anti-CD19 CAR T cells
[0306] After preparation of edited anti-CD19 CAR T cells, a flow cytometry-based cytotoxicity assay was used to validate the functional activity of CAR T cells. For a disclosure of anti-CD19 CARs see WO 2019 / 097305, the relevant disclosure of which is incorporated by reference for the purposes and subject matter mentioned herein. Combining anti-CD19 CAR T cells (TRAC- / β2M- / CD19 CAR+ and TRAC- / β2M- / CD70- / TET2- / FAS- / CD19 CAR+) with CD19-expressing cancer cell lines (target cells) Raji (ATCCccl-86 )co-cultivate. Target cells were labeled with 5 μM efluor670 (eBiosciences), washed and differentiated with TRAC- / β2M- / anti-CD19 CAR+ or TRAC- / β2M- / CD70- / TET2- / FAS- / anti-CD19 CAR+ Ratios (0.01, 0.05, 0.1, 0.5, 1:1 T cells:target cells) were co-cultured. Target cells were seeded in a 96-well U-bottom plate at 100,000 cells / well. The co-cultures were incubated for 48 hours. ...
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