Table 2.
Examples of prominent studies using CRISPR system for genetic modification of T cells to produce CAR-T cells.
| Reference | Target Antigen and co-stimulation | Target Genetic Locus | Methods for Gene Editing | Frequency of CAR+ T Cells after Knock-in | Potency Assays in vitro and in vivo |
|---|---|---|---|---|---|
| Eyquem et al. Nature 2017 (45) | CD19 CD28 zeta |
TRAC | sgRNA and Cas9 mRNA AAV-mediated HDR |
Up to 40%(10e6 AAV dose) | In vitro culture with Nalm-6/fLuc/GFP or NIH-3T3/CD19 |
| B2M | sgRNA-Cas9 mRNA AAV-mediated HDR |
14% | In vivo Nalm-6/fluc/GFP xenogra fted in NSG male mice | ||
| Feucht et al, Nature Medicine 2019 (46) | CD19 CD28 Zeta (+ITAM-mutated versions) |
TRAC | sgRNA and Cas9 mRNA AAV-mediated HDR |
60-75% |
In vitro culture with Nalm-6/fLuc/GFP or NIH/3T3/CD19 In vivo Nalm-6/fLuc/GFP xenograftedin NSG male mice |
| Wiebking et al, Haematologica 2021 (47) | CD19 CD28 zeta |
TRAC | sgRNA-Cas9RNP AAV-mediated HDR |
>70% |
In vitro co-culture cytotoxicity assays & cytokine production from supernatants (ELISA) In vivo Nalm-6/fLuc/GFP xenograft in NSG mice |
| Roth et al. Cell 2020 (49) | Different chimeric receptors (pool) + TCR e.g. TGFβR2-41BB |
TRAC | SgRNA-Cas9 RNP dsDNA-mediated HDR |
5-6% | In vitro expansion, co-culture killing assay and in vivo solid tumor A375 melanoma xenograft in NSG mice |
| Ode et al. Cancers 2020 (50) | IL13Rα2 CD28 | TRAC | sgRNA-Cas9RNP dsDNA-mediated HDR |
20% (but low expression level) | none |
| Kath et al, Biorxiv preprint 2021 (51) In press Mol Ther Meth Clin Dev 2022 |
CD19 CD28 zeta |
TRAC
AAVS1 |
sgRNA-Cas9RNP dsDNA-medai ted HDR sgRNA-Cas9RNP dsDNA-medai ted HDR |
25-68%(enhanced by drug co- treatments) 10-15% |
In vitro co-culture cytotoxicity assays & intracellular staining of effector cytokineproduction In vivo Nalm-6/fLuc/GFP xenograft in NRG mice |
| Muller et al. Frontiersin Immunology 2021 (52) | HLA-A2 CD28 zeta |
TRAC | sgRNA-Cas9RNP dsDNA-mediated HDR |
ca. 8-10% (increased during expansion up to 90%) |
In vitro assays for Treg function (phenotyping, activation status, proliferation suppression) In vivo mouse model of GvHD and xenogeneic GvHD |
| Jing et al. Small Methods 2021 (53) | CD19 or CD19/CD22 CD28 mutZeta or Zeta |
TRAC | sgRNA-Cas9 RNP Minicircle pDNA-mediated HDR sgRNA-Cas9 RNP AAV-mediated HDR |
10-18% (with two Cas9-target sequences in donor template & recombinant Cyclin D protein) No details regarding Kl rates |
In vitro expansion, co-culture cytotoxicity assays. (Nalm-6/fLuc/GFP) In vivo Nalm-6/fLuc/GFP xenograft in NSG mice |