Optimization of a universal allogeneic CAR-T cells combining CRISPR and transposon-based technologies for treatment of Acute Myeloid Leukemia
Despite the potential of CAR-T therapies for hematological malignancies, their efficacy in patients with relapse and refractory Acute Myeloid Leukemia has been limited. The aim of our study has been to develop and manufacture a CAR-T cell product that addresses some of the current limitations. We initially compared the phenotype of T cells from AML patients and healthy young and elderly controls. This analysis showed that T cells from AML patients displayed a predominantly effector phenotype, with increased expression of activation (CD69 and HLA-DR) and exhaustion markers (PD1 and LAG3), in contrast to the enriched memory phenotype observed in healthy donors. This differentiated and more exhausted phenotype was also observed, and corroborated by transcriptomic analyses, in CAR-T cells from AML patients engineered with an optimized CAR construct targeting CD33, resulting in a decreased in vivo antitumoral efficacy evaluated in xenograft AML models. To overcome some of these limitations we have combined CRISPR-based genome editing technologies with virus-free gene-transfer strategies using Sleeping Beauty transposons, to generate CAR-T cells depleted of HLA-I and TCR complexes for allogeneic approaches. Our optimized protocol allows one-step generation of edited CAR-T cells that show a similar phenotypic profile than non-edited CAR-T cells, with equivalent in vitro and in vivo antitumoral efficacy. Moreover, genomic analysis of edited CAR-T cells revealed a safe integration profile of the vector, with no preferences for specific genomic regions, with a highly specific editing of the HLA-I and TCR, without significant off-target sites. Finally, production of edited CAR-T cells at a larger scale allowed the generation and selection of enough HLA-IKO/TCRKO CAR-T cells that would be compatible with clinical applications. In summary, our results demonstrate that CAR-T cells from AML patients, although functional, present phenotypic and functional features that could compromise their antitumoral efficacy, compared to CAR-T cells from healthy donors. The combination of CRISPR technologies with transposon-based delivery strategies allow the generation of HLA-IKO/TCRKO CAR-T cells, compatible with allogeneic approaches, that would represent a promising option for AML treatment. Overall design: As mentioned in the paper, the my96(45) CAR, containing the 45aa hinge from CD8a, was selected for the rest of the studies. Using our 4-1BB aforementioned second-generation CAR construct, we sequenced the generated CAR-T cells (CD33-CAR-T) from 4 AML patients that were compared to CD33-CAR-T cells from 3 young (adult) and  3 elderly (senior) healthy donors. RNA-seq libraries from these CAR-T cells were prepared following MARS-seq protocol adapted for bulk RNA-seq (23,24) with minor modifications. RNA-seq libraries quantification was done with Qubit 3.0 Fluorometer (Life Technologies) and size profiles were examined using Agilent's 4200 TapeStation System. Libraries were sequenced in an Illumina NextSeq500 at a sequence depth of 10 million reads per sample. Samples were aligned to the Human genome (GRCh38) with STAR (v2.6.1). Gene expression was quantified with quant3p (github.com/ctlab/quant3p). Downstream analyses were performed in R (v3.6.2).