CRISPR/Cas-based Human T cell Engineering: Basic Research and Clinical Application.

Engineering human T cells for the treatment of cancer, viral infections and autoimmunity has been a long-standing dream of many immunologists and hematologists. Although primary human T cells have been genetically engineered for decades, this process was challenging, time consuming and mostly limited to transgene insertions mediated by viral transduction. The absence of widely accessible tools to efficiently and precisely engineer T cells genetically in a targeted manner limited their applicability as a living drug. This fundamentally changed with the discovery of CRISPR/Cas9 and its adaptation to human T cells. CRISPR/Cas9 has made T cell engineering widely accessible and accelerated the development of engineered adoptive T cell therapies. Only 6 years after the discovery of CRISPR/Cas9 as a biotechnological tool the first CRISPR engineered T cells have been administered to patients with refractory cancers in a phase I clinical trial. Novel Cas proteins - natural and engineered ones - are rapidly emerging. These offer for instance increased flexibility, activity and/or specificity. Moreover, sophisticated protein engineering and fusions of Cas with deaminases or reverse transcriptases enable genomic DNA editing without the need for a double strand cut. Thus, the "CRISPR tool box" for experimental use as well as for novel therapeutic approaches is rapidly expanding. In this review, we will summarize the current state of CRISPR/Cas-based engineering in human T cells for basic research and its clinical applications.

Targeted T cell receptor gene editing provides predictable T cell product function for immunotherapy.

Adoptive transfer of T cells expressing a transgenic T cell receptor (TCR) has the potential to revolutionize immunotherapy of infectious diseases and cancer. However, the generation of defined TCR-transgenic T cell medicinal products with predictable in vivo function still poses a major challenge and limits broader and more successful application of this "living drug." Here, by studying 51 different TCRs, we show that conventional genetic engineering by viral transduction leads to variable TCR expression and functionality as a result of variable transgene copy numbers and untargeted transgene integration. In contrast, CRISPR/Cas9-mediated TCR replacement enables defined, targeted TCR transgene insertion into the TCR gene locus. Thereby, T cell products display more homogeneous TCR expression similar to physiological T cells. Importantly, increased T cell product homogeneity after targeted TCR gene editing correlates with predictable in vivo T cell responses, which represents a crucial aspect for clinical application in adoptive T cell immunotherapy.