CRISPR-Cas9 Engineering for Improvement and Development of Blood Cell-Based Gene Therapy

In the past few years, the CRISPR-Cas9 system has emerged as one of the most valuable and versatile technologies for efficient gene editing. Its advantageous features offer a wide range of applications, including the development of human therapy for the treatment of cancer and genetic diseases. In this context, blood cells are ideal candidates for CRISPR modification as their isolation is less invasive than the procedures required for other tissues. These cells can be suitably expanded and engineered ex vivo, then subjected to careful analysis in standardized conditions to ensure successful genetic modification and cellular fitness, and finally, infused into the patient. In the first project of this thesis, CRISPR-Cas9 was employed to enhance the effector function of NK-92 cells for leukemia treatment (AML and B-ALL) in combination or absence of a chimeric antigen receptor (CD19-CAR and CD276-CAR). Up to three different inhibitory checkpoints (CBLB, NKG2A, and TIGIT) were knocked out in the NK-92 cell lines. These targets were selected given the relevance of their inhibitory pathways in cancer immunotherapy and the fact that both receptors and ligands are highly expressed in NK-92 and cancer cells, respectively. The resulting knock-out cells were further tested by in vitro assays against malignant cell lines to corroborate the potential anticancer benefit derived from the corresponding genetic modification. While NKG2A knock-out did not boost the killing performance, CBLB and TIGIT knock-outs showed promising enhanced cytotoxicity against AML. Future experiments in animal models would conclude whether the implementation of these genetic improvements can boost NK-92 cell-based immunotherapy against leukemia in vivo. In the second project, the main objective was the generation of a protocol for the automation of efficient CRISPR-Cas9 gene editing of HSPCs in the GMP-compatible device CliniMACS® Prodigy for the treatment of β-hemoglobinopathies following the same gene editing strategy as the CTX001 treatment. Together with our partners from Miltenyi Biotec, we pursued a thorough protocol optimization to achieve efficient gene editing, and it led to a clinical-scale proof-of-concept study that resulted in suitable BCL11A editing and restoration of HbF expression. The generated protocol will support the development of novel treatments for patient care as it can be easily transferred to other genetic diseases, and will potentially increase the accessibility of gene therapy in the near future