CRISPR-Cas9 Genome Editing of Primary Chronic Lymphocytic Leukemia Cells

Chronic lymphocytic leukemia (CLL) is a common B-cell neoplasia that exhibits a very heterogeneous course. Over the last decade, multiple molecular studies have described the genomic, epigenomic and transcriptional landscape of the disease. These studies have led to the identification of more than 60 recurrently mutated genes as well as a large number of deregulated genes and regulatory elements. While the prognostic significance of some of these alterations is well described, their specific contributions to the pathogenesis of the disease remains largely unknown. Attempts to address this experimentally have employed genetically engineered mouse models and human cell lines. Although mouse models have been helpful to recapitulate disease aspects that can be only properly studied in vivo, significant limitations exist that prevent extrapolation of data from mice to human. Moreover, cell lines are commonly obtained from patients with end-stage, refractory diseases and are frequently EBV-positive, showing great discrepancies with primary cells, including high proliferation rate and absence of spontaneous apoptosis. In addition, they do not faithfully recapitulate the transcriptional and chromatin landscape of CLL primary cases. Thus, there is a need of better cellular models that recapitulate these disease aspects.

An attractive solution to this problem would be the use of primary human CLL cells as a platform for ex vivo genetic manipulation, applying CRISPR-Cas9 genome editing technology. However, this is technically challenging, as (i) CLL cells are critically dependent on stimulatory signals coming from the tumor microenvironment, particularly in the lymph nodes (LN), and (ii) primary cells are resistant to most gene transfer methods. In order to overcome these limitations, we took advantage of a cell culture system that is reminiscent of a LN environment and induces vigorous proliferation of CLL primary cells for several weeks, allowing retroviral gene transfer with high efficiency and minimal toxicity (Mangolini, M. et al., 2022, Nat Commun, in press). To avoid undesired off-target effects induced by the constitutive expression of a CRISPR-Cas9 viral plasmid, we used transient expression systems. As an initial proof of concept, we designed sgRNA targeting the CD19 gene, a pan-B cell marker highly expressed in CLL cells, and assessed the transfection efficiency and cell viability when introducing the Cas9-CD19 sgRNA ribonucleoprotein complex using the Neon Transfection System (Thermo Fisher Scientific). Cryopreserved CLL primary cells (from peripheral blood samples) were expanded for 3 days in vitro and were subsequently electroporated with optimized conditions. Cell viability and electroporation efficiency were assessed 24 hours later and demonstrated a remarkable low percentage of apoptotic cells, with only 10% of cell death compared to non-electroporated cells (measured by flow cytometry using the LIVE/DEAD fixable Aqua dead cell stain, n= 6 cases); while high transfection efficiency was accomplished, with over 80% of cells containing the fluorescent Cas9-ATTO550 sgRNA ribonucleoprotein. Finally, 72 hours after electroporation we observed a downregulation greater than 85% of CD19 levels by flow cytometry (n=6 cases).

After successful CD19 downregulation, we aimed at targeting the cell cycle regulatory protein Cyclin D2 (CCND2), which is upregulated by CLL cells at proliferation centers and the most upregulated cyclin in our cell culture system. Two independent sgRNAs were electroporated with the Cas9 protein into 3 CLL primary cases, with a minimum effect on cell viability after 24 hours. CCND2 knock-down (KD) efficiency was of 70% and 60% respectively in all cases studied, assessed by qRT-PCR. Interestingly, despite being exposed to cell culture conditions promoting vigorous proliferation, CCND2 KD blocked cell proliferation, and a cell growth reduction of 50-70% was obtained with both sgRNAs compared with non-targeting controls already at 48 hours.

Altogether, these results demonstrate the feasibility to genetically modify primary CLL cells using the CRISPR-Cas9 system. This strategy overcomes the current dependency on cell lines or mouse models and represents a powerful tool to unveil the role of candidate genes involved in CLL pathogenesis.

No relevant conflicts of interest to declare.