Authors
J Komen2; B Recolin2; G Ciotta3; A Goeppert4; M Coen2; C Scott1;
1 Oncology Safety, Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, Waltham, United States; 2 Oncology Safety, Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, Cambridge, UK; 3 Discovery Biology, Discovery Sciences, R&D, AstraZeneca, Cambridge, UK; 4 Precision Medicine, Oncology, R&D, AstraZeneca, Cambridge, UK
Abstract
Understanding the biological role of oncology drug targets in hematopoietic stem cells is critical given the high incidence of bone marrow (BM) adverse events which often limit the therapeutic utility of anti-cancer agents.
Assessment of BM toxicity (hemetox) risk is conducted in the discovery phase through application of a novel in vitro human hematopoietic stem/progenitor cells (HSPCs) model. Viability of human CD34+ HSPCs driven to differentiate into either erythroid, myeloid, or megakaryocytic cell-lineages using specialist liquid culture medium is assessed in the presence of drug. This assay format allows a higher throughput than the standard Colony Forming Unit (CFU) assay, however it remains a challenge to mechanistically dissect the contribution of on or off-target activity to the viability phenotype.
To address this gap, we aim to use CRISPR to knock-out (KO) drug targets to evaluate the role of the target in HSPC differentiation and proliferation. In this proof of concept (POC) study, we have targeted the non-essential PTPRC gene encoding CD45 using a CRISPR ribonucleoprotein approach combined with electroporation.
We achieved highly consistent and efficient KO of CD45 HSPCs as demonstrated on both DNA (>90%, TIDE analysis) and protein level (>80%, flow cytometry). The CD45 KO HSPCs retained the capability to differentiate into the erythroid and myeloid lineages in our 2D bone marrow (BM) assay as anticipated for a non-essential gene, showing successful POC.
Currently, we are applying this methodology to better understand haematopoietic safety liabilities of drug targets in the oncology portfolio. Such an approach will allow us to understand the impact of genetic knockdown of a given target on stem cell proliferation and differentiation into various hematopoietic lineages whilst simultaneously evaluate potential poly-pharmacology of compounds. Ultimately this will identify key risks to avoid/design away from early in the drug discovery phase, which is critical information for the development of safer drugs.
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