Optimization of CRISPR-Cas9 knock-out of CD33 in human hematopoietic stem / progenitor cells for allogeneic transplantation in patients with acute myeloid leukemia

Abstract

Background & Aim Chimeric antigen receptor T-cells (CART) for acute myeloid leukemia (AML) are limited by hematological toxicity due to shared antigens in AML blasts & healthy hematopoietic stem cells (HSPC). We developed a treatment paradigm combining transplantation with CD33neg HSPC, followed by anti-CD33 CART; CD33negHSPC are preserved while CD33pos AML blasts are eradicated. We describe clinical scale-up of manufacture of HSPC-CD33neg for a pilot clinical trial of this approach for relapsed AML, focusing on electroporation method, guide RNA source, cell culture media & cryomedia. Methods, Results & Conclusion Human CD34+ cells were isolated from frozen mobilized peripheral blood (donated by the Transfusion Medicine Department at the University of Pennsylvania). Experiments described herein used the Lonza 4D device at small and medium-scale (20uL or 100uL cuvettes, respectively). We compared in vitro transcribed (IVT) guide RNA (gRNA) with synthetic modified gRNA (synRNA) from two vendors, described here as synRNA-1 and synRNA-2. IVT gRNA demonstrated better knock-out (KO) efficiency than both synRNAs (CD33 surface expression baseline 97.65%; IVT gRNA 0.87-1.33%; synRNA-1 2.15-2.25%, synRNA-2 5.76-7.36%). Two different electroporation settings showed similar CD33 KO efficiency. Cell viability was improved with synRNA compared with IVT gRNA and caused less cytokine production (29 cytokine multiplex) by HSPCs 24 hours after electroporation. Residual Cas9 protein was detected by ELISA and levels were consistently lower when IVT gRNA was used compared with synRNA (IVT 37.1- 78.55 fg/cell; synRNA 56.0 - 150.1 fg/cell), with no reduction in Cas9 levels observed over 48 hours of culture. We evaluated culture media and found better cell viability with StemSpan SFEM compared to X-Vivo15. No significant differences in post-thaw recovery were observed between 3 different cryomedia. Product potency, defined as hematopoietic colony formation on Methocult H4435 after 14 days, was higher with StemSpan SFEM compared with StemLine HSPC expansion media, but potency was equivalent regardless of gRNA source. We conducted an unbiased off-target editing screen using iGUIDE (an adapted version of GUIDEseq) on cells from one donor. There was no statistically significant enrichment for off-target events in cancer-associated genes compared with randomly selected locations, using a Fisher's Exact test. We have now optimized a GMP-compliant system for CRISPR-based editing of human HSPC, poised for translation to clinical scale.