Gene Modification of Human Hematopoietic Stem Cells with Suicide Genes As a Safety Control
Authors: Truscott Laurel Christine , Chiou Tzu Ting , Kao Roy L. , De Oliveira Satiro N. 
Published: November 2019

Abstract
Background:Gene-modified human hematopoietic stem cells (HSC) have been used as tools to introduce genes into organisms to correct metabolic or cellular disorders caused by defective or absent genes, or to prevent or treat diseases. We propose the innovative concept of using gene-modification of HSC to enable persistent generation of multilineage immune effectors able to directly target cancer cells. Stable transgene integration has been done by modification of HSC using retroviral or lentiviral vectors. In early clinical trials, there was a high incidence of retroviral-mediated insertional mutagenesis, prompting the need for alternative approaches. Despite current research data suggesting lentiviral vectors are relatively safer, concerns regarding malignant transformation, abnormal hematopoiesis and autoimmunity still exist, making the co-delivery of a suicide gene a necessary safety measure. We have compared two potential suicide gene systems for this use: the herpes simplex virus thymidine kinase (HSV-sr39TK) and a truncated epidermal growth factor receptor (EGFRt). To enable future clinical applications, those transgenes were co-delivered with anti-CD19 chimeric antigen receptor (CAR).
We hypothesize gene-modified HSC can be successfully targeted and ablated using a suicide gene system, and HSV-sr39TK and EGFRt will both be effective suicide gene systems.
Methods: Third generation self-inactivating lentiviral vector constructs were used to co-deliver an anti-CD19 CAR and HSV-sr39TK or EGFRt. Each suicide gene's efficacy was tested using cytotoxicity assays. Jurkat and primary cells expressing HSV-sr39TK were incubated with varying concentrations for ganciclovir. For the EGFRt suicide gene system, an antibody-dependent cell-mediated cytotoxicity (ADCC) assay was used with EGFRt-transduced Jurkat and primary cells as targets, with the target cells incubated with leukocytes and varying ratios of the EGFR-specific monoclonal antibody cetuximab. For both assays, the cells were stained and analyzed by flow cytometry to determine the percentage of surviving cells. For in vitro assays, gene-modified HSC were differentiated into myeloid cells over ten days to allow transgene expression before cytotoxicity challenges. Gene-modified HSC were also engrafted into immunodeficient NSG mice for in vivo experiments using treatments with intraperitoneal ganciclovir 50mg/kg/day over 5 days or intraperitoneal cetuximab 1mg/mouse/day over 12 days. Persistence of gene-modified cells was assessed by flow cytometry and ddPCR of animal tissues.
Results:For the HSV-sr39TK transgene, primary human T-cells and myeloid cells differentiated from gene-modified human HSC had consistently decreased rates of survival for the HSV-sr39TK transduced cells when incubated with varying concentrations of ganciclovir, as compared to both the non-transduced and control-transduced cells, with remaining survival of gene-modified cells of 20% in the assays employed. For the EGFRt transgene, cytotoxicity was significantly increased (p<0.0001) in target cells expressing EGFRt after 4-hour incubation of leukocytes with target cells and cetuximab 1µg/mL, compared with either EGFRt+ cells without cetuximab, and non-transduced cells with or without cetuximab. This was seen at all effector to target ratios and average remaining gene-modified cells also approached 20% in the assays employed. Mice humanized with gene-modified HSC presented significant ablation of gene-modified cells after treatment with either ganciclovir for the HSV-sr39TK transgene, or cetuximab for the EGFRt transgene, with successful ablation of gene-modified cells. Remaining detected gene-modified cells in both models were close to background on flow cytometry and one to two logs of decrease of vector copy numbers by ddPCR in mouse tissues.
Conclusions: Ganciclovir-mediated killing of HSV-sr39TK transduced cells was shown to be effective in cells differentiated from gene-modified human HSC. Cetuximab ADCC of EGFRt-modified cells also determined effective killing. These results give proof of principle for CAR-modified HSC regulated by suicide gene, and further studies are needed to enable full clinical translation of this approach. Different ablation approaches, such as inducible caspase 9 or co-delivery of inert cell surface markers (truncated CD20, truncated EGFR) should be evaluated.

Disclosures
No relevant conflicts of interest to declare.