Multiplex Deletion of Myeloid Antigens CD33 and CLL-1 by CRISPR/Cas9 in Human Hematopoietic Stem Cells Highlights the Potential of Next-Generation Transplantation for AML Treatment
Multiplex Engineering: Knocking out two or more distinct genes such as CD33 and CLL1 through gene editing offers a promising new approach to treating patients with AML using our novel eHSC platform.
Multiplex engineering is a method in which multiple genetic targets are engineered within the same cells in the same manufacturing process. Multiplex engineering could allow removal or modification of two or more distinct genes, thus allowing for targeted therapies directed at two or more separate targets to be used in combination or in sequence, which could be particularly valuable to prevent escape mechanisms involving tumor cells down-regulating target expression.
We have developed several techniques for multiplex engineering HSCs.
Sequential Cas9 Editing
Sequential Cas9 editing is a technique where HSPCs are subject to two separate Cas9 edits separated by a defined time period in order to allow the first edit to complete before applying the second edit. This separation is important to avoid translocation errors, which are gene repairs resulting in one DNA segment joining other DNA segments from different parts of the same chromosome or segments of other chromosomes. We have demonstrated that we can efficiently knock out expression of both CD33 and CLL-1 from HSPCs using this technique.
Base Editing involves converting a specific DNA base into another at a targeted genomic
locus. As such, base editing does not require a cut, lowering the risk of translocation errors. We have demonstrated that we can efficiently knock out expression of both CD33 and CLL-1 from HSPCs using a single base editing step.
VOR33-CLL1 + VCAR33-CLL1 Treatment System
Editing multiple antigens using our eHSC platform will allow us to push the frontiers of CAR-T therapies. Knocking out CD33 and CLL-1 through gene editing offers a promising new approach to treating patients with AML. Our research demonstrates that multiplex genome editing of allogeneic hematopoietic stem cells may represent another exciting strategy to efficiently and safely edit multiple genes in blood stem cells, allowing the potential use of multi-targeted blood cancer therapies.
This Treatment System is made up of the VOR33-CLL1 multiplex-engineered eHSC, and the VCAR33-CLL1 multi-specific CAR-T.
A multiplex approach may provide advantages in two areas. Firstly, target expression can vary in tumor cells from the same patient, a phenomenon known as tumor heterogeneity. Applying therapies such as a multi-specific CAR-T may reduce that concern. Secondly, it is theoretically possible for tumor cells to downregulate expression of a target to avoid being killed, a phenomenon known as tumor escape. Again, pursuing multiple targets simultaneously may reduce the effectiveness of the tumor escape mechanism.