The transplant of autologous or allogeneic hematopoietic stem cells (HSCs) has the proven ability to treat a wide array of malignant and non-malignant hematological diseases. The preparative regimen, however, routinely entails aggressive and genotoxic treatment with total body irradiation and/or chemotherapy, which brings severe and even life-threatening complications that limit its broader application. Previous experimental studies have established that depletion of recipient HSCs is an essential requirement of these conditioning regimens in allowing successful engraftment of the composite donor HSCs. Animal and clinical studies have also indicated that alloreactive anti-HSC donor T cells additionally facilitate stem cell engraftment, but this is often accompanied by the risks of GvHD. This has prompted the consideration of alternative conditioning methods for the depletion of HSCs with less toxic side-effects, such as anti-c-kit and anti-CD45 antibody-directed treatments. In this way, more precise HSC targeting may also be achieved by the application of short-lived, genetically engineered chimeric antigen receptor (CAR)-T cells for stem cell transplantation conditioning.

We developed a novel and controllable CAR-T approach for recipient HSC targeting via genetic modification using the non-viral piggyBac™ (PB) transposon system. As opposed to viral vector delivery systems, the relatively large carrying capacity of PB allows the stable introduction of at least three separate genes encoded within the same tri-cistronic transgene cassette. This includes a second-generation CAR that targets either human c-kit (CD117) or prominin-1 (CD133), markers known to be antigenically expressed on the surface of HSCs. In addition, a drug resistance element serves as a selection gene that, in combination with a non-genotoxic drug, provides an effective method of CAR-T cell purification during manufacture. Importantly, a small molecule drug-inducible safety switch gene is also included to facilitate rapid in vivo clearance of the CAR-T cells after depletion of recipient HSCs and prior to donor HSC transplant. Lastly, as a result of the manufacturing process, the majority of the CAR-T cells express chemokine receptors such as CXCR4 that can allow more selective trafficking to the bone marrow (BM) for eradication of resident HSCs.

To select a lead candidate from a panel of anti-HSC CAR constructs, CD3/CD28 stimulated T cells from human peripheral blood were first electroporated with mRNA encoding each of the CAR candidates directed against either c-kit or CD133. CAR surface expression was confirmed in transfected T cells by flow cytometry. In vitro functional assays were performed by co-culturing mRNA-transfected CAR-T cells with mouse or human cell lines (EML-C1, TF-1 and K562), expressing either c-kit or CD133, as well as mouse and human primary BM cells. Lead CAR candidates were identified from their specific activation of the CAR-T cells through degranulation according to CD107a expression and secretion of IFNγ. Furthermore, those CARs were also capable of selectively depleting c-kit or CD133 positive cells. Interestingly, some mRNA-transfected CAR-T cells retained effector activity against target c-kit+ TF-1 cells even in the presence of its soluble ligand, stem cell factor. Next, lead CAR candidates were co-expressed with the selection and drug-inducible safety switch genes in the same tri-cistronic transgene and then stably delivered to T cells using PB. The manufacturing process yielded CAR-T cells that were mainly of the T memory stem cell (Tscm) phenotype, as determined by positive expression of CD62L and CD45RA, and also expressed high levels of the CXCR4 chemokine receptor. Similar to the mRNA-transfected CAR-T cells, these stably-transposed cells were capable of extensive effector capabilities including specific depletion of c-kit or CD133 expressing target cells.

Future studies will evaluate PB-produced lead anti-HSC CAR-T cells in immune-deficient NSG mice with pre-established xenogeneic human hematopoietic chimerism, along with standard busulfan or radiation conditioning controls. This approach constitutes a novel targeted biological therapy that is envisaged to lead the way towards minimally toxic transplant regimens for depletion of endogenous HSCs in the BM and to procure their replacement with engrafted allogeneic or gene-corrected stem cells.


Timberlake: Poseida Therapeutics, Inc.: Employment. Richter: Poseida Therapeutics, Inc.: Employment. Wang: Poseida Therapeutics, Inc.: Employment. Cam: Poseida Therapeutics, Inc.: Employment. Tan: Poseida Therapeutics, Inc.: Employment. Ostertag: Poseida Therapeutics, Inc.: Employment, Equity Ownership. Shedlock: Poseida Therapeutics, Inc.: Employment. Down: Poseida Therapeutics, Inc.: Employment.

Author notes


Asterisk with author names denotes non-ASH members.