An Inducible Caspase 9 Suicide Gene to Improve the Safety of Mesenchymal Stromal Cell Therapies.

Abstract 1444

Poster Board I-467

Given their immunomodulatory properties and potential for tissue differentiation, mesenchymal stromal cells (MSCs) are attractive vehicles for the treatment of graft-versus-host disease and autoimmune disorders and for regenerative cellular therapies. While MSCs have been infused in hundreds of patients to date with minimal reported side effects, follow-up is limited and little is known of their longer term potential for autonomous growth or unwanted activity or differentiation. Several in vitro and animal models have recently raised safety concerns, including reports of spontaneous osteosarcoma formation in culture, and ectopic ossification and calcification foci in the myocardium and lung. In light of those concerns, we sought to develop a system that could control the growth and survival of MSCs used therapeutically.

We previously described a suicide gene system for T lymphocytes which was based on an inducible caspase-9 (iCasp9) protein that can be activated using a specific chemical inducer of dimerization (CID), which has been safely tested in a phase I study. Because caspase 9 should induce apoptosis even in differentiated and non-dividing cells, we tested this approach in MSC before and after induction of differentiation. MSCs isolated from healthy donors were transduced with a retroviral vector encoding iCasp9 together with a truncated CD19 (ΔCD19), to allow selection of transduced cells. After a single transduction, 47% ± 6% of the cells were iCasp9/CD19-positive, a percentage that was stable over more than two weeks in culture, suggesting no growth modulation of MSC by the construct. Transduced cells were readily selected by an immunomagnetic column to >97% purity. The phenotype of the iCasp9/CD19-positive cells was identical to that of untransduced cells, with >98% cells positive for CD73, CD90 and CD105, and negative for hematopoietic markers. Non-transduced MSCs had a spontaneous rate of apoptosis in culture of approximately 18% (±7%), and this was not increased following iCasp9-ΔCD19 transduction and selection (apoptotic rate 15% ± 6%, P = 0.47). Addition of CID to MSC cultures after transduction and selection with iCasp9-ΔCD19 resulted in the apoptotic death of 93% ± 1% of iCasp9-positive cells within 24 hrs (P < 0.0001 compared to control), while iCasp9-negative cells retained an apoptosis index similar to that of non-transduced controls (20% ± 7%, P = 0.99 and P = 0.69 vs. non-transduced controls with or without CID, respectively). Furthermore, iCasp9-positive MSCs injected subcutaneously in immunodeficient mice were selectively eliminated after administration of CID to the animals, as assessed by in vivo imaging, without systemic toxicity. Hence, iCasp9-positive MSCs can be selectively killed by exposure to this drug in vitro or in vivo. Moreover, addition of CID to cultures of MSCs differentiated to adipocytes, osteoblasts or chondroblasts also resulted in >90% apoptosis as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.

In summary, we have demonstrated that MSCs can express an inducible caspase gene without affecting their phenotype, survival or capacity to differentiate, that the transduced cells can be selected with clinical grade procedures and maintain their basic physiology, and that both the MSC and their differentiated progeny can be selectively eliminated in vitro and in vivo by exposure to a small dimerizer molecule. This approach may provide an added margin of safety to the increasing clinical applications of MSCs and their progeny.