An inducible caspase 9 suicide gene to improve the safety of mesenchymal stromal cell therapies

Abstract

Mesenchymal stromal cells (MSCs) have been infused in hundreds of patients to date, with minimal reported side effects. However, follow-up is limited and long-term side effects are unknown. Because several animal models have raised safety concerns, we sought to develop a system allowing control over the growth and survival of MSCs used therapeutically. We have previously described a suicide system based on an inducible caspase-9 (iCasp9) protein that is activated using a specific chemical inducer of dimerization (CID), analogs of which have been safely tested in a phase I study. Here, we show that MSCs can be easily transduced with this system and selected to high purity (greater than 97%) with clinical grade immunomagnetic procedures. The transduced cells maintain their basic physiology, including expression of surface antigens (such as positivity for CD73, CD90, and CD105, and negativity for hematopoietic markers) and their potential to differentiate into diverse connective tissue lineages (adipocytes, osteoblasts, and chondroblasts). Those cells and their differentiated progeny can be selectively eliminated in vitro or in vivo within 24 hours after exposure to pharmacological levels of CID, with evidence of apoptosis in more than 95% of iCasp9-positive cells. In conclusion, we have developed directed MSC killing to provide a necessary safety mechanism for therapies using progenitor cells. We believe that this approach will become of increasing value as clinical applications for MSCs develop further.

Figure 1. MSCs isolated from healthy individuals

A. The mononuclear adherent fraction isolated from bone marrow was homogenously positive for CD73, CD90 and CD105 and negative for hematopoietic markers. B. These cells were able to differentiate into adipocytes (left, oil red and methylene blue), osteoblasts (center, alkaline phosphatase-bromochloroindolyl phosphate/nitroblue tetrazolium and methylene blue) and chondroblasts (right, anti-type II collagen antibody-Texas red and DAPI) when cultured in specific media.

Figure 2. Human MSCs are readily transduced with iCasp9-ΔCD19 and maintain their phenotype

A. MSCs underwent a single round of transduction with iCasp9-ΔCD19 retrovirus. The percentage of CD19-positive cells, a surrogate for successful transduction with iCasp9, remains constant for more than 2 weeks. B. Successfully transduced and non-transduced cells retain the characteristic MSC surface phenotype; positive for CD73, CD90 and CD105 and negative for hematopoietic markers.

Figure 3. Human MSCs expressing iCasp9 are selectively driven to apoptosis in vitro after exposure to a small molecule chemical inducer of dimerization

A. After transduction of MSCs with iCasp9, the chemical inducer of dimerization (CID) was added at 50 nM to cultures in complete medium. Apoptosis was evaluated 24 hours later by FACS analysis, after cell harvest and staining with annexin V-PE and 7-AAD. Ninety-three percent of the iCasp9-CD19-positive cells (iCasp pos/CID) became annexin positive versus only 19% of the negative population (iCasp neg/CID), a proportion comparable to non-transduced control MSC exposed to the same compound (Control/CID, 15%) and to iCasp9-CD19-positive cells unexposed to CID (iCasp pos/no CID, 13%), and similar to the baseline apoptotic rate of non-transduced MSCs (Control/no CID, 16%). B. Magnetic immunoselection of iCap9-CD19-positive cells can be achieved to high degree of purity. More than 95% of the selected cells become apoptotic after exposure to CID.

Figure 4. Human MSCs expressing iCasp9 retain the differentiation potential of unmodified MSCs and their progeny is killed by exposure to CID in vitro

Human MSCs were immunomagnetically selected for CD19 (thus iCasp9) expression, with a purity greater than 91%. After culture in specific differentiation media, iCasp9-positive cells were able to give rise to adipocytic (A, oil red and methylene azur), osteoblastic (B, alkaline phosphatase-BCIP/NBT and methylene blue) and chondroblastic lineages (C, alcian blue and nuclear red) lineages. These differentiated tissues are driven to apoptosis by exposure to 50 nM CID (D–H). Note numerous apoptotic bodies (arrows), cytoplasmic membrane blebbing (inset) and loss of cellular architecture (D and E); widespread TUNEL positivity in chondrocytic nodules (F–H), and adipogenic (I–K) and osteogenic (L–N) cultures, in contrast to that seen in untreated iCasp9-transduced controls (adipogenic condition shown, O–Q) (F, I, L, O, DAPI; G, J, M, P,TUNELFITC; H, K, N, Q, overlay).

Figure 5. Human MSCs expressing iCasp9 are selectively killed in vivo after exposure to CID

A. Human MSCs were singly transduced with the eGFP-firefly luciferase (FFLuc) gene or doubly transduced with eGFP-FFLuc and iCasp9-ΔCD19 genes. The eGFP-positive or eGFP and CD19 double-positive fractions were isolated by fluorescence activated cell sorting, with a purity > 95%. NOD-SCID animals were injected subcutaneously on day −7 with eGFP-FFLuc-positive iCasp9-positive MSCs in the right flank and control eGFP-positive MSCs in the left flank. Localization of the MSCs was tracked by the Xenogen-IVIS Imaging System. Seven days (day 0) after MSC implantation, animals were given two intraperitoneal injections of 50 μg of CID, 24 hours apart. iCasp-positive MSCs were quickly killed by the drug, as demonstrated by the disappearance of their luminescence signal. Negative control cells were unaffected by the drug, while iCasp9 positive cells persisted in the absence of drug. B. In another set of experiments, a 1:1 mixture of singly and doubly transduced MSCs was injected subcutaneously in the right flank and the mice received CID as above. The subcutaneous pellet of MSCs was harvested at different time points, genomic DNA was isolated and qPCR was used to determine copy numbers of the eGFP-FFLuc and iCasp9-ΔCD19 genes. Under these conditions, the ratio of the iCasp9 to eGFP gene copy numbers is proportional to the fraction of iCasp9-positive cells among total human cells (see Methods for details). The ratios were normalized so that time zero corresponds to 100% of iCasp9-positive cells. We observed a progressive decrease in the percentage of iCasp9-positive cells down to ~0.7% of the original population after one week. C. Serial examination of animals after subcutaneous inoculation of MSCs (prior to CID injection) shows evidence of spontaneous apoptosis in both cell populations (as demonstrated by a fall in the overall luminescence signal to ~20% of the baseline). This has been previously observed after systemic and local delivery of MSCs in xenogeneic models.