Evaluation of Genotoxicity of Chromatin Modifying Agents Expanded Hematopoietic Graft in a Non-Human Primate Model.

Abstract 2994

The suboptimal numbers of hematopoietic stem cells (HSC) present in human umbilical cord blood (CB) grafts results in a significant delay in blood cell reconstitution or graft failure, particularly in adult patients following transplantation. Using chromatin modifying agents (CMAs), including 5aza-2-deoxycytidine (5azaD) and trichostatin A (TSA), we developed an ex-vivo expansion strategy that permits 7-fold expansion of transplantable HSC (Araki et al. Blood 2007, Exp Hematol 2009). Here we performed validation studies in non-human primate (NHP) baboons to examine the potential genotoxicity of CMA-expanded bone marrow (BM) graft. To our knowledge, there have been no reports suggesting 5azaD exacerbate chromosomal instability in human. We have demonstrated that the sequential addition of low-dose 5azaD (5 micromolar) and TSA (5 ng/ml) to human CB CD34+ cells does not result in a persistent depletion of Dnmt1 transcripts and protein levels. In this global methylation analysis using a LINE-1 assay indicates that DNA demethylation, which occurs with CMA treatment in culture, is transient: LINE-1 methylation of CD34+ cells in the absence or presence of CMA was 78.5% ± 2.2% and 55.4 % ± 2.2% respectively at day 3 while methylation levels of treated CD34+ cells returns to 69.7% ± 2.1% by day 9. In an attempt to evaluate the genotoxic potential of low-dose 5azaD/TSA, we transplanted baboons with CMA-expanded autologous BM grafts following a myeloablative dose of IV busulfan (4 mg/kg/day × 4 days). Although the initial pre-expansion CD34+ cell dose of CMA-expanded BM grafts was low (0.68, 2.64 and 5.18 × 10⁶ CD34+ cells/kg respectively), all three baboons engrafted following transplantation. Following peripheral blood (PB) cell count recovery we examined BM CD34 and colony-forming cell (CFC) counts. Of note, the absolute number of CD34+ cells in the BM following hematopoietic reconstitution (926.0 ± 142.8 /microliter) was 3.5 fold higher than pre-transplant levels (257.9 ± 56.2 /microliter). However, the absolute number of CFC in the BM was 2.2 fold lower than pre-transplant levels. BM biopsies performed on the 3 baboons serially prior to, and 56, 120 and 400 days post-transplant respectively to examine hematopoietic recovery, myeloid-to-erythroid ratio and cellularity did not demonstrate myelodysplasia or malignancy. There was tri-lineage hematopoiesis evident in the BM in all 3 baboons with 50 to 70% cellularity. Further evaluation of the safety of 5azaD/TSA-expanded grafts was performed using three independent methods. Firstly, 150 NOD/SCID mice who received 5azaD/TSA expanded human CB grafts have not displayed any signs of tumor formation after primary or secondary transplantation. Secondly, karyotyping of CMA-expanded human CB graft or baboon CD34+ cells prior to and following transplant (day 266) reveals no detectable chromosomal anomalies. Thirdly, we used Flow-FISH, an in situ flow cytometry-based assay to measure relative telomere lengths (RTL) of purified CD11a+ (97.59% ± 0.38%) leukocytes from baboon BM prior to and following transplantation. The majority of CD11a+ leukocytes are short-lived which indirectly reflects the telomere lengths of the respective HSC from which they are derived. Our results show that the RTL of Jurkat cells were 10.6% ± 0.37%, while human CB cell RTLs were 19.7% ± 1.2%. The pre- and post-transplant (day 56) BM cells of one baboon (PA7619) displayed (average) RTL values of 13.48% ± 0.76 % and 13.96% ± 0.68% respectively. In another animal (PA7963), average RTL values were determined serially using Flow-FISH on BM CD11a+ enriched cells at multiple time-points post-transplant up to day 417 which displayed mean RTL of 17.66 % ± 0.52% while pre-transplant RTL was 15.39% ± 0.62%. In summary, the mean RTL values of baboon leukocytes were stable and post-transplant values were comparable to pre-transplant levels. Importantly, year-long monitoring shows no evidence of hematologic malignancies as determined by PB counts, BM examination (including cytogenetics) and measurements of leukocyte RTL values in a clinically relevant NHP model. These studies offer preliminary evidence that CMA-mediated expansion of CD34+ cells transferred into primates (and human CB CD34+ cells transferred into mice) is not genotoxic and does not induce hematopoietic malignancies, of relevance to use of human CMA-expanded CB grafts in clinical trials.