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

摘要: Abstract 2994 The suboptimal numbers of hematopoietic stem cells (HSC) present in human umbilical cord blood (CB) grafts results a significant delay cell reconstitution or graft failure, particularly 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 transplantable HSC (Araki et al. Blood 2007, Exp Hematol 2009). Here performed validation studies non-human primate (NHP) baboons to examine the potential genotoxicity CMA-expanded bone marrow (BM) graft. To our knowledge, there have been no reports suggesting 5azaD exacerbate chromosomal instability human. We demonstrated sequential addition low-dose (5 micromolar) TSA ng/ml) CB CD34+ does not result persistent depletion Dnmt1 transcripts protein levels. In this global methylation analysis using LINE-1 assay indicates DNA demethylation, which occurs with CMA treatment culture, is transient: absence presence was 78.5% ± 2.2% 55.4 % respectively at day 3 while levels treated returns 69.7% 2.1% by 9. attempt evaluate genotoxic 5azaD/TSA, transplanted autologous BM myeloablative dose IV busulfan (4 mg/kg/day × 4 days). Although initial pre-expansion low (0.68, 2.64 5.18 10 6 cells/kg respectively), all three engrafted Following peripheral (PB) count recovery examined CD34 colony-forming (CFC) counts. Of note, absolute number (926.0 142.8 /microliter) 3.5 fold higher than pre-transplant (257.9 56.2 /microliter). However, CFC 2.2 lower biopsies on serially prior to, 56, 120 400 days post-transplant recovery, myeloid-to-erythroid ratio cellularity did demonstrate myelodysplasia malignancy. There tri-lineage hematopoiesis evident 50 70% cellularity. Further evaluation safety 5azaD/TSA-expanded independent methods. Firstly, 150 NOD/SCID mice who received 5azaD/TSA expanded displayed any signs tumor formation after primary secondary Secondly, karyotyping baboon transplant (day 266) reveals detectable anomalies. Thirdly, used Flow-FISH, situ flow cytometry-based measure relative telomere lengths (RTL) purified CD11a+ (97.59% 0.38%) leukocytes from majority are short-lived indirectly reflects respective they derived. Our show RTL Jurkat were 10.6% 0.37%, RTLs 19.7% 1.2%. pre- 56) one (PA7619) (average) values 13.48% 0.76 13.96% 0.68% respectively. another animal (PA7963), average determined Flow-FISH enriched multiple time-points up 417 mean 17.66 0.52% 15.39% 0.62%. summary, stable comparable Importantly, year-long monitoring shows evidence hematologic malignancies as PB counts, examination (including cytogenetics) measurements leukocyte clinically relevant NHP model. These offer preliminary CMA-mediated transferred into primates (and mice) induce malignancies, relevance use clinical trials. Disclosures: No conflicts interest declare.