758. A Nonhuman Primate Transplantation Model to Evaluate Gene Editing Strategies Aimed at Inducing Fetal Hemoglobin Production for the Treatment of Hemoglobinopathies

Abstract

Fetal hemoglobin (HbF) is the major form of hemoglobin present in newborns but is almost completely replaced by adult hemoglobin after birth, where it constitutes less than 1 percent of total hemoglobin. Hereditary persistence of HbF is linked to mutations at multiple genetic loci that regulate the switch from fetal to adult hemoglobin. The targeting of these genes using site-specific nucleases thus constitutes a promising approach for the treatment of hemoglobinopathies by increasing HbF production.We have developed a nonhuman primate (NHP) model to investigate gene editing strategies aimed at inducing HbF production following hematopoietic stem cell (HSC) transplantation. As proof of principle, we focused on the transcription factor B-cell lymphoma/leukemia 11A (BCL11A), which functions as suppressor of HbF in humans. We disrupted the Bcl11a coding region using Transcription Activator-Like Effector Nucleases (TALENs) and achieved on average 30% gene editing by electroporation of mRNA in NHP CD34+ cells. Erythroid differentiation of these cells in culture confirmed that HbF expression was increased in Bcllla-edited cells as compared to control cells. To determine if Bcl11a-edited HSCs could engraft and give rise to HbF-producing erythrocytes, we transplanted a NHP with autologous CD34+ electroporated with Bcl11a TALEN mRNA following conditioning by total body irradiation. Using next generation sequencing, we detected about 1 % disruption in vivo one week after transplant, to reach a set point of about 0.3% over the course of the experiment. We were able to track several clones that persisted at least 200 days post transplantation based on their mutation signatures, suggesting engraftment of Bcllla-modified cells. HbF production was monitored in this animal by flow cytometry analysis of peripheral blood and was compared with three transplanted controls and one untransplanted control. In all transplanted animals, we observed a rapid increase in the frequency of F cells, reaching 10% to 40%, and lasting for about 140 days. In contrast, F cell production in the untransplanted control remained constant and minimal (<0.5%). After returning to basal levels, we found significantly higher HbF levels (1-1.5%) in the animal transplanted with Bcl11a-edited cells as compared to all other transplanted animals. These findings were confirmed by real-time PCR analysis of hemoglobin transcripts, which showed a 5-to 10-fold increase in gamma to beta globin ratio in the animal transplanted with Bcl11a-edited cells as compared to all controls. We also initiated work demonstrating the targeted integration of the chemoselection cassette P140K/MGMT at the Bcl11a locus in NHP HSCs by co-delivery of TALEN mRNA with a donor template carried on an adeno-associated viral vector, offering the potential for in vivo selection of modified cells. In summary, our experiments establish the NHP as pre-clinical model to evaluate therapeutic gene editing strategies for the treatment of hemoglobinopathies.