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806 Persistence of CRISPR/Cas9-Edited Hematopoietic Stem and Progenitor Cells and Reactivation of Fetal Hemoglobin in Nonhuman Primates

Program: Oral and Poster Abstracts
Type: Oral
Session: 701. Experimental Transplantation: Basic Biology, Pre-Clinical Models: Hematopoietic Stem Cells and Alternate GVHD Tissues
Hematology Disease Topics & Pathways:
Diseases, sickle cell disease, Biological, Therapies, thalassemia, Hemoglobinopathies, Technology and Procedures, gene therapy, gene editing, Clinically relevant, transplantation, flow cytometry, stem cells
Monday, December 3, 2018: 3:00 PM
Grand Hall C (Manchester Grand Hyatt San Diego)

Olivier Humbert, PhD1*, Stefan Radtke, PhD1*, Ray R Carillo1*, Anai M Perez1*, Sowmya Somashekar Reddy1*, Lauren E Schefter1*, Christopher Lux, MD, PhD2, Sowmya Pattabhi, PhD3*, Olivier Negre, PhD4*, Ciaran M Lee, PhD5*, Jennifer Adair, PhD1,6*, Christopher Peterson, PhD1*, Gang Bao, PhD5*, David J. Rawlings, MD7*, Andrew M. Scharenberg, MD3* and Hans-Peter Kiem, MD, PhD8,9,10

1Fred Hutchinson Cancer Research Center, Seattle, WA
2Seattle Children's Research Institute, Seattle Children's Research Institute, Seattle, WA
3Seattle Children's Research Institute, Seattle Children's Hospital, Seattle, WA
4bluebird bio, inc., Cambridge, MA
5Bioengineering, Rice University, Houston, TX
6University of Washington, Seattle, WA
7Center for Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, WA
8Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA
9Department of Medicine, University of Washington, Seattle, WA
10Department of Pathology, University of Washington School of Medicine, Seattle, WA

Beta-thalassemia and sickle cell disease are monogenic disorders that are currently treated by allogeneic bone marrow (BM) transplantation although the challenges of finding a suitable matched-donor and the risk of graft vs host disease have limited the adoption of this otherwise curative treatment. A potentially promising approach for hemoglobinopathies aims to reactivate fetal hemoglobin (HbF) as a substitute for defective or absent adult hemoglobin by modifying the patient’s own hematopoietic stem and progenitor cells (HSPCs). Here, we evaluated CRISPR/Cas9-induced small deletions in HSPCs that are associated with hereditary persistence of fetal hemoglobin (HPFH) using our nonhuman primate (NHP) stem cell transplantation and gene therapy model.

The CRISPR/Cas9 nuclease platform was employed to recapitulate a natural genetic alteration identified in individuals with HPFH, consisting of a 13-nucleotide (nt) deletion in the gamma globin gene promoter. A first cohort of three rhesus macaques received 70-75% HPFH-edited BM-derived CD34+ HSPCs. All animals showed rapid hematopoietic recovery and peripheral blood (PB) editing levels stabilized at 12-30% for at least a year post transplantation (Figure 1). HbF production, determined by circulating F-cells, persisted at frequencies of 8-22% and correlated with in vivo PB editing. Robust engraftment of gene-edited HSPCs in the BM compartment was observed in all animals, with no measurable off-target activity or clonal expansion.

We have recently shown, that the CD34+CD90+CD45RA- phenotype is exclusively required for short- and long-term multilineage reconstitution, significantly reduces the target cell number for gene therapy/editing and is conserved between human and NHP hematopoietic cells (Radtke et al., STM, 2017). To explore this cell population further, we transplanted a second cohort of three animals by sort-purifying and solely editing this hematopoietic stem cell (HSC)-enriched CD34+CD90+CD45RA- phenotype, thus reducing the number of target cells by over 10-fold without impacting hematopoietic recovery, engraftment, or HbF reactivation. In vivo levels of gene-edited PB started at less than 5% because of the high number of co-infused unmodified progenitor cells, but rapidly increased to about 50% within 1 week (Figure 1) and stabilized at levels comparable to the CD34 cohort. This data supports our interpretation that CD34+CD90+CD45RA- cells are the main cell population relevant for long-term reconstitution and an excellent target for improved and efficient gene therapy/editing.

These results demonstrate robust engraftment and persistence of CD34+ HPSCs as well as HSC-enriched CD34+CD90+CD45RA- cells that have been CRISPR/Cas9-edited at the 13nt-HPFH site, with marked and stable HbF reactivation and no overt adverse effects in a NHP transplantation and gene therapy model. Most importantly, we validated our refined CD90+ target which reduces the need for editing reagents by 90% without compromising the gene modification and engraftment efficiencies. These are the first data in a clinically relevant large animal model to demonstrate the feasibility and clinical applicability of CRISPR/Cas9-mediated fetal hemoglobin reactivation. The successful targeting and engraftment of our HSC-enriched population should also have significant implications for gene therapy and editing of other genetic diseases.

Figure 1: Tracking of HPFH editing in transplanted animals. A) Editing efficiency was longitudinally determined by next generation sequencing of the targeted locus in PB white blood cells from 2 cohorts of transplanted rhesus animals. Frequency is represented as the proportion of all sequence reads containing an edited locus. B) Normalized frequency of the desired 13nt-HPFH deletion in the same animals as shown in A).

Disclosures: Negre: Bluebird Bio: Employment, Equity Ownership, Other: Salary. Adair: RX Partners: Honoraria; Miltenyi Biotec: Honoraria; Rocket Pharmaceuticals: Patents & Royalties: PCT/US2017/037967 and PCT/US2018/029983. Scharenberg: Generation Bio: Equity Ownership; Casebia Therapeutics: Employment; Alpine Immune Sciences: Equity Ownership. Kiem: Rocket Pharmaceuticals: Consultancy; Magenta: Consultancy; Homology Medicine: Consultancy.

*signifies non-member of ASH