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Gene editing following designer nuclease cleavage in the presence of a DNA donor template can revert mutations in disease-causing genes. For optimal benefit, reversion of the point mutation in HBB leading to sickle cell disease (SCD) would permit precise homology-directed repair (HDR) while concurrently limiting on-target non-homologous end joining (NHEJ)-based HBB disruption. In this study, we directly compared the relative efficiency of co-delivery of a novel CRISPR/Cas9 ribonucleoprotein targeting HBB in association with recombinant adeno-associated virus 6 (rAAV6) versus single-stranded oligodeoxynucleotides (ssODNs) to introduce the sickle mutation (GTC or GTG; encoding E6V) or a silent change (GAA; encoding E6optE) in human CD34+ mobilized peripheral blood stem cells (mPBSCs) derived from healthy donors. In vitro, rAAV6 outperformed ssODN donor template delivery and mediated greater HDR correction, leading to both higher HDR rates and a higher HDR:NHEJ ratio. In contrast, at 12–14 weeks post-transplant into recipient, immunodeficient, NOD, B6, SCID Il2rγ−/− Kit(W41/W41) (NBSGW) mice, a ∼6-fold higher proportion of ssODN-modified cells persisted in vivo compared to recipients of rAAV6-modified mPBSCs. Together, our findings highlight that methodology for donor template delivery markedly impacts long-term persistence of HBB gene-modified mPBSCs, and they suggest that the ssODN platform is likely to be most amenable to direct clinical translation. Keywords: sickle cell disease, gene editing, rAAV6, ssODN, homology-directed repair, Crispr/Cas9, hemoglobin disorders, NHEJ versus HDR, in vivo engraftment, NBSGW41 mice, CD34, hematopoietic stem cells, stem cell cures

Elements within the γ-hemoglobin promoters (HBG1 and HBG2) function to bind transcription complexes that mediate repression of fetal hemoglobin expression. Sickle cell disease (SCD) subjects with a 13-bp deletion in the HBG1 promoter exhibit a clinically favorable hereditary persistence of fetal hemoglobin (HPFH) phenotype. We developed TALENs targeting the homologous HBG promoters to de-repress fetal hemoglobin. Transfection of human CD34+ cells with TALEN mRNA resulted in indel generation in HBG1 (43%) and HBG2 (74%) including the 13-bp HPFH deletion (∼6%). Erythroid differentiation of edited cells revealed a 4.6-fold increase in γ-hemoglobin expression as detected by HPLC. Assessment of TALEN-edited CD34+ cells in vivo in a humanized mouse model demonstrated sustained presence of indels in hematopoietic cells up to 24 weeks. Indel rates remained unchanged following secondary transplantation consistent with editing of long-term repopulating stem cells (LT-HSCs). Human γ-hemoglobin expressing F cells were detected by flow cytometry approximately 50% more frequently in edited animals compared to mock. Together, these findings demonstrate that TALEN-mediated indel generation in the γ-hemoglobin promoter leads to high levels of fetal hemoglobin expression in vitro and in vivo, suggesting that this approach can provide therapeutic benefit in patients with SCD or β-thalassemia., Graphical Abstract

Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies. Here, we evaluated the therapeutic potential of hematopoietic stem and progenitor cells (HSPCs) edited with the CRISPR/Cas9 nuclease platform to recapitulate naturally occurring mutations identified in individuals who express increased amounts of HbF, a condition known as hereditary persistence of HbF. CRISPR/Cas9 treatment and transplantation of HSPCs purified on the basis of surface expression of the CD34 receptor in a nonhuman primate (NHP) autologous transplantation model resulted in up to 30% engraftment of gene-edited cells for >1 year. Edited cells effectively and stably reactivated HbF, as evidenced by up to 18% HbF-expressing erythrocytes in peripheral blood. Similar results were obtained by editing highly enriched stem cells, defined by the markers CD34(+)CD90(+)CD45RA(–), allowing for a 10-fold reduction in the number of transplanted target cells, thus circumventing issues associated with scale-up and considerably reducing the need for editing reagents. The frequency of engrafted, gene-edited cells persisting in vivo using this approach may be sufficient to ameliorate the phenotype for a number of genetic diseases.

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). Figure. Figure. 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.

The mammalian yolk sac is known to play a prominent role in emergence of the hematopoietic system. The extent of this contribution has been a subject of debate in recent years largely due to effects of the early circulation that obscures the site of origin of hematopoietic stem and progenitor cells. This review discusses the limitations of some of the standard assays currently employed to study hematopoietic stem and progenitor cell emergence and highlights several recently reported novel methods that address this problem from new perspectives. Two methods directly alter the circulation by either preventing it from occurring in the first place or by removing vascular connections between the embryo and the yolk sac. Other approaches have altered the ability of hematopoietic cells to interact with their environment, resulting in the lack of migration or an inability to bind to potential hematopoietic niches. A third set of experiments utilize lineage tracing techniques to follow the migration of early progenitors once they enter the circulation. Taken together, these novel methods provide new evidence for the contribution of yolk sac hematopoietic stem and progenitor cells to the adult hematopoietic system.

The relative contribution of yolk sac (YS)–derived cells to the circulating definitive hematopoietic progenitor cell (HPC) pool that seeds the fetal liver remains controversial due to the presence of systemic circulation and the onset of hematopoiesis within the embryo proper (EP) before liver seeding. Ncx1−/− embryos fail to initiate a heartbeat on embryonic day (E) 8.25, but continue to develop through E10. We detected normal numbers of primitive erythroid progenitors in Ncx1−/− versus wild type (WT) YS, but primitive erythroblasts did not circulate in the Ncx1−/− EP. While there was no significant difference in the number of definitive HPCs in Ncx1−/− versus WT YS through E9.5, the Ncx1−/− EP was nearly devoid of HPCs. Thus, primitive erythroblasts and essentially all definitive HPCs destined to initially seed the fetal liver after E9.5 are generated in the YS between E7.0-E9.5 and are redistributed into the EP via the systemic circulation.

Hemoglobinopathies are common and morbid inherited conditions caused by mutations in hemoglobin genes that generate pathologic globin molecules or reduced expression of wild type hemoglobin. A subset of individuals with hemoglobinopathy-associated beta globin mutations have a mild clinical phenotype and are found to carry additional disease modifying mutations including small deletions or point mutations that induce fetal hemoglobin expression. This condition is referred to as hereditary persistence of fetal hemoglobin. Recent progress in the arenas of gene editing and hematopoietic stem cell (HSC) expansion has raised the possibility of treating hemoglobinopathies through direct modification of genes in HSCs to increase the expression of fetal hemoglobin in the erythroid lineage. To date, most attempts to induce fetal hemoglobin have targeted the expression of transcription factors implicated in regulation of fetal hemoglobin production, such as Bcl11a, an approach which could lead to unintended and potentially deleterious effects on cellular phenotype. Here, we are evaluating the application of mRNA transfection of TALENs to directly modify transcription factor binding sites within the hemoglobin locus in HSCs to achieve efficient, stable expression of fetal hemoglobin. We have designed TALENs to induce small deletions in the beta hemoglobin promoter at the binding site of KLF1, postulated to play a role in localizing the active chromatin hub to and preferentially expressing adult beta hemoglobin, as well as other globin locus target regions implicated in the regulation of fetal hemoglobin production. By combining TALEN-mediated gene modification with novel HSC expansion approaches involving the recently described small molecule UM729, we hope to realize both efficient HSC modification and expansion with a sufficient level of fetal hemoglobin production in erythrocytes to achieve a functional cure for a wide range of beta hemoglobinopathies.

The placenta was recently unveiled as an important hematopoietic organ, harboring a large pool of HSCs during midgestation. Yet, it has not been defined whether the placenta can generate HSCs de novo. By using the Runx1-LacZ and Ncx1 knockout mouse models we show that the placenta is a site of HSC generation and identify the cellular niches in which placental HSCs reside. Runx1 is essential for the emergence of definitive HSCs and remains expressed in HSCs throughout fetal development and adult life. Analysis of Runx1LacZ/+ and Runx1LacZ/LacZ placental sections nominated the large vessels of the placenta and the chorioallantoic mesenchyme as putative sites of HSC origin. Once formed, LacZ+ candidate HSCs convened in the labyrinth vessels. Co-staining of Runx1LacZ/+ placentas with an antibody specific for phosphorylated Ser 10 at histone 3, a marker of mitosis, showed mitotically active definitive hematopoietic cells in the labyrinth vessels, suggesting that the labyrinth is a microenvironmental niche capable of stimulating HSC expansion. In wild-type placentas, CD41+ nascent hematopoietic cells were found in the same vascular sites as in the Runx1-LacZ placentas but never in the mesenchyme. Instead, placental stroma was populated by F4/80+CD45+/−CD41- macrophages, suggesting that the placenta harbors two distinct hematopoietic lineages that are supported by different microenvironments. To verify that the CD41+ nascent HSCs were generated de novo in the placenta, we analyzed Ncx1−/− embryos, which lack heartbeat due to lack of the sodium-calcium exchange pump 1. In the absence of circulation, trafficking of hematopoietic cells between tissues is abolished. Strikingly, CD41+ HSCs emerge in the large vessels of the placenta in Ncx1−/− mutants. In some sections CD41+ cells formed clusters that were still connected to the vessels of the placenta and umbilical cord. These findings imply that formation of HSCs extends to a much larger anatomical area than was previously thought, including the placenta. Importantly, the placentas in both Ncx1−/− and control embryos (E8.5–9.5) generated mixed hematopoietic outgrowth including definitive progenitors in OP-9 co-culture, as verified by expression of c-kit, CD41 and CD45. When the differentiation of the definitive progenitors was assessed on methylcellulose, Ncx1−/− tissues demonstrated similar potential as Ncx1+/− hematopoietic organs (yolk sac, aorta gonad mesonephros (AGM) and placenta), yielding erythroid, myeloid and mixed colonies and B220+ lymphoid cells. These studies reveal that definitive hematopoietic cells with both myeloerythroid and lymphoid potential are generated de novo within the placental vasculature. Furthermore, the placental labyrinth provides a unique hematopoietic niche that is conducive for proliferation of hematopoietic cells and, unlike the AGM or the yolk sac, serves as a supportive niche for a large pool of HSCs prior to liver colonization.

18 Circulation Plays an Essential Role in Distributing Hematopoietic Progenitors from the Yolk Sac to the Embryo Proper; Lessons from the Ncx1-Null Mouse.

The yolk sac is the lone site of primitive hematopoiesis. The role of the yolk sac in generating definitive hematopoietic progenitors, however, has remained controversial. One complicating factor preventing an accurate investigation of this subject has been the onset of early circulation which alters the localization of hematopoietic progenitors. An Ncx1 knockout mouse (which fails to initiate a heartbeat) is used here to investigate the temporal and spatial distribution of definitive hematopoietic progenitor cells (HPCs: adult type BFU-E, CFU-GM, CFU-GEMM) in an environment lacking circulation. Embryos were harvested from timed pregnancies from Ncx1 heterozygote crosses beginning at the onset of circulation 8.5 days post conception (E8.5) and ending 36 hours later (E10). Developmental age was determined by somite pair number. All embryos were carefully separated from their yolk sacs and both samples were digested and plated in methylcellulose using a previously published definitive hematopoietic progenitor colony assay. Colonies were counted at seven days and then collected for genotyping. In embryos of all genotypes at E8.5, definitive HPCs were enriched more than 28 fold in the yolk sac compared to the embryo proper (EP), but following redistribution by a functional circulation in E10 wild type and heterozygous embryos, the ratio drops to a 3 fold enrichment in the yolk sac (similar to the 3–5 fold enrichment previously reported at this age; KM & JP, Blood 2003). Ncx1 null embryos lacking circulation produced few HPCs in the EP and never redistribute the HPCs as late as E10 resulting in a 72 fold enrichment in the yolk sac even at E10. Whole mount ζ-hemoglobin mRNA staining was carried out to visualize the distribution of blood cells in the yolk sac and embryo proper in the presence and absence of circulation. The staining pattern in null embryos confirms that cells from the yolk sac blood band remain in the yolk sac and are not found in the EP. Our findings support a model in which the primitive and definitive hematopoietic progenitors are generated in the yolk sac and are only redistributed to the EP upon the onset of circulation. Not only is the yolk sac an important source of definitive HPCs, it is the developing embryo’s primary source through E10.

SummaryThe mouse placenta was unveiled as an important reservoir for hematopoietic stem cells (HSCs), yet the origin of placental HSCs was unknown. By tracking developing HSCs by expression of Runx1-lacZ and CD41, we have found that HSCs emerge in large vessels in the placenta. Analysis of Ncx1−/− embryos, which lack a heartbeat, verified that HSC development is initiated in the placental vasculature independent of blood flow. However, fewer CD41+ hematopoietic cells were found in Ncx1−/− placentas than in controls, implying that some HSCs/progenitors colonize the placenta via circulation and/or HSC emergence is compromised without blood flow. Importantly, placentas from Ncx1−/− embryos possessed equal potential to generate myelo-erythroid and B and T lymphoid cells upon explant culture, verifying intact multilineage hematopoietic potential, characteristic of developing HSCs. These data suggest that, in addition to providing a niche for a large pool of HSCs prior to liver colonization, the placenta is a true site of HSC generation.