ALBERT

Description: Ablation of Bcl11A could be a viable approach for the treatment of β-hemoglobinopathies such as β-thalassemia and sickle cell disease (SCD), since patients with Bcl11A haploinsufficiency have persistently high levels of fetal hemoglobin (HbF) (up to 30%), which are associated with development of minimal to no disease symptoms. Genome editing by engineered zinc-finger nucleases that target either the exon 2 (exon ZFN) or the GATA motif of the erythroid specific enhancer (enhancer ZFN) of Bcl11A has been shown to increase HbF level in erythroid progeny from mobilized peripheral hematopoietic stem and progenitor cells (PB-CD34+ HSPCs). However, peripheral mobilization of CD34+ cells is associated with high risk and currently is not an option for SCD patients. Therefore, we investigated the efficacy of genome editing of Bcl11A in bone marrow derived CD34+ cells (BM-CD34+ HSPCs). We first established a clinically compatible large-scale process to isolate CD34+ HSPCs from human bone marrow aspirates and to transiently express the ZFN protein by mRNA electroporation. The CD34+ isolation process resulted in ~ 95% pure CD34+ cells with greater than 90% viability. Both the exon and the enhancer ZFN drove 50-60% Bcl11A gene editing, resulting in a robust elevation of HbF in the erythroid progeny. Notably, the BM-CD34+ HSPCs were found to contain a small population (10 to 25%) of CD34+CD19+ pro-B cells that were refractory to ZFN transfection under our current electroporation condition. Since CD34+CD19+ pro-B cells are not expected to contribute to reconstituting the hematopoietic system other than B-cell lineage, the Bcl11A editing efficiency in the multipotent BM-CD34+ HSPC could be even higher. The engraftment abilities of Bcl11A edited BM-CD34+ cells were then investigated in an immunodeficient NOD/scid/gamma (NSG) mouse model. At a dose of 1 million cells per mouse, treatment with either the exon ZFN or the enhancer ZFN did not detectably impact engraftment or multi-lineage reconstitution compared with untreated cells. However, Bcl11A marking in engrafted human cells was found to be markedly higher in the mice treated by the enhancer ZFN than that by the exon ZFN. The exon ZFN resulted in a strong bias towards in-frame mutations across multi-lineages with the strongest effect observed in the B-cell lineage, suggesting that a threshold level of Bcl11A is required for efficient hematopoietic reconstitution and that cells fully lacking it due to disruption of the coding sequence are at a disadvantage. In contrast, the enhancer ZFN resulted in comparable Bcl11A marking across all lineages with no apparent selection for cells with a functional GATA sequence. Collectively, these data indicate that genome editing of the erythroid specific enhancer of Bcl11A in BM-CD34+ promotes HbF reactivation in the erythroid progeny while maintaining the engraftment and multi-lineage repopulating activities of edited BM-CD34+ HSPCs, which supports further clinical development of this approach for the treatment of SCD. Disclosures Tan: Biogen: Employment, Equity Ownership. Chang:Biogen: Employment, Equity Ownership. Smith:Biogen: Employment, Equity Ownership. Chen:Biogen: Employment, Equity Ownership. Sullivan:Biogen: Employment, Equity Ownership. Zhou:Biogen: Employment, Equity Ownership. Reik:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Urnov:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Rebar:Sangamo BioSciences: Employment. Danos:Biogen: Employment, Equity Ownership. Jiang:Biogen: Employment, Equity Ownership.

Description: Sickle cell disease (SCD) is one of the most common inherited blood disorders and is caused by a mutation at the adult beta globin gene resulting in substitution of valine for glutamic acid at position 6 in the encoded protein. While SCD can be cured by hematopoietic stem cell transplant (HSCT), complete donor chimerism is not required to achieve clinical benefits. Stable mixed chimerism of 10-15% in bone marrow or peripheral blood nucleated cells with 〉70% donor-derived RBCs has been reported to achieve transfusion independence and a symptom-free state in a SCD patient. It has also been proposed that SCD can be treated by reactivating developmentally silenced fetal gamma globin to form fetal hemoglobin (alpha2gamma2, HbF), which inhibits polymerization of HbS. The effect of HbF is predicted to be maximal when HbF content per cell exceeds 10 pg (~30% of total Hb). Furthermore, pathology is prevented when protective F cells (〉30% HbF per cell) constitute 〉70% of total RBCs. We hypothesize that in a gene therapy setting, if 〉15% of SCD patients' autologous HSCs are programmed to produce protective F cells during erythropoiesis, it will translate into 〉70% protective F cells in circulation and provide significant alleviation of clinical symptoms. Genome wide association studies have identified BCL11A as a major modifier of HbF levels. Subsequent studies have shown that BCL11A plays a critical role in the fetal to adult globin developmental switch and in repressing fetal globin expression in adult erythroid cells. Conditional inactivation of BCL11A in adult erythroid cells leads to high levels of pan-cellular fetal globin expression and correction of hematologic and pathologic defects in a humanized SCD mouse model. Previously, we have reported that zinc finger nucleases (ZFNs) targeting BCL11A either in the coding region or the GATAA motif in the erythroid-specific enhancer efficiently disrupt the BCL11A locus in human primary CD34+ cells following electroporation of ZFN-encoding mRNA. Elevated fetal globin expression in bulk erythroid cultures was observed following disruption. To determine what percentage of HSPCs have been modified and whether the HbF/F cell content has reached the hypothesized therapeutic level, we analyzed erythroid cells clonally derived from ZFN-transfected CD34+ cells. Genotype of each clonal culture was determined by deep sequencing and globin production was analyzed by a highly sensitive UPLC method. We found that up to 80% of the BFU-Es had both BCL11A alleles edited, half of which had KO/KO alleles (either out of frame mutations for coding region or elimination of the GATAA motif in the enhancer). BCL11A coding KO/KO cells expressed on average 79.1% ± 12.2% fetal globin (Mean ± SD) whereas GATAA motif enhancer region KO/KO cells expressed approximately 48.4% ± 14.1% fetal globin, in comparison with 14.5% ± 9.6% in WT/WT cells . These levels of fetal globin should be sufficiently high to confer protection against HbS polymerization in sickle cells. WT/KO cells in both coding and enhancer editing experiments showed an intermediate phenotype with fetal globin averaging 26.9%± 9.9% and 25.79% ± 12.6%, respectively. Interestingly, when background (WT/WT) fetal globin level was subtracted, the fetal globin levels in WT/KO cells are comparable to those observed in patients with BCL11A haploinsufficiency, which average 14.6%± 10.3%. Together, our data demonstrate that genome editing of BCL11A using highly efficient ZFNs can lead to clinically relevant levels of fetal globin expression in KO/KO erythroid cells. If the frequency of KO/KO BFU-Es we observed in vitro reflects the frequency of KO/KO HSCs in bone marrow after autologous transplantation, genome editing of BCL11A has the potential to provide significant clinical benefit for patients with SCD. Disclosures Chang: Biogen: Employment, Equity Ownership. Sullivan:Biogen: Employment, Equity Ownership. Liu:Biogen: Employment, Equity Ownership. Yang:Biogen: Employment, Equity Ownership. Sun:Biogen: Employment, Equity Ownership. Vieira:Biogen: Employment, Equity Ownership. Zhang:Biogen: Employment. Hong:Biogen: Employment, Equity Ownership. Chen:Biogen: Employment, Equity Ownership. Smith:Biogen: Employment, Equity Ownership. Tan:Biogen: Employment, Equity Ownership. Reik:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Urnov:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Rebar:Sangamo BioSciences: Employment. Danos:Biogen: Employment, Equity Ownership. Jiang:Biogen: Employment, Equity Ownership.

Description: Inappropriately low reticulocytosis may exacerbate malarial anemia, but the under-lying mechanism is not clear. In this study, naive and infected mice were treated with recombinant murine erythropoietin (EPO), and the upstream events of erythropoiesis affected by blood-stage Plasmodium chabaudi AS were investigated. Malaria infection, with or without EPO treatment, led to a suboptimal increase in TER119+ erythroblasts compared with EPO-treated naive mice. Furthermore, a lower percentage of TER119+ erythroblasts in infected mice were undergoing terminal differentiation to become mature hemoglobin-producing erythroblasts. The impaired maturation of erythroblasts during infection was associated with a shift in the transferrin receptor (CD71) expression from the TER119+ population to B220+ population. Moreover, the suboptimal increase in TER119+ erythroblasts during infection coincided with a blunted proliferative response by splenocytes to EPO stimulation in vitro, although a high frequency of these splenocytes expressed EPO receptor (EPOR). Taken together, these data suggest that during malaria, EPO-induced proliferation of early EPOR-positive erythroid progenitors is suppressed, which may lead to a suboptimal generation of TER119+ erythroblasts. The shift in CD71 expression may result in impaired terminal maturation of these erythroblasts. Thus, inadequate reticulocytosis during malaria is associated with suppressed proliferation, differentiation, and maturation of erythroid precursors.

Description: Beta hemoglobinopathies resulting from dysfunctional or deficient adult beta-globin expression are some of the most prevalent inherited blood disorders in the world. Upregulation of developmentally-silenced fetal gamma-globin would replace adult beta-globin to ameliorate disease symptoms. One of the approaches to reactivate fetal globin expression in erythroid cells is through gene editing by zinc finger or CRISPR-Cas9 nucleases to disrupt the expression of a transcription factor BCL11A, which mediates fetal globin silencing. As BCL11A-deficiency leads to hematopoietic stem cells (HSCs) defects, the current editing approaches target the BCL11A erythroid-enhancer region located in intron 2 of the BCL11A gene to selectively reduce BCL11A expression in erythroid cells. Instead of targeting BCL11A, we sought to identify novel cis-regulatory elements at the beta-globin locus for targeted gene editing to achieve fetal globin reactivation. From a lenti-CRISPR mediated saturated mutagenesis screen covering the beta-globin locus using Human Umbilical Cord Blood-Derived Erythroid Progenitor (HUDEP)-2 cells, multiple fetal hemoglobin (HbF)-inducing genomic domains were identified. Most of the hits were concentrated at the gamma-globin (HBG1/2) promoters, clustered at known hereditary persistence of fetal hemoglobin (HPFH) mutation hotspots. In-depth genotype to phenotype analysis further defined the indels responsible for HbF induction in these subdomains. We interrogated multiple families of nucleases and guide RNA (gRNA) combinations with or without single-stranded oligodeoxynucleotides (ssODN) to guide editing outcome. gRNAs were selected based on their HbF induction potential (up to 40%) when introduced into mobilized peripheral blood (mPB) CD34+ hematopoietic stem and progenitor cells (HSPCs) as ribonucleoprotein (RNP) complexes. HSPCs transfected with RNPs targeting either the BCL11A erythroid-enhancer or the HBG1/2 proximal regions were then injected into NBSGW mice to study the editing in SCID-repopulating cells (SRC) and their multilineage reconstitution potential. All groups achieved high levels of human chimerism (〉70% hCD45+/hCD45+mCD45) and comparable monocytes, granulocytes, B lymphocytes, and hCD34+ HSPCs reconstitution. However, BCL11A-edited cells showed selective reduction in erythroid lineage (CD235a+) output, up to 4-fold lower than untreated or HBG1/2 promoter-edited HSPCs. Sequencing analysis from lineage-specific sorted cells further revealed reduced editing levels at BCL11A erythroid-specific enhancer in the erythroid compartment compared to unfractionated bone marrow (BM) or other human lineages (70% in erythroid vs. 90% in all other lineages). Furthermore, the nonproductive fraction of indels that did not disrupt the BCL11A GATAA motif was significantly enriched in erythroid cells (22% in erythroid vs. 8% in other lineages). Ex vivo erythroid cultures suggests BCL11A erythroid-enhancer editing may lead to slightly increased apoptosis during erythroid differentiation. In contrast, HBG1/2 promoter-edited cells had similar editing levels and indel patterns across all lineages with no significant lineage skewing. When chimeric BM from HBG1/2 promoter-edited groups were cultured in erythroid conditions, ex vivo-derived erythroid cells had significantly elevated levels of HbF compared to controls. When CD235a+ cells were sorted without further culture from chimeric BM of mice engrafted with HBG1/2 promoter-edited cells, significantly increased levels of HbF were detected by UPLC (up to 30%) compared to controls (~6%). Thus, long-term HSCs have been edited productively at the HBG1/2 promoters. These cells were able to generate erythroid progenitors that harbor HbF-inducing indels, which in turn, gave rise to erythroid cells in vivo with a clinically-relevant levels of HbF in a xenotransplantation model. Together, our data suggest that BCL11A-edited cells have an erythroid differentiation defect or survival disadvantage in NBSGW mouse model that warrants further investigation. In contrast, editing of the HBG1/2 promoters in mPB CD34+ cells achieved sustained HbF expression in erythroid lineage while maintaining multilineage differentiation potential. Targeting of the HBG1/2 promoters in HSPCs may be an attractive strategy for the development of potential gene editing medicines for beta hemoglobinopathies. Disclosures Chang: Editas Medicine Inc.: Employment, Equity Ownership. Sanchez:Editas Medicine Inc.: Employment, Equity Ownership. Heath:Editas Medicine Inc.: Employment, Equity Ownership. deDreuzy:Editas Medicine Inc.: Employment, Equity Ownership. Haskett:Editas Medicine Inc.: Employment, Equity Ownership. Vogelaar:Editas Medicine Inc.: Employment. Gogi:Editas Medicine Inc.: Employment, Equity Ownership. Da Silva:Editas Medicine Inc.: Employment, Equity Ownership. Wang:Editas Medicine Inc.: Employment, Equity Ownership. Sadowski:Editas Medicine Inc.: Employment, Equity Ownership. Gotta:Editas Medicine Inc.: Employment, Equity Ownership. Siwak:Editas Medicine Inc.: Employment, Equity Ownership. Viswanathan:Editas Medicine Inc.: Employment, Equity Ownership. Loveluck:Editas Medicine Inc.: Employment, Equity Ownership. Chao:Editas Medicine Inc.: Employment, Equity Ownership. Tillotson:Editas Medicine Inc.: Employment, Equity Ownership. Chalishazar:Editas Medicine Inc.: Employment, Equity Ownership. Dass:Editas Medicine Inc.: Employment, Equity Ownership. Ta:Editas Medicine Inc.: Employment, Equity Ownership. Brennan:Editas Medicine Inc.: Employment, Equity Ownership. Tabbaa:Editas Medicine Inc.: Employment, Equity Ownership. Marco:Editas Medicine Inc.: Employment, Equity Ownership. Zuris:Editas Medicine Inc.: Employment, Equity Ownership. Reyon:Editas Medicine Inc.: Employment, Equity Ownership. Isik:Editas Medicine Inc.: Employment, Equity Ownership. Friedland:Editas Medicine Inc.: Employment, Equity Ownership. Ta:Editas Medicine Inc.: Employment, Equity Ownership. Harbinski:Editas Medicine Inc.: Employment, Equity Ownership. Giannoukos:Editas Medicine Inc.: Employment, Equity Ownership. Teixeira:Editas Medicine Inc.: Employment, Equity Ownership. Wilson:Editas Medicine Inc.: Employment, Equity Ownership. Albright:Editas Medicine Inc.: Employment, Equity Ownership. Jiang:Editas Medicine Inc.: Employment, Equity Ownership.

Description: Human embryonic stem cells are a promising tool to study events associated with the earliest ontogenetic stages of hematopoiesis. We describe the generation of erythroid cells from hES (H1) by subsequent processing of cells present at early and late stages of embryoid body (EB) differentiation. Kinetics of hematopoietic marker emergence suggest that CD45+ hematopoiesis peaks at late D14EB differentiation stages, although low-level CD45- erythroid differentiation can be seen before that stage. By morphologic criteria, hES-derived erythroid cells were of definitive type, but these cells both at mRNA and protein levels coexpressed high levels of embryonic (ϵ) and fetal (γ) globins, with little or no adult globin (β). This globin expression pattern was not altered by the presence or absence of fetal bovine serum, vascular endothelial growth factor, Flt3-L, or coculture with OP-9 during erythroid differentiation and was not culture time dependent. The coexpression of both embryonic and fetal globins by definitive-type erythroid cells does not faithfully mimic either yolk sac embryonic or their fetal liver counterparts. Nevertheless, the high frequency of erythroid cells coexpressing embryonic and fetal globin generated from embryonic stem cells can serve as an invaluable tool to further explore molecular mechanisms.

Description: Abstract 1027 Red blood cells (RBC) survive shear forces in the microvasculature because trans-membrane complexes embedded in the lipid bilayer attach their membrane to the cytoskeleton assuring its flexibility. The expression of clinically relevant red blood cell antigens present on these complexes is determined by genetic polymorphisms and their developmental regulation. Therefore, flow cytometry studies of blood group antigens may provide insights both on potential immunogenicity and on membrane structure of ex-vivo generated EBs. Blood group antigen profiles of EBs expanded ex vivo from one AB (three experiments), three CB, the H1 hESC line and one iPS line derived from mononuclear cells from a healthy donor were compared by flow cytometry using commercially available antibodies recognizing antigens present on proteins in the 4.1R [Duffy (Fya and Fy3), Kell (Kell prot, K/k, Kpa/Kpb, Jsb) and glycophorin C (GPC, Ge2)] and ankyrin R [glycophorin A (GPA, CD235a, M and EnaFS) RhAG and band 3 (Wrb)] complexes and on other important membrane proteins [glycophorin B (GPB, s and U), urea transporter (Kidd, Jk3), the complement receptor (CD35) and inhibitors of complement-mediated lysis (CD55 and CD59)]. Controls included DNA genotyping (CB, AB and H1-hESC) (HEA-Bead Chip, Immunocor, Norcross, GA) and immunophenotyping of blood red cells from the same AB and CB. Antigen expression similar to that observed on in vivo generated RBC was considered normal. EBs were generated from AB and CB at day 10 in HEMAser cultures whereas EBs from hESC and iPS were derived using previously optimized protocols. The maturation state was determined by morphological analyses and CD36/CD235a profiles. Irrespective of the stem cell source, the immunophenotype of ex-vivo expanded EBs was consistent with that predicted by genotyping. However, source specific differences in the magnitude of antigen expression and in the changes with maturation were observed (see Figure). Immature EBs from AB expressed normal levels of the antigens present on both the 4.1R (Duffy, Kell, GPC) and ankyrin R (GPA, M/N, EnaFS, RhAG and band 3) complexes. With maturation, expression of 4.1R-associated antigens remained normal while that of ankyrin R associated antigens varied (M decreased and RhAG increased). EBs from CB expressed normal levels of antigens present on the ankyrin R complex and of some of those present on the 4.1R complex (Duffy, Kell protein and GPA). However, expression of epitopes on Kell protein varied with some antigens expressed at normal levels (k and Jsb) and others (Kpa/Kpb) at levels 2x greater than normal. With maturation, CB-derived EBs maintained normal levels of ankyrin R associated antigens while those associated with complex 4.1R became barely detectable. EB from hESC expressed unbalanced levels of proteins associated with both ankyrin R (2x levels of GPA and barely detectable levels of RhAG) and 4.1R [3x levels of Duffy and 2x levels of Jsb (Kell) with normal levels of k and Kpb (Kell) antigens] complexes. The variegation in expression of different epitopes on the same protein observed with CB- and hESC-derived EBs likely reflect altered structural conformation of the complexes rather than differences in protein concentration on the membrane. EBs from iPS, as those from AB, expressed normal levels of antigens present on Ankyrin R and 4.1R complexes which increased with maturation. Irrespective of stem cell sources, EBs expressed normal levels of GPB and Kidd. EBs from AB expressed normal levels of the complement regulatory proteins tested which in the case of CD59 CD59 decreased with maturation. EBs from CB expressed normal levels of CD35 and CD59 but 2x levels of CD55 with expression of CD35 and CD55 decreasing with maturation. EBs from iPS expressed 2x levels of CD35 and CD55 and expression of these antigens was not affected by maturation. The observation that blood group antigenic profiles of ex-vivo generated EBs are consistent with those predicted by DNA-genotyping suggests that these cells are unlikely to be immunogenic for known epitopes. However, the antigen profiles of ankyrin R and 4.1R complexes were normal only for AB and iPS-derived EBs raising the possibility that antigenic deviations seen in EBs derived from CB and hESC may have immunologic or functional consequences in vivo. Disclosures: No relevant conflicts of interest to declare.

Description: We describe here a fundamentally novel way to develop a disease therapeutic: combining genome-wide association studies (GWAS) with targeted genome editing to create, in a clinically compliant setting, a disease-ameliorating genotype in the patient's own cells. In β-thalassemia, elevated levels of fetal hemoglobin (HbF) lessen or eliminate disease symptoms, thus making a reversal of HbF silencing in patients an appealing therapeutic strategy. Loss-of-function variants in the erythroid-specific enhancer of the fetal globin repressor, BCL11A, elevate HbF; rare individuals carrying a monoallelic knockout of BCL11A exhibit no known hematologic abnormality and up to 30% circulating HbF. We previously reported de novo knockout of BCL11A using targeted genome editing with engineered zinc finger nucleases (ZFNs) yielding up to 40% HbF in erythroid progeny of edited human CD34 cells in vitro. We now find that the targeted ablation of a single, specific GATAA motif in the BCL11A intronic enhancer does not affect in vitro erythroid differentiation, but reproducibly (n=6) activates fetal globin transcription in erythroid progeny of modified CD34 cells; importantly, at similar levels of on-target marking in CD34+ cells, these effects on fetal globin mRNA are comparable to those resulting from ZFN-driven coding knockout of BCL11A itself. We demonstrate reproducible (n=8), high-efficiency (up to 82%; average, 69%) ZFN-driven marking at the enhancer in peripheral blood mobilized human CD34 cells at clinical production scale (〉1e8 cells) in a GMP-compliant setting for which we use a clinical-grade electroporation device to deliver nuclease-encoding transcribed mRNA ex vivo. Using erythroid colony assay genotyping we find that up to 70% of the cells in the resulting population are biallelically modified at the target locus, while ~10% remain wild-type, and find comparably high levels of marking in research-scale preparations of CD34 cells from patients with β-thalassemia. We observe robust long-term (18-24 week) engraftment and multilineage differentiation of genome-edited cells in immunodeficient mice, similar to control cells, and equivalent modification at the targeted enhancer locus at all timepoints in both differentiated (CD19+, CD3+, CD33+) and more primitive progenitor (CD34+CD38low) cells of human origin purified from bone marrow of long-term-engrafted animals. Our findings support clinical development of enhancer editing as a treatment of the β hemoglobinopathies with autologous hematopoietic stem cell transplant. Disclosures Urnov: Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Reik:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Vierstra:University of Washington: Patents & Royalties: Patent applications have been filed based on this work. Chang:Biogen: Employment, Equity Ownership. Zhou:Sangamo BioSciences: Employment, Equity Ownership. Mich:Sangamo BioSciences: Employment, Equity Ownership. Adrian:Cellerant Therapeutics: Equity Ownership; Sangamo BioSciences: Employment, Equity Ownership. Fox:Sangamo BioSciences: Employment, Equity Ownership. Tan:Biogen: Employment, Equity Ownership. Craig:Sangamo BioSciences: Employment, Equity Ownership. Rebar:Sangamo BioSciences: Employment. Stamatoyannopoulos:University of Washington: Patents & Royalties: Patent applications have been filed based on this work.. Jiang:Biogen: Employment, Equity Ownership.

Description: Abstract 2535 Poster Board II-512 We used a high throughput approach to determine the chromatin profiles of the human β and α globin loci and their upstream and downstream regions in human undifferentiated ES cells, ES cell-derived erythroid cells, human fetal and adult origin erythroid cells and in primary cells and cell lines of endo-meso and ectodermal origins. All DNase I hypersensitive sites of the b-locus were absent in undifferentiated human ES cells except for HS2 of the b-globin locus control region. The chromatin profiles of the β and α globin loci of ES cell-derived erythroid cells were identical to those of fetal liver erythroid cells except that the hypersensitive site of the embryonic globin gene was more prominent. DNase I hypersensitive site 2 of the b-globin LCR, a potent enhancer, was present in all the cell lines and primary lineages we studied, providing direct evidence that it is ubiquitous. Several new erythroid specific DHSs were detected upstream of 5′HS7 of the β-LCR, raising the possibility that they play a role in the regulation of the β globin locus. The region downstream to 3′HS1 was depleted of DHSs except for the previously identified DHS mapping near the breakpoint of HPFH 1. Since DHSs are absent near the breakpoints of deletional HPFHs and db thalassemias and since enhancers are typically DHS positive, our results argue against the hypothesis of imported enhancers in the pathogenesis of deletional HPFH and db thalassemia mutants. All the previously identified erythroid specific DHSs of the α globin locus were absent in human ES cells. The α globin locus of ES cells, however, displayed three very prominent DHSs, which were located almost symmetrically about 40 Kb apart from each other and they were constitutively formed in all the lineages and cell lines we have studied; the 3′ and 5′ DHSs carried CTCF sites by ChIP-Seq assay raising the possibility that they mark the sites of chromatin insulators. Overall these results demonstrate the power of the new high throughput chromatin profiling approaches and their ability to uncover features of chromatin that may be of regulatory relevance. Disclosures: No relevant conflicts of interest to declare.

Description: Beta-thalassemia (β-thal) and sickle cell disease (SCD) are monogenic diseases caused by mutations in the adult β-globin gene. A bone marrow transplant (BMT) is the only curative treatment, but its application is limited since (i) HLA-matched donors can be found for 75% of alleles modified). In vitro differentiation of these ZFN-treated CD34+ HSCs into erythroid cells resulted in potent elevation of γ-globin mRNA and protein levels without significant effects on erythroid development. Importantly, a similar and specific elevation of γ-globin levels was observed with RBC progeny of genome-edited CD34+ cells obtained from SCD and β-thal patients. Notably, in the latter case a normalization of the β-like to α-globin ratio to ∼1.0 was observed in RBCs obtained from genome-edited CD34s from two individuals with β-thalassemia major. To deploy this strategy in a clinical setting, we developed protocols that yielded comparably high levels of target gene editing in mobilized adult CD34+ cells at large scale (〉108 cells) using a clinical-grade electroporation device to deliver mRNA encoding the ZFN pair. Analysis of modification at the most likely off-target sites based on ZFN binding properties, combined with the maintenance of target genome editing observed throughout erythroid differentiation (and in isolated erythroid colonies) demonstrated that the ZFNs were both highly specific and well-tolerated when deployed at clinical scale. Finally, to assess the stemness of the genome-edited CD34+ HSCs we performed transplantation experiments in immunodeficient mice which revealed long term engraftment of the modified cells (〉16 weeks, ∼25% human chimerism in mouse bone marrow) with maintenance of differentiation in vivo. Moreover, ex vivo erythroid differentiation of human precursor cells isolated from the bone marrow of transplanted animals confirmed the expected elevation of γ-globin. Taken together, these data suggest that a therapeutic level of γ-globin elevation can be obtained by the selective disruption, at the genome level, of specific regulators of the fetal to adult globin developmental switch. The ability to perform this modification at scale, with full retention of HSC engraftment and differentiation in vivo, provides a foundation for advancing this approach to a clinical trial for the hemoglobinopathies. Disclosures: Reik: Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Lee:Sangamo BioSciences: Employment. Truong:Sangamo BioSciences: Employment. Wood:Sangamo BioSciences: Employment. Zhang:Sangamo BioSciences: Employment. Luong:Sangamo BioSciences: Employment. Chan:Sangamo BioSciences: Employment. Liu:Sangamo BioSciences: Employment. Miller:Sangamo BioSciences: Employment. Paschon:Sangamo BioSciences: Employment. Guschin:Sangamo BioSciences: Employment. Zhang:Sangamo BioSciences: Employment. Giedlin:Sangamo BioSciences: Employment. Rebar:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment. Urnov:Sangamo BioSciences: Employment.

Description: In vivo selection may provide a means to increase the relative number of cells of donor origin in recipients with hemopoietic chimerism. We have tested whether in vivo selection using chemical inducers of dimerization (CIDs) can direct the expansion of transduced normal donor erythrocytes in recipients with chimerism using a mouse model of pyruvate kinase deficiency. Marrow cells from normal CBA/N mice were transduced with a vector (F36VmplGFP) that promotes cell growth in the presence of CIDs. Transduced cells were then transplanted into minimally conditioned, pyruvate kinase-deficient recipients (CBA-Pk-1slc/Pk-1slc) to establish stable chimerism. CID administration resulted in expansion of normal donor erythrocytes and improvement of the anemia. The preferential expansion of normal erythrocytes also resulted in a decrease in erythropoietin levels, reducing the drive for production of pyruvate kinase-deficient red blood cells. CID-mediated expansion of genetically modified erythrocytes could prove a useful adjunct to transplantation methods that achieve erythroid chimerism. (Blood. 2004;103:4432-4439)