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Organic molecules having emission in the NIR(II) region are emergent and receiving enormous attention. Unfortunately, attaining accountable organic emission intensity around the NIR(II) region is hampered by the dominant internal conversion operated by the energy gap law, where the emission energy gap and the associated internal reorganization energy λ

In this study, the chromophore 3,4,9,10-perylenetetracarboxylic diimide (PDI) is anchored with phenyl substituents at the imide N site, followed by thionation, yielding a series of thione products

We demonstrate the construction of wavelength λ-ratiometric images that allow visualizing the distribution of microscopic dynamics within living cells and tissues by using the newly developed principle of fluorescence response. The bent-to-planar motion in the excited state of incorporated fluorescence probes leads to elongation of the π-delocalization, resulting in microviscosity-dependent but polarity-insensitive interplay between well-separated blue and red bands in emission spectra. This allows constructing the exceptionally contrasted images of cellular dynamics. Moreover, the application of probes with increased affinity toward biological membranes allowed detecting the differences in dynamics between the plasma membrane and intracellular membrane structures. Such λ-ratiometric microviscosity imaging was extended for mapping the living tissues and observing their inflammation-dependent changes.

Beta thalassemia is one of the most common recessive hematological disorders in the world with more than 200 mutations identified to date. These mutations reduce or completely abrogate beta globin expression. As beta globin pairs with alpha globin to form adult hemoglobin (HbA, α2β2), reduced or absent beta globin results in excessive alpha globin chains, which form toxic aggregates. These aggregates cause maturation blockade and premature death of erythroid precursors, and hemolysis of red blood cells (RBC), leading to varying degrees of anemia. Patients with the most severe form of beta thalassemia, namely beta thalassemia major, are transfusion-dependent, i.e., requiring life-long RBC transfusions accompanied by the burden of iron chelation therapy. EDIT-301 is an experimental autologous cell therapy in which CD34 + cells are genetically modified to promote gamma globin expression. EDIT-301 is currently in clinical development for sickle cell disease, and IND enabling stage for transfusion-dependent beta thalassemia (TDT). Gamma globin decreases the alpha to beta globin chain imbalance in beta thalassemia by pairing with the over-abundant alpha globin chains to form fetal hemoglobin (HbF, α2γ2). Gamma globin induction, and consequently HbF induction, for EDIT-301 is achieved through AsCas12a ribonucleoprotein (RNP)-mediated editing of the distal CCAAT box region of the HBG1 and HBG2 promoters, where naturally occurring hereditary persistence of fetal hemoglobin (HFPH) mutations exist. We chose this target over BCL11A based on previous preclinical data demonstrating that BCL11A editing reduces erythroid output in NBSGW mice. An engineered AsCas12a RNP edits the HBG1 and HBG2 promoter distal CCAAT box with high efficiency and specificity. We have previously shown that on-target editing of >80% was achieved in mobilized peripheral blood (mPB) CD34 + cells from normal donors with no detectable off-target editing both at research scale and at clinical manufacturing scale. Edited normal donor CD34 + cells led to long-term, polyclonal, multilineage engraftment without lineage skewing in immunocompromised mice and sustained robust HbF production in their erythroid progeny. To test whether EDIT-301 may be an efficacious therapy for TDT, mPB CD34 + cells from individuals with TDT were electroporated with the engineered AsCas12a RNP targeting the HBG1 and HBG2 promoters. AsCas12a RNP edited mPB CD34 + cells from individuals with TDT as efficiently as CD34 + cells from normal donors. Importantly, EDIT-301 has the potential to address the underlying pathophysiology of TDT, i.e., the maturation blockade and premature death of erythroid precursors. Erythroid differentiation of edited beta thalassemia CD34 + cells showed significant improvement in erythroid maturation and health. Specifically, ~70% edited erythroblasts reached late erythroblast stage compared to ~53% unedited erythroblasts; ~56% edited erythroid cells underwent terminal maturation and enucleated compared to ~28% of unedited erythroid cells; and non-viable erythroblasts decreased from ~33% to ~22% after editing. The improved erythropoiesis was accompanied by significantly increased total hemoglobin content per cell. These data strongly support that editing of the HBG1 and HBG2 promoter CCAAT box using engineered AsCas12a RNP can reverse the dyserythropoiesis associated with beta thalassemia and increase the hemoglobin production. In summary, we have provided strong preclinical data supporting the development of EDIT-301 for the treatment of TDT. Edited mPB CD34 + cells retained their ability to engraft without lineage skewing, resulted in robust HbF induction long-term, improved erythropoiesis, and increased hemoglobin content in TDT erythroid cells. These data support that a single administration of EDIT-301 may have the potential to safely and effectively reverse dyserythropoiesis and ameliorate anemia in individuals with TDT long-term. Clinical studies to demonstrate the safety and efficacy of EDIT-301 in the treatment of TDT are currently being planned. Disclosures Sousa: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Janoudi: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. deDreuzy: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Natural killer (NK) cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. Effector function of allogeneic NK cells can be diminished by the lack of functional persistence, as well as tumor-intrinsic immunosuppressive mechanisms, such as production of TGF-β, a pleiotropic cytokine that inhibits immune effector function. Gene editing is the power tool to modify NK cells to potentially overcome these biological limitations. Here, we developed a next-generation iPSC-derived NK cell therapy using CRISPR-AsCas12a gene editing to enhance NK cell function by deleting the CISH and TGFβR2 genes. We hypothesized that knockout of CISH, a negative regulator of IL-2/IL-15 signaling, would improve NK cell effector function, while knockout of the TGF-β receptor gene, TGFβR2, would render NK cells resistant to TGF-β mediated suppression. NK cells are typically isolated from either cord blood or peripheral blood of healthy donors, but recent advances with induced pluripotent stem cells (iPSCs) allows a nearly unlimited supply of iPSC-derived natural killer cells (iNK). In this study, we used CRISPR-Cas12a to generate edited iPSC lines that were differentiated into TGFβ R2-/-/CISH-/- double knockout (DKO) iNK cells. Using flow cytometry-based assays we demonstrate that DKO iNK cells phosphorylated less SMAD2/3 relative to unedited control iNK cells in response to IL-15 and TGF-β, while CISH KO NK cells showed enhanced pSTAT3 upon IL-15 stimulation. Additionally, DKO iNKs produced higher levels of cytotoxic cytokines including IFN-γ and TNF-α in response to PMA/ionomycin stimulation. We next explored the ability of these DKO iNKs in controlling 3D SKOV-3 ovarian tumor spheroids in vitro over 5 days of co-culture. Both freshly generated and cryopreserved DKO iNKs demonstrated significantly better tumor killing as compared to unedited control iNKs. Importantly, there was no difference in tumor killing between freshly generated and cryopreserved DKO iNKs, suggesting that the freeze/thaw process does not impact functional capacity. We utilized the SKOV3-luc IP tumor model to evaluate the in vivo efficacy of cryopreserved iNKs cells. Here, NSG mice with established SKOV3-luc tumors were treated IP with unedited control iNKs or DKO iNKs. DKO iNK cell treatment induced robust anti-tumor efficacy resulting in a significant 7.2- fold and 3.2-fold reduction in tumor burden as compared to vehicle and unedited iNK cell treatment, respectively, at 9 days post-iNK cell dosing. In summary, we demonstrated that TGFβ R2-/-/CISH-/- DKO iPSCs differentiated into iNK cells have potent anti-tumor activity that is maintained after cryopreservation. Together, the increased overall effector function of TGFβ R2-/-/CISH-/- DKO human iNK cells support their development as a potent allogeneic cell-based medicine for cancer. This potential medicine is being investigated with other gene edits for future advancement to clinic. Disclosures Gerew: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Sexton: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Wasko: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Khan: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Current cell and gene therapy medicines for oncology have reshaped how cancer is treated. Specifically, chimeric antigen receptor (CAR)-T cells have demonstrated that cell therapy can achieve durable remissions in hematologic malignancies. However, CAR-T cell therapies have limited efficacy in solid tumors and are often associated with severe toxicity, highlighting the need for novel cell therapies that are safer and more efficacious. With their intrinsic killing capacity of tumor cells and few, if any, treatment related toxicities, natural killer (NK) cell therapies represent an attractive alternative therapy option to CAR-T cells. In addition, NK cells can be generated from allogeneic donors and given to patients off-the-shelf without causing graft versus host disease. Of the various sources of donor types to generate NK cells from, induced pluripotent stem cells (iPSCs) have the unique advantage of being a renewable source. A clone with any desired edits to enhance the effector function of NK cells can be derived, fully characterized, and expanded indefinitely, to generate large quantities of a naturally allogeneic medicine, therefore streamlining the manufacturing process and increasing scalability. Here, a bicistronic cargo encoding CD16 and a membrane-bound IL-15 (mbIL-15) was knocked into iPSCs at the GAPDH locus using an engineered and highly active AsCas12a. The promoter at the GAPDH locus drives robust constitutive expression of inserted cargos and avoids the promoter silencing that often occurs during differentiation with other strategies. CD16 and mbIL-15 were selected as Knock-Ins (KI) to specifically enhance NK cell therapy in two areas, namely NK cell deactivation caused by CD16 downregulation, and the reliance of co-administration of cytokines such as IL-15 or IL-2 for persistence. CD16 (FcRyIII) can bind the Fc portion of IgG antibodies triggering the lysis of targeted cells. This mechanism of cytotoxicity is known as antibody dependent cellular cytotoxicity (ADCC), and is an innate immune response largely mediated by NK cells through CD16. ADCC is severely impaired when surface CD16 is cleaved by a metalloprotease known as ADAM17. By having CD16 expressed from the GAPDH locus, there is consistent CD16 protein expression to replace what is shed. This hypothesis was demonstrated by performing flow cytometry before and after a cytotoxicity assay. WT cells showed a marked reduction in the surface level expression of CD16 compared to CD16 KI cells after tumor cell exposure. Using a lactate dehydrogenase (LDH) release assay as a measure of cytotoxicity, only the iNK cells expressing the CD16 construct showed statistically significant increases in cytotoxicity when trastuzumab was added. Furthermore, to better model a solid tumor, a 3D tumor spheroid killing assay was utilized where CD16 KI cells showed an increase in ADCC capacity. The benefit of increased effector function via CD16 KI cannot be fully realized without iNK cells persisting. IL-2 or IL-15 is needed for NK maintenance but the administration of either cytokine is associated with acute clinical toxicities. mbIL-15 allows NK cells to survive for a prolonged period without the support of homeostatic cytokines. An in vitro persistence assay was performed that demonstrated IL-15 KI cells showed an increase in persistence compared to WT cells. Specifically, during the three-week in vitro assay, WT cells became undetectable by Day 14 while IL-15 KI NK cells remained stable over time. In summary, to overcome two shortfalls of NK cell therapies, a bicistronic construct encoding CD16 and a mbIL-15 was knocked into the GAPDH locus of iPSCs. The strong GAPDH promoter drove constitutive expression of CD16 that mitigated CD16 shedding, enhanced ADCC of iNK cells, which can be used in combination with any ADCC enabling IgG1 and IgG3 antibodies, such as trastuzumab and rituximab, for tumor-specific targeting. In addition, mbIL-15 KI allowed iNK cells to persist without exogenous cytokine administration and thus can circumvent exogeneous cytokine-induced clinical toxicities. CD16 and mbIL-15 double KI iNKs, with enhanced ADCC and increased cytokine-independent persistence, can potentially be developed into a safe and efficacious therapy for the treatment of a variety of liquid and solid tumors with high unmet medical needs. Disclosures Allen: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pattali: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Izzo: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Getgano: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko: Editas Medicine: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Blaha: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zuris: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Sickle cell disease (SCD) is an inherited blood disorder affecting approximately 100,000 individuals in the United States. As fetal hemoglobin (HbF) has been shown to be protective against clinical manifestation of SCD, we are developing EDIT-301, an autologous cell therapy comprising CD34+ cells genetically modified using a Cas12a ribonucleoprotein (RNP) to promote HbF expression to treat SCD. Fetal hemoglobin induction for EDIT-301 is achieved by disrupting the HBG1 and HBG2 promoter distal CCAAT-box region where naturally occurring mutations are found to be associated with elevated HbF expression. Cas12a was selected over Cas9 due to the more productive and sustainable (NHEJ derived) indel profile, as well as high specificity. Using Cas12a RNP, on-target editing of ~90% was achieved in mobilized peripheral blood CD34+ cells (mPB-CD34+ cells) from both healthy and SCD donors at research scale with no detectable off targets. Editing of CD34+ cells led to an average of 43% and 54% of HbF expression in the erythroid progeny of normal donor and SCD donor cells respectively in a pancellular fashion (~93% population). The robust HbF induction in SCD red blood cells (RBCs) resulted in significant phenotypic and functional improvement including reduced sickling and increased deformability under hypoxia ex vivo. Using a microfluidic assay that replicated blood flow in microvasculature under varying oxygen conditions, SCD RBCs derived from RNP electroporated CD34+ cells showed improved rheological behavior. The rheology improvement under hypoxia was strongly correlated with the increased levels of HbF in each sample. Infusion of the modified CD34+ cells from normal donors into NBSGW mice resulted in long-term multi lineage and polyclonal reconstitution. Editing levels at 16 weeks post infusion were > 90% in all human lineages tested, demonstrating the efficient editing of SCID-repopulating hematopoietic stem cells (HSCs). Consistent with the high editing levels, human erythroid cells from the bone marrow of mice that received Cas12a-RNP treated cells demonstrated pancellular (~90% F+ RBCs) HbF expression averaging 40-50% of total hemoglobin compared to ~5% HbF observed in the control group. We have developed a consistent large-scale process using functionally closed, semi-automated systems suitable for use in clinical manufacturing. We have shown robust editing of normal donor CD34+ cells and SCID-repopulating HSCs with the clinical scale process. Editing levels of >90% were detected after long term engraftment in mice. In summary, we have demonstrated successful on-target editing of mPB CD34+ cells derived from both normal and SCD donors using a Cas12a RNP, which coincided with robust HbF induction and a phenotypic reduction of sickling in the SCD erythroid progeny, as well as improved rheological behavior. Editing of the HBG1 and HBG2 promoters using this RNP was highly specific with no measurable off-target. In vivo, cells from normal donors readily engrafted and reconstituted all blood cell lineages at levels comparable to unedited cells. Finally, a robust large-scale manufacturing process has been developed to supply material for the clinical setting. Based on these results, we are completing the activities required to assess EDIT-301 in the clinic as treatment for SCD. Disclosures De Dreuzy: Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Heath:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Sousa:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Janoudi:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. An:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Albright:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Teixeira:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Monesmith:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Chang:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company.

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.

Regulatory regions harbor multiple transcription factor recognition sites; however, the contribution of individual sites to regulatory function remains challenging to define. We describe a facile approach that exploits the error-prone nature of genome editing-induced double-strand break repair to map functional elements within regulatory DNA at nucleotide resolution. We demonstrate the approach on a human erythroid enhancer, revealing single TF recognition sites that gate the majority of downstream regulatory function.

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.

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)

To the editor: The groundbreaking innovation of reprogramming adult cells to pluripotency has opened new avenues for patient-tailored treatments. The successful treatment of a humanized sickle cell anemia mouse model with induced pluripotent stem cells (iPSCs) from autologous skin illustrates this principle.1 Human iPSCs can be directed to undergo hematopoietic differentiation in a fashion similar to human embryonic stem cells (hESCs).2–6 However, the globin expression profile of iPSC-derived erythroid cells has not been fully explored and only partial results were mentioned in one study.4 An important question is whether the age of the cells used to derive iPSCs influences developmentally regulated globin expression. We studied globin expression in erythroid cells derived from 6 different human iPSC lines generated in our laboratory with lentiviral vectors expressing OCT4, NANOG, LIN28, and SOX2 as described.7 Three lines (iPSC-MHF2) were derived from human fetal fibroblasts isolated at 20 weeks' gestation (GM05387, Coriell Institute for Medical Research), and the other 3 (iPSC-OI12) were derived from human mesenchymal stem cells (MSCs) isolated from the spine of a 15-year-old patient.8 Two lines, iPSC-MHF2 C2 and iPSC-OI12 C7, were verified to have normal male karyotype (46X,Y). The ability of each iPSC clone used in this study to give rise to progenies of all 3 germ layers was established by teratoma assays in immunodeficient mice and staining of histologic sections for human microtubule associated protein–2 (MAP-2; ectoderm), human α–smooth muscle actin (SMA; mesoderm), or human α-fetoprotein (AFP; endoderm), as described9 (supplemental Figure 1). The iPSC lines were induced to undergo hematopoietic differentiation as previously described for human ESCs.10 As was previously reported in hESC and iPSC lines,5,10 these 6 iPSC lines exhibited a wide range of hematopoietic differentiation potential, as demonstrated by the percentage expression of hematopoietic markers such as CD45 and glycophorin-A (Figure 1A) and by the formation of hematopoietic colonies (Figure 1B). In 2 of the lines with robust erythroid development (Figure 1C), we studied the globin expression pattern at the mRNA (Figure 1D) and protein levels (Figure 1E). We found that despite the original adult status of the cells from which they were derived, these iPSC-derived erythroid cells expressed mostly embryonic (ϵ) and fetal (γ) globins, similar to ESC-derived erythroid cells,10 consistent with complete reprogramming at the globin locus. This globin phenotype, if maintained in vivo, has an important implication for the treatment of hemoglobinopathies using patient-specific iPSC-derived hematopoietic cells in that correcting the defective β-globin gene may not be necessary. Furthermore, our data highlight the fact that iPSCs are highly heterogeneous in their hematopoietic potential as previously reported by Choi and colleagues.5 While this variation could reflect differential silencing and/or expression of reprogramming factors, it is similar to that observed in human ESC-derived cells, and demonstrates that large numbers of individual iPSC lines need to be screened before drawing conclusions related to the effects of cell origin on differentiation potential. Further studies are needed to dissect these issues in iPSC-derived hematopoietic cells before their anticipated use in humans. Figure 1 iPSC-derived cells' hematopoietic surface marker expression, clonogenic potential, and globin expression pattern. (A) Hematopoietic surface marker expression by iPSC-derived cells. iPSCs were induced to undergo hematopoietic differentiation by embryoid ...

Although the influence of microenvironmental “niche” on the function of a variety of stem cells is undisputed, the details of hematopoietic stem cell/niche interactions at the cellular and molecular level have sparked a continuous debate. We studied the microanatomic partitioning of transplanted normal and α4 integrin-deficient Lin−kit+ cells in trabecular and compact bone before and after irradiation and present robust quantitative data on both. We found that (1) the microanatomic distribution of normal highly enriched progenitor cells is random in nonirradiated recipients based on area distribution analyses, (2) in contrast, in irradiated hosts normal cells distribute preferentially near the endosteum, (3) the overall cell seeding efficiency was higher in trabecular versus compact bone both before and after irradiation, and (4) α4 integrin-deficient cells not only lodge with reduced overall efficiency confirming previous data, but fail to preferentially partition themselves into endosteal regions in irradiated hosts, as normal cells do. A similar phenotype was observed with cells rendered Gi-protein signaling incompetent by pertussis toxin treatment, supporting an active stromal-derived factor 1 (SDF-1) gradient near endosteum after irradiation.

Blockade of CD49d-mediated lymphocyte trafficking has been used therapeutically for certain autoimmune diseases, such as multiple sclerosis (MS). In addition to negative effects on the trafficking of mature lymphocytes to sites of inflammation, CD49d blockade in mice and monkeys rapidly mobilizes hematopoietic stem/progenitor cells (HSPCs) capable of short- and long-term engraftment. Here we aimed to ascertain the effects of treatment with antifunctional anti-CD49d antibody in humans (MS patients receiving infusions of the CD49d-blocking antibody natalizumab) on levels of circulating HSPCs after a single dose of antibody or after long-term treatment. On average, 6-fold elevated levels of circulating CD34+ cells and colony-forming unit-culture (CFU-C) were achieved within 1 day of the first dose of natalizumab, and similar levels were continuously maintained under monthly natalizumab infusions. The blood of natalizumab-treated subjects also contained SCID-repopulating cells. The fate of these circulating HSPCs and their clinical relevance for MS patients remains to be determined.

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.

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.

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.

The laminins are a group of extracellular matrix proteins with constitutive expression in all tissues, including bone marrow stroma. A functional role for the nonintegrin laminin receptor p67 has been described for cancer metastasis and lymphocyte trafficking. Expression of p67 was also reported for other subsets of mature leukocytes and for malignant hematopoietic or nonhematopoietic cells. We explored p67 expression on normal hematopoietic progenitor cells (HPCs) and its putative role in bone marrow retention of transplanted HPCs. We found p67 expression on a subset of primary human CD34+ cells coexpressing erythroid markers. Of importance, p67 recognizes early erythroid progenitors, since sorted p67+ cells were significantly enriched for burst-forming units–erythroid (BFU-Es) and depleted of colony-forming units–granulocyte/macrophage (CFU-GMs). Blockade of p67 binding of donor cells, using antifunctional antibody, reduced bone marrow homing of BFU-Es. These studies identify p67 as a novel phenotypic marker for erythroid HPCs of functional importance for lineage-specific homing/retention among adult transplanted HPCs.

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.

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 Recent insights into the regulation of γ-globin transcription by a network of transcription factors and regulatory elements both inside and outside the β-globin locus have revealed a set of new molecular targets, the modulation of which is expected to elevate γ-globin levels for potential therapeutic intervention. To this end, we and others have established that designed zinc finger nucleases (ZFNs) transiently introduced into stem cells ex vivo provide a safe and efficient way to permanently ablate the expression of a specific target gene in hematopoietic stem cells (HSC) by introduction of mutations following target site cleavage and error-prone DNA repair. Here we report the development and comparison of different ZFNs that target various regulators of γ-globin gene transcription in human HSCs: Bcl11a, Klf1, and specific positions in the γ-globin promoters that result in hereditary persistence of fetal hemoglobin (HPFH). In all cases these target sites / transcription factors have previously been identified as crucial repressors of γ-globin expression in humans, as well as by in vitro and in vivo experiments using human erythroid cells and mouse models. ZFN pairs with very high genome editing activity in CD34+ HSCs were identified for all targeted sites (>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.

Abstract 4730 Krüppel-like factors (KLFs) are a conserved family of Cys2His2 zinc finger proteins which are important components of eukaryotic cellular transcriptional machinery that controls many biological processes including erythroid differentiation and development. As a transcriptional activator and a tumor suppressor, KLF6 was also involved in hematopoiesis. Klf6−/− mice is embryonic lethal by embryonic day 12.5 and associated with markedly reduced hematopoiesis as well as poorly organized yolk sac vascularization. Moreover, the expression of erythroid differentiation markers including Klf1, Gata1 and Scl are delayed and hematopoietic differentiation is impaired in klf6−/− ES cells. However, the detailed mechanism that KLF6 regulates hematopoiesis is not fully understood. To characterize the role of KLF6 in hematopoiesis, we firstly detected the dynamic expression pattern of KLF6 during erythroid differentiation by mRNA-seq in undifferentiated human embryonic stem cells (hESC), three primary erythroid cells at different developmental stages including ES-derived erythroid cells (ESER), fetal- and adult-type erythroid cells (FLER, PBER). The transcriptome analysis showed that KLF6 expressed at significantly higher level in ESER cells compared with that in other cells. Meanwhile, chromatin immunoprecipitation (ChIP) studies in human K562 cells demonstrated the enrichment of KLF6 on the promoter region of embryonic epsilon-globin gene. These results probably indicate that KLF6 play an important role in primitive hematopoiesis. To clarify whether the erythroid-specific enhancers in the genomic region of KLF6 participate in the regulation of primitive hematopoiesis, we extensively screened the erythroid-specific DNaseI hypersensitive sites (DHSs) in the KLF6 locus, from 70 kb upstream of the transcription start site to 20 kb downstream of the poly(A) site, from DNase-seq data in four erythroid cells including ESER, FLER, PBER, K562 and seven non-erythroid cells. The enhancer activity of these erythroid-specific DHSs was comprehensively characterized by dual-luciferase reporter assay in K562 cells as well as non-erythroid HeLa and HEK293 cells. Three erythroid-specific enhancers located 18–24 kb upstream of human KLF6 were finally characterized, which not only helps to understand the higher expression of KLF6 in ESER, but also hints that KLF6 could participate in primitive hematopoiesis through erythroid-specific enhancers. In conclusion, we depicted the dynamic expression pattern of KLF6 during erythroid differentiation, characterized three erythroid-specific enhancers in KLF6 gene locus, and disclosed the potential role of KLF6 in primitive hematopoiesis. Next, the overexpression and depletion of KLF6 in K562 cells will be executed to further explore whether the abnormal KLF6 will affect the expression and functions of globin genes as well as erythroid-specific transcription factors. Chromosome conformation capture (3C) analysis will be performed to evaluate the interactions between the erythroid-specific enhancers and the cis-regulatory elements of hematopoiesis related genes. Moreover, we will establish morpholino-based klf6 knockdown zebrafish model and study the target genes, interacting networks and pathways in which KLF6 involved. Collectively, these results will address the detailed cis- and trans- regulatory functions and molecular mechanism of KLF6 in regulating hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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.

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.

Blockade of CD49d-mediated lymphocyte trafficking has been used therapeutically for certain autoimmune diseases. In addition to effects on mature lymphocytes, in mice and monkeys CD49d blockade also mobilizes immature hematopoietic cells from bone marrow (BM), capable of complete long-term engraftment despite a partial BM seeding defect. The aim of these studies was to ascertain effects on the biology of HSPC mobilization of single or chronic CD49d blockade in Multiple Sclerosis (MS) patients receiving disease-modifying treatment with the anti-functional anti-CD49d antibody, Natalizumab. These studies represent the first observations on HSPC mobilization by anti-CD49d antibodies in humans and on chronic exposure to a mobilizing agent. Circulating CD34+ cells (1.8±0.4/μL) and CFU-C (638±128/mL) were normal in MS patients (n=9) prior to the first Natalizumab infusion (normal controls: 1.3±0.1/μL CD34+ cells, 608±129/mL CFU-C). In chronically treated patients (i.e patients who had received ≥5 prior doses of Natalizumab) HSPC numbers were significantly elevated 30 days after the last infusion (CD34+ cells 9.0±1.2/μL, CFU-C 3243±332/mL, n=10, p