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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

Finding a relationship between kinetics and thermodynamics may be difficult. However, semi-empirical rules exist to compensate for this shortcoming, among which the Bell-Evans-Polanyi (B-E-P) principle is an example for reactions involving bond breakage and reformation. We expand the B-E-P principle to a new territory by probing photoinduced structure planarization (PISP) of a series of dibenz[b,f]azepine derivatives incorporating bent-to-planar and rotation motion. The latter involves twisting of the partial double bond character, thereby inducing a barrier that is substituent dependent at the para N-phenyl position. The transition-state structure and frequency data satisfy and broaden the B-E-P principle to PISP reactions without bond rearrangement. Together with dual emissions during PISP, this makes possible harnessing of the kinetics/thermodynamics relationship and hence ratiometric luminescence properties for excited-state structural transformations.

The development of high-performance near-infrared organic light-emitting diodes is hindered by strong non-radiative processes as governed by the energy gap law. Here, we show that exciton delocalization, which serves to decouple the exciton band from highly vibrational ladders in the S0 ground state, can bring substantial enhancements in the photoluminescence quantum yield of emitters, bypassing the energy gap law. Experimental proof is provided by the design and synthesis of a series of new Pt(ii) complexes with a delocalization length of 5–9 molecules that emit at 866–960 nm with a photoluminescence quantum yield of 5–12% in solid films. The corresponding near-infrared organic light-emitting diodes emit light with a 930 nm peak wavelength and a high external quantum efficiency up to 2.14% and a radiance of 41.6 W sr−1 m−2. Both theoretical and experimental results confirm the exciton–vibration decoupling strategy, which should be broadly applicable to other well-aligned molecular solids. Pt(ii) complexes allow the fabrication of efficient near-infrared organic light-emitting diodes that operate beyond the 900 nm region.

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.

BackgroundNatural killer (NK) cells have emerged as an alternative cell type for clinical utility given the low propensity for graft-versus-host disease, thereby making NK cells a potential off-the-shelf cell therapy. One critical pathway NK cells use to target tumor cells is through expression of Fc gamma receptor III alpha (CD16). Antibodies that bind tumor antigens are recognized by CD16 on NK cells, promoting NK-mediated tumor cell killing. High-affinity CD16 variants in the human population correlate with better clinical outcome and anti-tumor response. One mechanism tumors use to evade NK cell recognition is through down-regulation of CD16 expression on the NK cell surface. After being activated, CD16 is cleaved by A Disintigrin and Metalloprotease-17 (ADAM-17). By using a highly-active engineered AsCas12a to knock-in high-affinity CD16 (hCD16KI) at the GAPDH locus, hCD16 is constitutively expressed, continuously replacing hCD16, thereby allowing for repeated ADCC mediated killing.Methods iPSCs were edited at the GAPDH locus with an engineered AsCas12a along with the CD16 donor construct. The bulk edited population was then plated at clonal density and single clones were selected and screened. iPSC clones were then differentiated into NK cells. A 3D tumor spheroid killing assay was used to demonstrate NK cell cytotoxicity against an ovarian cancer cell line (SKOV-3). In addition, a serial killing assay was used to better model NK cell serial killing.ResultsBi-allelic CD16KI iPSC clones were successfully generated. These iPSCs exhibited normal morphology and were able to differentiate into iNK cells. hCD16KI iNK cells showed normal differentiation and surface marker expression, such as CD45/CD56, compared to unedited iNK cells. CD16KI iNK cells demonstrated significantly increased cytotoxicity in the presence of antibody against tumor cells when compared with unedited iNK cells, as measured by reduction in tumor spheroid size in a 3D tumor spheroid killing assay. Importantly, enhanced surface expression of hCD16 on iNK cells after tumor exposure was detected, demonstrating the replenishment of cleaved hCD16. Notably, hCD16KI iNK cells demonstrated prolonged and enhanced tumor cell killing after being subjected to repeated tumor cell exposure in a serial killing assay.ConclusionsThis work demonstrates a powerful new method to drive high-level constitutive hCD16 expression on the surface of iNK cells through transgene knock-in at the GAPDH locus using an engineered AsCas12a. The high level constitutive hCD16 expression enhances ADCC of iNK cells and enables enhanced serial tumor killing and is expected to exert enhanced anti-tumor activity in the clinic.

7-Aminoquinoline (7AQ) and various amino derivatives thereof (-NHR) have been strategically designed and synthesized to study their excited-state proton-transfer (ESPT) properties. Due to the large separation between the proton donor -NHR and the acceptor -N- site, ESPT in 7AQ derivatives, if available, should proceed under protic solvent catalysis. ESPT is found to be influenced by the acidity of -NHR and the basicity of the proton-acceptor -N- in the quinoline moiety. The latter is varied by the resonance effect at the quinoline -N- site induced by the -NHR substituent. For those 7AQ derivatives undergoing ESPT, increased quinoline basicity results in a faster rate of ESPT, implying that proton donation from methanol to the quinoline moiety may serve as a key step in the process. Our studies also indicate the existence of an equilibrium between cis and trans arrangements of -NHR in terms of its hydrogen-bond (H-bond) configuration with methanol, whereby only the cis-H-bonded form undergoes methanol-assisted ESPT. With one exception, the interconversion between cis and trans configurations is much faster than the rate of ESPT, yielding amino-type (normal form) and imine-type (proton-transfer tautomer) emissions with distinct relaxation dynamics.

Indeno[1,2-b]fluorene-based [2,2]cyclophanes with 4n/4n and 4n/[4n+2] π-electron systems were prepared, and their structures were identified by X-ray crystallography. With short π-π distances around 3.0 A, [2.2](5,11)indeno[1,2-b]fluorenophane and its precursor [2.2](5,11)indeno[1,2-b]fluorene-6,12-dionophane exhibit remarkable transannular interactions, leading to their unusual electrochemical and photophysical properties. With the aid of femtosecond transient absorption spectroscopy, the transition from the monomeric excited state to the redshifted H-type dimeric state was first observed, correlating to the calculated excitonic energy splitting and the steady-state absorption spectra induced by charge-transfer-mediated superexchange interaction.

Diindeno-fused dibenzo[a,h]anthracene 6 and diindeno-fused dibenzo[c,l]chrysene 9 contain the key moieties 1,4-quinodipropene (1,4-QDP) and 2,6-naphthoquinodipropene (2,6-NQDP), respectively, and they both have an open-shell singlet ground state. The latter compound exhibits a strong biradical character and interesting properties, including a low ΔET-S (2.44 kcal mol-1 ), a small HOMO-LUMO gap (1.06 eV), a wide photoabsorption range (250-1172 nm), and a large two-photon absorption cross-section (σ=1342±56 GM). This work verifies that 6 has a slightly larger HOMO-LUMO gap and ΔET-S than its helical isomer diindeno[2,1-f:1',2'-j]picene (DIP), but is a much stronger two-photon absorber, verifying the important effect of geometry on the photophysical properties.

Current cell and gene therapy medicines for oncology have reshaped how cancer is treated. Chimeric antigen receptor (CAR)-T cells have demonstrated that cell therapy can achieve durable remissions in hematologic malignancies. CAR-T cell therapies, however, have limited efficacy in solid tumors and are associated with severe toxicity, highlighting the need for safer and more efficacious novel cell therapies. With their intrinsic tumor killing capacity, few treatment-related toxicities, and the ability to be given to patients off-the shelf, natural killer (NK) cells are an attractive alternative therapy option to CAR-T cells. While most NK cell therapies are produced from healthy donor cells, deriving NK cells from induced pluripotent stem cells (iPSCs) has the unique advantage that a clone with any desired edits can be generated. We aim to leverage our iPSC platform in combination with our proprietary gene editing technologies to create highly differentiated off-the-shelf treatments for solid tumors. Using our proprietary engineered AsCas12a, we generated double knocked-in (DKI) iPSC clones in which a bicistronic cargo encoding CD16 and a membrane-bound IL-15 (mbIL-15) was knocked into the GAPDH locus to increase the effector function and persistence of iNKs. Constitutive surface expression of CD16 and mbIL-15 by the DKI iNKs was demonstrated. DKI iNKs showed significantly increased natural and antibody dependent cellular cytotoxicity when compared to wild type (WT) iNKs in a SKOV3 tumor spheroid assay in vitro. Furthermore, in the absence of exogeneous cytokines, DKI iNKs persistence in vitro was dramatically improved over WT iNKs. The anti-tumor efficacy of the DKI iNKs in vivo was evaluated using a SKOV3 ovarian cancer model. Tumor bearing mice were treated with WT or DKI iNKs intraperitoneally in combination with trastuzumab or treated with trastuzumab alone. No exogenous cytokines were administered. DKI iNKs combined with a single dose or multiple doses of trastuzumab exerted greater tumor control compared to WT iNKs with trastuzumab, or trastuzumab alone. A single dose of DKI iNKs combined with three doses of trastuzumab induced tumor clearance in 6 out of 8 mice and significantly prolonged survival. Importantly, DKI iNKs were detected in the peritoneum of the treated animals for greater than 3 months, demonstrating that the mbIL-15 maintained iNK survival for a prolonged period of time in the absence of exogeneous cytokine support. In summary, knocking-in CD16 and mbIL-15 to the GAPDH locus of iPSCs dramatically increased the persistence of the DKI iNKs which exhibited robust anti-tumor activities in a solid tumor mouse model. These data demonstrate that our platform enables the development of off-the-shelf iNK cell medicines that may be highly effective for treating solid tumors. Citation Format: Alexander G. Allen, Samia Q. Khan, Kaitlyn M. Izzo, Mrunali Jagdale, Alexandra Gerew, Nadire R. Cochran, Jared Getgano, Stephen Sherman, Laura Blaha, Mark Shearman, Kate Zhang, Kai-Hsin Chang. AsCas12a gene-edited iPSC-derived NK cells constitutively expressing CD16 and membrane-bound IL-15 demonstrate prolonged persistence and robust anti-tumor activities in a solid tumor mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 562.

Background Natural killer (NK) cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. However, 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. We developed a next-generation allogeneic NK cell therapy using CRISPR-Cas12a gene editing to enhance NK cell function through knockout of the CISH and TGFBR2 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, TGFBR2, would render NK cells resistant to TGF-β mediated suppression. Methods NK cells were expanded from CD3-PBMC starting material in the presence of 20 ng/mL IL-15 for 14 days. A variety of methods were performed to assess the effects of CRISPR-Cas12a gene editing on primary human NK cells including NGS to assess editing efficiency, flow cytometry, in vitro spheroid killing assays and an in vivo NSG tumor model. These methods were performed consistent with protocols widely accepted in the field. Results Following editing optimization, we achieved greater than 80% in/dels at both targets in NK cells in both single and double gene knockout (KO, DKO) contexts. Using flow cytometry-based assays we demonstrated that TGFBR2 KO NK cells phosphorylated less SMAD2/3 relative to unedited control NK cells in response to TGF-β, while CISH KO NK cells showed enhanced pSTAT3 and pSTAT5 upon IL-15 stimulation. We next explored the ability of these single knockouts in controlling 3D SK-OV-3 ovarian tumor spheroids and PC-3 prostate tumor spheroids in vitro over 5 days of co-culture. Consistently, both single knockouts demonstrated improved cytotoxicity against tumor targets in the presence of exogenous TGF-β (p Conclusions In summary, using CRISPR-Cas12a we demonstrated highly efficient gene editing of primary human NK cells at two unique targets designed to augment NK cell anti-tumor activity. Together, the increased overall effector function of CISH/TGFBR2 DKO primary human NK cells support their development as a potent allogeneic cell-based medicine for cancer. This potential medicine, termed EDIT-201, is being advanced to clinical study.

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.

The encapsulation and/or surface modification can stabilize and protect the phosphorescence bio‐probes but impede their intravenous delivery across biological barriers. Here, a new class of biocompatible rhenium (ReI) diimine carbonyl complexes is developed, which can efficaciously permeate normal vessel walls and then functionalize the extravascular collagen matrixes as in situ oxygen sensor. Without protective agents, ReI‐diimine complex already exhibits excellent emission yield (34%, λ em = 583 nm) and large two‐photon absorption cross‐sections (σ 2 = 300 GM @ 800 nm) in water (pH 7.4). After extravasation, remarkably, the collagen‐bound probes further enhanced their excitation efficiency by increasing the deoxygenated lifetime from 4.0 to 7.5 µs, paving a way to visualize tumor hypoxia and tissue ischemia in vivo. The post‐extravasation functionalization of extracellular matrixes demonstrates a new methodology for biomaterial‐empowered phosphorescence sensing and imaging., Biocompatible rhenium (ReI) diimine carbonyl complexes functionalize the extravascular collagen matrixes as in situ oxygen sensor. After tail‐vein delivery and vessel penetration, the probe can bind extravascular collagens and achieve stabilization enhanced two‐photon phosphorescence yields. Increased deoxygenated phosphorescence lifetime from 4.0 to 7.5 µs paves a way to visualize tumor hypoxia and tissue ischemia in vivo.

To develop an effective and sustainable cell therapy for sickle cell disease (SCD), we investigated the feasibility of targeted disruption of the BCL11A gene, either within exon 2 or at the GATAA motif in the intronic erythroid-specific enhancer, using zinc finger nucleases in human bone marrow (BM) CD34+ hematopoietic stem and progenitor cells (HSPCs). Both targeting strategies upregulated fetal globin expression in erythroid cells to levels predicted to inhibit hemoglobin S polymerization. However, complete inactivation of BCL11A resulting from bi-allelic frameshift mutations in BCL11A exon 2 adversely affected erythroid enucleation. In contrast, bi-allelic disruption of the GATAA motif in the erythroid enhancer of BCL11A did not negatively impact enucleation. Furthermore, BCL11A exon 2-edited BM-CD34+ cells demonstrated a significantly reduced engraftment potential in immunodeficient mice. Such an adverse effect on HSPC function was not observed upon BCL11A erythroid-enhancer GATAA motif editing, because enhancer-edited CD34+ cells achieved robust long-term engraftment and gave rise to erythroid cells with elevated levels of fetal globin expression when chimeric BM was cultured ex vivo. Altogether, our results support further clinical development of the BCL11A erythroid-specific enhancer editing in BM-CD34+ HSPCs as an autologous stem cell therapy in SCD patients.

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.

With the aim of generalizing the structure-properties relationship of bending heterocyclic molecules that undergo prominent photoinduced structural planarization (PISP), a series of new dihydrodibenzo[ac]phenazine derivatives in which one nitrogen atom is replaced by oxygen (PNO), sulfur (PNS), selenium (PNSe), or dimethylmethanediyl (PNC) was strategically designed and synthesized. Compounds PNO, PNS, and PNSe have significantly nonplanar geometries in the ground state, which undergo PISP to give a planarlike conformer and hence a large emission Stokes shift. A combination of femtosecond early relaxation dynamics and computational approaches established an R*→I* (intermediate)→P* sequential kinetic pattern for PNS and PNSe, whereas PNO undergoes R*→P* one-step kinetics. The polarization ability of the substituted heteroatoms, which is in the order O

Indeno[1,2-b]fluorene-based [2,2]cyclophanes with 4n/4n and 4n/[4n+2] π-electron systems were prepared, and their structures were identified by X-ray crystallography. With short π-π distances around 3.0 Å, [2.2](5,11)indeno[1,2-b]fluorenophane and its precursor [2.2](5,11)indeno[1,2-b]fluorene-6,12-dionophane exhibit remarkable transannular interactions, leading to their unusual electrochemical and photophysical properties. With the aid of femtosecond transient absorption spectroscopy, the transition from the monomeric excited state to the redshifted H-type dimeric state was first observed, correlating to the calculated excitonic energy splitting and the steady-state absorption spectra induced by charge-transfer-mediated superexchange interaction.

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 towards biological membranes allowed detecting the differences in dynamics between plasma membrane and intracellular membrane structures. Such λ-ratiometric microviscosity imaging was extended for mapping the living tissues and observing their inflammation-dependent changes.

Oxygen sensors play important roles in areas such as determining the oxygen content in exhaust gas and optimizing combustion. Resistive oxygen sensors made of an n -type semiconductor, doped ceria (CeO 2 ), have recently received much attention due to their relatively compact and simple structures. In the present study, alumina substrate was first coated with electrodes by screen printing. Porous ceria films were then coated on the electrodes using different mixtures of two undoped ceria powders synthesized by spray-pyrolysis and precipitation, respectively. The ratios of the two powders were varied to produce differences in the heat-treated porosity of the ceria films. The resistive oxygen sensors exhibited a 3-D interconnected pore structure that could be optimized by varying the powder ratio for sensing properties. The dynamic response to the change in oxygen partial pressure of the optimized porous structure at 1073 and 1123 K was >1.8 times faster than those of non-optimized structures. Details on the processing, optimization of compositions, and responses to variation in oxygen partial pressure are provided. The mechanisms for the formations of the powders and the porous coatings are also described.

Hexyloxy-phenyl (C 6 OP), hexyl-phenyl (HexP), isopropylphenyl (iProP), and 3,4-dimethylphenyl (3,4dMP) groups were introduced into the pyrrole ring of dithienylpyrrole-based conducting polymers backbone. The four conjugated polymers were synthesized using electrochemical polymerization and were used as electrochromic materials to study the effects of units of substitution on the electrochromic properties in various supporting electrolytes. Spectroelectrochemical studies demonstrated that the four poly(2,5-dithienylpyrrole) derivatives (PSNS) could be reversibly oxidized and reduced, with accompanying obvious color changed from yellow to blue. The maximum contrast (ΔT%) of the PSNS-C 6 OP film was measured as 28.4% in TBAClO 4 -based supporting electrolyte, and the maximum coloration efficiency (η) of the PSNS-HexP film was calculated to be 204.2 cm 2 C −1 in TBABF 4 -based supporting electrolyte. Electrochromic switching studies showed that PSNS-iProP was faster to change color from the dedoped to the doped state than were PSNS-C 6 OP, PSNS-HexP, and PSNS-3,4dMP.

To explore the mechanisms controlling erythroid differentiation and development, we analyzed the genome-wide transcription dynamics occurring during the differentiation of human embryonic stem cells (HESCs) into the erythroid lineage and development of embryonic to adult erythropoiesis using high throughput sequencing technology. HESCs and erythroid cells at three developmental stages: ESER (embryonic), FLER (fetal), and PBER (adult) were analyzed. Our findings revealed that the number of expressed genes decreased during differentiation, whereas the total expression intensity increased. At each of the three transitions (HESCs–ESERs, ESERs–FLERs, and FLERs–PBERs), many differentially expressed genes were observed, which were involved in maintaining pluripotency, early erythroid specification, rapid cell growth, and cell–cell adhesion and interaction. We also discovered dynamic networks and their central nodes in each transition. Our study provides a fundamental basis for further investigation of erythroid differentiation and development, and has implications in using ESERs for transfusion product in clinical settings.

© 2014 by the American Association for the Advancement of Science; all rights reserved. To study the evolutionary dynamics of regulatory DNA, we mapped >1.3 million deoxyribonuclease I-hypersensitive sites (DHSs) in 45 mouse cell and tissue types, and systematically compared these with human DHS maps from orthologous compartments. We found that the mouse and human genomes have undergone extensive cis-regulatory rewiring that combines branch-specific evolutionary innovation and loss with widespread repurposing of conserved DHSs to alternative cell fates, and that this process is mediated by turnover of transcription factor (TF) recognition elements. Despite pervasive evolutionary remodeling of the location and content of individual cis-regulatory regions, within orthologous mouse and human cell types the global fraction of regulatory DNA bases encoding recognition sites for each TF has been strictly conserved. Our findings provide new insights into the evolutionary forces shaping mammalian regulatory DNA landscapes.

SummaryHuman trisomies can alter cellular phenotypes and produce congenital abnormalities such as Down syndrome (DS). Here we have generated induced pluripotent stem cells (iPSCs) from DS fibroblasts and introduced a TKNEO transgene into one copy of chromosome 21 by gene targeting. When selecting against TKNEO, spontaneous chromosome loss was the most common cause for survival, with a frequency of ∼10−4, while point mutations, epigenetic silencing, and TKNEO deletions occurred at lower frequencies in this unbiased comparison of inactivating mutations. Mitotic recombination events resulting in extended loss of heterozygosity were not observed in DS iPSCs. The derived, disomic cells proliferated faster and produced more endothelia in vivo than their otherwise isogenic trisomic counterparts, but in vitro hematopoietic differentiation was not consistently altered. Our study describes a targeted removal of a human trisomy, which could prove useful in both clinical and research applications.

A resistive oxygen sensor with extremely good temperature independence within a temperature range of 923–1123 K was fabricated from precipitated ceria–zirconia solid solution powders. Experimental results identified the as-precipitated powders as a nanocrystalline ZrO 2 -doped CeO 2 (ZDC) solid solution phase. The amount of Ce 3+ in CeO 2 film was increased by doping with ZrO 2 , identified by XPS. The electrical resistance of ZDC film showed a more significant decreasing rate than that of the undoped CeO 2 at lower oxygen partial pressure. An n -type semiconductance of ZDC films was obtained under oxygen partial pressures from 1.0 to −15 atm. We selected two compositions of the ZDC films whose activation energies were approximately equal (∼1.69 eV at 1.0 atm and ∼1.66 eV at 0.01 atm) for the fabrication of temperature independent resistive oxygen sensors. ZDC films containing 10 at% and 5 at% ZrO 2 (10ZDC and 5ZDC) were used as OMM (oxygen partial pressure measurement material) and TCM (temperature compensating material), respectively. This study confirms the conductive behaviors of ZDC at various oxygen partial pressures as a function of temperature, as well as the suitability of ZDC films for application as temperature independent oxygen sensors. The dynamic response of the resistive oxygen sensor with TCM also revealed that the sensor possessed a relatively good sensibility and stability.

In the present study, nanocrystalline ceria powder and 10 mol% zirconia-doped ceria (10ZDC) powder were prepared by co-precipitation method from cerium nitrate and zirconium nitrate precursors. The resulting particles with irregular shapes were printed onto alumina substrate for the investigation of oxygen gas sensing behavior. The coating morphology of 10ZDC with a network structure revealed a better connection enhancing the sensing properties. Zirconia doping tended to inhibit the grain growth and decrease the lattice constant of ceria. Such effects may improve the electrical conductivity of 10ZDC under different oxygen partial pressures and shorten the response time of 10ZDC to the change of oxygen partial pressure.

Because of the imbalance in the supply and demand of red blood cells (RBCs), especially for alloimmunized patients or patients with rare blood phenotypes, extensive research has been done to generate therapeutic quantities of mature RBCs from hematopoietic stem cells of various sources, such as bone marrow, peripheral blood, and cord blood. Since human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can be maintained indefinitely in vitro, they represent potentially inexhaustible sources of donor-free RBCs. In contrast to other ex vivo stem-cell-derived cellular therapeutics, tumorigenesis is not a concern, as RBCs can be irradiated without marked adverse effects on in vivo function. Here, we provide a comprehensive review of the recent publications relevant to the generation and characterization of hESC- and iPSC-derived erythroid cells and discuss challenges to be met before the eventual realization of clinical usage of these cells.

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.

Nanofibers of polysulfone (PSU) were prepared by electro-spinning from 10∼20 wt.% PSU solutions in N,N’-dimethyl acetamide (DMAc) mixed with 0.0∼0.1 wt.% LiCl. With increasing PSU concentration, the morphology of fibers electrospun were bead, mixture of bead-fiber and fiber, and smooth fibers when PSU concentration was 10, 12–15, and 18–20 wt.%, respectively. The bead sizes decreased and fiber diameters increased as PSU concentration was increased. The fiber diameter decreased with increases of the LiCl concentration and the distance from spinneret to collection plate. The fiber diameter also decreased with decreasing solution feeding rate. The fiber diameter distribution electrospun from 20 wt.% PSU solutions was much broader than those electrospun from 18 wt.% PSU solution. For 18 wt.% PSU solution, the average fiber diameter (AFD) decreased when the applied voltage V was increased from 7 to 12 kV. However, for 20 wt.% PSU solutions, the AFD increased when V was increased from 7 to 12 kV. The different morphology of fibers electrospun from 18 and 20 wt.% PSU solutions was attributed to the much higher viscosity of 20 wt.% PSU solution than 18 wt.% PSU solution.

Despite a growing body of literature concerning the hematopoietic differentiation of human embryonic stem cells (hESCs), the full hematopoietic potential of the majority of existing hESC lines remains unknown. In this study, the hematopoietic response of five NIH-approved hESC lines (H1, hSF6, BG01, BG02, and BG03) was compared. Our data show that despite expressing similar hESC markers under self-renewing conditions and initiating mesodermal differentiation under spontaneous differentiation conditions, marked differences in subsequent hematopoietic differentiation potential among these lines existed. A high degree of hematopoietic differentiation was attained only by H1 and BG02, whereas this process appeared to be abortive in nature for hSF6, BG01, and BG03. This difference in hematopoietic differentiation predisposition was readily apparent during spontaneous differentiation, and further augmented under hematopoietic-inducing conditions. This predisposition appeared to be intrinsic to the specific hESC line and independent of passage number or gender karyotype. Interestingly, H1 and BG02 displayed remarkable similarities in their kinetics of hematopoietic marker expression, hematopoietic colony formation, erythroid differentiation, and globin expression, suggesting that a similar, predetermined differentiation sequence is followed. The identification of intrinsic and extrinsic factors governing the hematopoietic differentiation potential of hESCs will be of great importance for the putative clinical utility of hESC lines.

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.

Objective Cytokines released during inflammatory processes have been proposed to play a central role in mediating mechanism(s) leading to anemia. Here, we used CpG-ODN to investigate the effects of a pro-inflammatory response on the pathophysiological processes leading to anemia. Methods Naive and erythropoietin (EPO)-treated mice were injected for 2 days with 100 μg CpG-ODN or control ODN and the effects on the course of red blood cell (RBC) and reticulocyte counts, RBC turnover, and EPO-stimulated maturation of erythroid cells were analyzed. To study the effect of CpG-ODN on erythroid cell maturation in vitro, we obtained primary EPO-responsive cells by treating mice with thiamphenicol (15 mg/g body weight). Results CpG-ODN-treated mice developed anemia, which persisted for 5 days and was associated with a 50% reduction in EPO-stimulated differentiation of EPOR + cells to TER119 + erythroblasts. CpG-ODN-induced suppression required accessory cells, including antigen presenting cells, which activated other cells to produce pro-inflammatory cytokines. In vitro neutralization of IFN-γ, but not IL-12, TNF-α, IFN-α, IL-1α, or IL-1β, abrogated the erythropoietic suppression induced by CpG-ODN. The anemia observed in CpG-ODN-treated mice was also associated with reduced RBC survival in vivo, as demonstrated by a sevenfold to eightfold higher turnover of biotinylated RBC compared to control ODN–treated mice. In vivo IFN-γ neutralization confirmed that IFN-γ contributed to erythropoietic suppression but not reduced RBC survival. Conclusions Together, these results demonstrate that CpG-ODN anemia is associated with suppressed erythropoiesis and decreased RBC survival. Importantly, CpG-ODN-induced IFN-γ was found to be the major factor mediating erythropoietic suppression but not decreased RBC survival.

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.

It has been proposed that the basis of severe malarial anaemia, a major cause of morbidity and mortality in endemic areas, is multifactorial. Inappropriately low reticulocytosis is observed in malaria patients suggesting that insufficient erythropoiesis is a major factor. Clinical studies provide conflicting data concerning the production of adequate levels of erythropoietin (EPO) during malaria. Plasmodium chabaudi AS causes non-lethal infection in resistant C57BL/6 mice, and lethal infection in susceptible A/J mice. In P. chabaudi AS infected C57BL/6 and A/J mice, which experience varying degrees of severity of anaemia, kidney EPO production is appropriate to the severity of anaemia and is regulated by haematocrit level. Neutralisation of endogenous EPO during infection leads to lethal anaemia while timely administration of exogenous EPO rescues mice although reticulocytosis is suppressed in proportion to the parasitemia level. Characterisation of alterations in splenic erythroid compartments in naive and P. chabaudi AS infected A/J mice revealed that infection, with or without EPO treatment, leads to sub-optimal increases in TER119+ erythroblasts compared to EPO-treated naive mice. A lower percentage of TER119+ erythroblasts in infected mice undergo terminal differentiation to become mature haemoglobin-producing cells. Furthermore, there is a shift in transferrin receptor (CD71) expression from TER119+ cells to a non-erythroid population. Deficiencies in the number and maturation of TER119+ erythroblasts during infection coincide with blunted proliferation to EPO stimulation in vitro by splenocytes, although a high frequency express EPO receptor (EPOR). Together, these data suggest that during malaria, EPO-induced proliferation of early EPOR+ erythroid progenitors is suppressed, leading to sub-optimal generation of TER119+ erythroblasts. Moreover, a shift in CD71 expression may result in impaired terminal maturation of erythroblasts. Thus, suppressed proliferation, differentiation, and maturation of erythroid precursors in association with inadequate reticulocytosis may be the basis of insufficient erythropoiesis during malaria.

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)