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Gene editing with the CRISPR-Cas9 system could revolutionize hematopoietic stem cell (HSC)-targeted gene therapy for hereditary diseases, including sickle cell disease (SCD). Conventional delivery of editing tools by electroporation limits HSC fitness due to its toxicity; therefore, efficient and non-toxic delivery remains crucial. Integrating lentiviral vectors are established for therapeutic gene delivery to engraftable HSCs in gene therapy trials; however, their sustained expression and size limitation preclude their use for CRISPR-Cas9 delivery. Here, we developed a Cas9 protein delivery non-integrating lentiviral system encoding guide RNA and donor DNA, allowing for transient endonuclease function and inclusion of all editing tools in a single vector (all-in-one). We demonstrated efficient one-time correction of the SCD mutation in the endogenous βs-globin gene up to 42% at the protein level (p < 0.01) with the Cas9 protein delivery non-integrating lentiviral all-in-one system without electroporation. Our findings improve prospects for efficient and safe genome editing., Graphical abstract, Uchida et al. develop a Cas9 protein delivery non-integrating lentiviral system encoding guide RNA and donor DNA, allowing for transient endonuclease function and inclusion of all editing tools in a single vector (all-in-one). One-time correction of the sickle cell disease mutation is demonstrated by this non-integrating lentiviral delivery without electroporation.

Gene editing of the erythroid-specific BCL11A enhancer in hematopoietic stem and progenitor cells (HSPCs) from patients with sickle cell disease (SCD) induces fetal hemoglobin (HbF) without detectable toxicity, as assessed by mouse xenotransplant. Here, we evaluated autologous engraftment and HbF induction potential of erythroid-specific BCL11A enhancer–edited HSPCs in 4 nonhuman primates. We used a single guide RNA (sgRNA) with identical human and rhesus target sequences to disrupt a GATA1 binding site at the BCL11A +58 erythroid enhancer. Cas9 protein and sgRNA ribonucleoprotein complex (RNP) was electroporated into rhesus HSPCs, followed by autologous infusion after myeloablation. We found that gene edits persisted in peripheral blood (PB) and bone marrow (BM) for up to 101 weeks similarly for BCL11A enhancer– or control locus–targeted (AAVS1-targeted) cells. Biallelic BCL11A enhancer editing resulted in robust γ-globin induction, with the highest levels observed during stress erythropoiesis. Indels were evenly distributed across PB and BM lineages. Off-target edits were not observed. Nonhomologous end-joining repair alleles were enriched in engrafting HSCs. In summary, we found that edited HSCs can persist for at least 101 weeks after transplant and biallelic-edited HSCs provide substantial HbF levels in PB red blood cells, together supporting further clinical translation of this approach.

Lentiviral addition of βT87Q-globin, a modified β-globin with an anti-sickling mutation, is currently being used in gene therapy trials for sickle cell disease (SCD) and β-thalassemia patients. βT87Q-globin interferes with sickle hemoglobin (HbS) polymerization. Here, we generated the SCD mutation in an immortalized human erythroid cell line (HUDEP-2) to investigate the anti-sickling activity of βT87Q-globin. Sickle HUDEP-2 (sHUDEP-2) cells produced robust HbS after differentiation and sickled under deoxygenated conditions, comparable with SCD CD34+ progeny. Lentiviral transduction provided 9.5–26.8 pg/cell βT87Q-globin (R2 = 0.83) in a vector copy number (VCN)-dependent manner, resulting in a significant reduction of sickling ratios (R2 = 0.92). Interestingly, βT87Q-globin transduction markedly reduced endogenous βS-globin (R2 = 0.84) to an undetectable level (0.4–16.8 pg/cell) in sHUDEP-2 cells, as well as endogenous β-globin in human CD34+ cell-derived erythroid cells. RNA sequencing (RNA-seq) analysis with βT87Q-transduced sHUDEP-2 and human CD34+-derived cells revealed activation of inflammation- and proliferation-related programs, suggesting minimal changes in background gene expression except for βT87Q-globin expression and endogenous β/βS-globin suppression. In summary, using sHUDEP-2 and CD34+-derived cells, we demonstrated that lentiviral addition of βT87Q-globin strongly reduced endogenous β-/βS-globin expression, resulting in an anti-sickling effect. Our findings should be helpful to understand the anti-sickling effects of therapeutic genes in SCD gene therapy.

New technologies of induced pluripotent stem cells (iPSCs) and genome editing have emerged, allowing for the development of autologous transfusion therapies. We previously demonstrated definitive β‐globin production from human embryonic stem cell (hESC)‐derived erythroid cell generation via hemangioblast‐like ES‐sacs. In this study, we demonstrated normal β‐globin protein production from biallelic corrected sickle cell disease (SCD) iPSCs. We optimized our ES/iPS‐sac method for feeder cell‐free hESC maintenance followed by serum‐free ES‐sac generation, which is preferred for electroporation‐based genome editing. Surprisingly, the optimized protocol improved yields of ES‐sacs (25.9‐fold), hematopoietic‐like spherical cells (14.8‐fold), and erythroid cells (5.8‐fold), compared with our standard ES‐sac generation. We performed viral vector‐free gene correction in SCD iPSCs, resulting in one clone with monoallelic and one clone with biallelic correction, and using this serum‐free iPS‐sac culture, corrected iPSC‐generated erythroid cells with normal β‐globin, confirmed at DNA and protein levels. Our serum‐free ES/iPS‐sac protocol with gene correction will be useful to develop regenerative transfusion therapies for SCD., We have successfully corrected the sickle mutation in induced pluripotent stem cells (iPSCs) derived from a sickle cell disease patient using an improved serum‐free method for the generation of ES/iPS‐sacs, a hemangioblast‐like structure containing hematopoietic stem and progenitor cells that can be differentiated into erythroid cells containing mostly definitive globins, including γ‐ and β‐globins.

Humanized animal models are central to efforts aimed at improving hematopoietic stem cell (HSC) transplantation with or without genetic modification. Human cell engraftment is feasible in immunodeficient mice; however, high HSC doses and conditioning limit broad use of xenograft models. We assessed human CD45+ chimerism after transplanting varying doses of human CD34+ HSCs (2 × 105 to 2 × 106 cells/mouse) with or without busulfan (BU) pretransplant conditioning in c-kit mutant mice that do not require conditioning (non-obese diabetic [NOD]/B6/severe combined immunodeficiency [SCID]/ interleukin-2 receptor gamma chain null (IL-2rγ−/−) KitW41/W41 [NBSGW]). We then tested a range of BU (5–37.5 mg/kg) using 2 × 105 human CD34+ cells. Glycophorin-A erythrocyte chimerism was assessed after murine macrophage depletion using clodronate liposomes. We demonstrated successful long-term engraftment of human CD34+ cells at all cell doses in this model, and equivalent engraftment using 10-fold less CD34+ cells with the addition of BU conditioning. Low-dose BU (10 mg/kg) was sufficient to allow human engraftment using 2 × 105 CD34+ cells, whereas higher doses (≥37.5 mg/kg) were toxic. NBSGW mice support human erythropoiesis in the bone marrow; however, murine macrophage depletion provided only minimal and transient increases in peripheral blood human erythrocytes. Our xenograft model is therefore useful in HSC gene therapy and genome-editing studies, especially for modeling in disorders, such as sickle cell disease, where access to HSCs is limited. Keywords: busulfan, c-kit, erythropoiesis, gene therapy, hematopoietic stem cell transplantation, immunodeficient mice, xenograft transplantation

Hematopoietic stem cell (HSC) gene therapy is curative for various hereditary diseases; however, high-efficiency transduction in HSCs remains crucial to improve the prospects for hemoglobinopathies. We previously optimized lentiviral transduction in human CD34+ cells with serum-free medium containing minimal cytokines, allowing efficient transduction (∼50%) and robust xenograft engraftment. In this study, we further improved lentiviral transduction in human CD34+ cells. High-density culture conditions (4e6/mL) resulted in ∼5-fold more efficient transduction in CD34+ cells (p < 0.01) compared with standard cell density (1e5/mL). After co-culturing vector-exposed CD34+ cells with non-transduced CD34+ cells, high-density culture conditions enhanced lentiviral gene marking in the non-transduced population (p < 0.01) compared with low-density conditions, suggesting that increasing cell-to-cell contact allows more efficient transduction. Two adjuvants, poloxamer 407 (100 μg/mL) and prostaglandin E2 (10 μM), were added to high-density CD34+ cells, resulting in ∼4-fold more efficient transduction (p < 0.01) without significant toxicity compared with no adjuvant control. In summary, we developed a highly efficient lentiviral transduction method in high-density CD34+ cell culture with poloxamer 407 and prostaglandin E2, allowing overall ∼10-fold improvement in transduction efficiency and consistently achieving more than 90% transduction and an average vector copy number of ∼10. Our optimized transduction method should improve gene therapy approaches using lentiviral vectors targeting HSCs. Keywords: lentiviral vector, hematopoietic stem cells, transduction efficiency

Hematopoietic stem cell gene therapy for hemoglobin disorders, including sickle cell disease, requires high-efficiency lentiviral gene transfer and robust therapeutic globin expression in erythroid cells. Erythropoietin is a key cytokine for erythroid proliferation and differentiation (erythropoiesis), and truncated human erythropoietin receptors (thEpoR) have been reported in familial polycythemia. We reasoned that coexpression of thEpoR could enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous nonhuman primate transplantation models. We generated thEpoR by deleting 40 amino acids from the carboxyl terminus, allowing for erythropoietin-dependent enhanced erythropoiesis of gene-modified cells. We then designed lentiviral vectors encoding both thEpoR and B cell lymphoma/leukemia 11A (BCL11A)–targeting microRNA-adapted short hairpin RNA (shmiR BCL11A) driven by an erythroid-specific promoter. thEpoR expression enhanced erythropoiesis among gene-modified cells in vitro. We then transplanted lentiviral vector gene-modified CD34+ cells with erythroid-specific expression of both thEpoR and shmiR BCL11A and compared to cells modified with shmiR BCL11A only. We found that thEpoR enhanced shmiR BCL11A–based fetal hemoglobin (HbF) induction in both xenograft mice and rhesus macaques, whereas HbF induction with shmiR BCL11A only was robust, yet transient. thEpoR/shmiR BCL11A coexpression allowed for sustained HbF induction at 20 to 25% in rhesus macaques for 4 to 8 months. In summary, we developed erythroid-specific thEpoR/shmiR BCL11A–expressing vectors, enhancing HbF induction in xenograft mice and rhesus macaques. The sustained HbF induction achieved by addition of thEpoR and shmiR BCL11A may represent a viable gene therapy strategy for hemoglobin disorders.

Background Ex vivo production of hematopoietic stem/precursor cells (HSPCs) represents a promising versatile approach for blood disorders. Methods To derive definitive HSPCs from human embryonic stem cells (ESCs), we differentiated mesodermally specified embryoid bodies (EBs) on gelatin-coated plates in serum/feeder-free conditions. Results Seven-day EB maturation followed by an 8-day differentiation period on OP9 cells provided the highest number of definitive (CD34+ CD235a−, 69%, p p p p Conclusions In summary, definitive HSPCs can be derived from ESCs through a dynamic cellular process from an organoid-like structure, where erythroid progeny are capable of producing adult hemoglobin and lymphoid progeny shows a diverse TCR repertoire.

Hematopoietic stem cell gene therapy for hemoglobin disorders, including sickle cell disease, requires high-efficiency lentiviral gene transfer and robust therapeutic globin expression in erythroid cells. Erythropoietin is a key cytokine for erythroid proliferation and differentiation (erythropoiesis), and truncated human erythropoietin receptors (thEpoR) have been reported in familial polycythemia. We reasoned that coexpression of thEpoR could enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous nonhuman primate transplantation models. We generated thEpoR by deleting 40 amino acids from the carboxyl terminus, allowing for erythropoietin-dependent enhanced erythropoiesis of gene-modified cells. We then designed lentiviral vectors encoding both thEpoR and B cell lymphoma/leukemia 11A (

One of the most promising objectives of clinical hematology is to derive engraftable autologous hematopoietic stem cells (HSCs) from human induced pluripotent stem cells (iPSCs). Progress in translating iPSC technologies to the clinic relies on the availability of scalable differentiation methodologies. In this study, human iPSCs were differentiated for 21 days using STEMdiff™, a monolayer-based approach for hematopoietic differentiation of human iPSCs that requires no replating, co-culture or embryoid body formation. Both monolayer and suspension cells were functionally characterized throughout differentiation. In the supernatant fraction, an early transient population of primitive CD235a+ erythroid cells first emerged, followed by hematopoietic progenitors with multilineage differentiation activity in vitro but no long-term engraftment potential in vivo. In later stages of differentiation, a nearly exclusive production of definitive erythroid progenitors was observed. In the adherent monolayer, we identified a prevalent population of mesenchymal stromal cells and limited arterial vascular endothelium (VE), suggesting that the cellular constitution of the monolayer may be inadequate to support the generation of HSCs with durable repopulating potential. Quantitative modulation of WNT/β-catenin and activin/nodal/TGFβ signaling pathways with CHIR/SB molecules during differentiation enhanced formation of arterial VE, definitive multilineage and erythroid progenitors, but was insufficient to orchestrate the generation of engrafting HSCs. Overall, STEMdiff™ provides a clinically-relevant and readily adaptable platform for the generation of erythroid and multilineage hematopoietic progenitors from human pluripotent stem cells.HighlightsRobust, scalable and clinically-relevant monolayer-based culture system for hematopoietic differentiation of human iPSCs.Successive emergence of primitive erythroid cells, definitive multilineage HSPCs and erythroid progenitors in the culture supernatant.Abundant mesenchymal cells and limited arterial vascular endothelium in the culture monolayer.CHIR/SB molecules increase arterial vascular endothelium formation, suppress primitive hematopoiesis and promote definitive multilineage and erythroid progenitors.

In vitro erythroid differentiation from primary human cells is valuable to develop genetic strategies for hemoglobin disorders. However, current erythroid differentiation methods are encumbered by modest transduction rates and high baseline fetal hemoglobin production. In this study, we sought to improve both genetic modification and hemoglobin production among human erythroid cells in vitro. To model therapeutic strategies, we transduced human CD34+ cells and peripheral blood mononuclear cells (PBMCs) with lentiviral vectors and compared erythropoietin-based erythroid differentiation using fetal-bovine-serum-containing media and serum-free media. We observed more efficient transduction (85%–93%) in serum-free media than serum-containing media (20%–69%), whereas the addition of knockout serum replacement (KSR) was required for serum-free media to promote efficient erythroid differentiation (96%). High-level adult hemoglobin production detectable by electrophoresis was achieved using serum-free media similar to serum-containing media. Importantly, low fetal hemoglobin production was observed in the optimized serum-free media. Using KSR-containing, serum-free erythroid differentiation media, therapeutic adult hemoglobin production was detected at protein levels with β-globin lentiviral transduction in both CD34+ cells and PBMCs from sickle cell disease subjects. Our in vitro erythroid differentiation system provides a practical evaluation platform for adult hemoglobin production among human erythroid cells following genetic manipulation. Keywords: lentiviral vector, hematopoietic stem cells, erythroid cells, erythroid differentiation, adult hemoglobin, gene therapy, genome editing

Stress erythropoiesis and chronic inflammation in subjects with sickle cell disease (SCD) may have an impact on the bone marrow (BM) haematopoietic stem and progenitor cell (HSPC) quality and yield necessary for effective autologous, ex vivo HSPC gene therapy. BM from 19 subjects with SCD and five volunteers without SCD (non-SCD) was collected in different anticoagulants and processed immediately (day 0) or the following day (day 1). Inflammatory, contamination and aggregation markers within the mononuclear layer, and CD34, CD45 and Glycophorin-A (GPA) expression on HSPCs after CD34(+) selection were analysed by conventional and imaging flow cytometry. Compared to non-SCD BM, multiple markers of inflammation, contamination (red cells, P < 0·01; platelets, P < 0·01) and aggregates (platelet/granulocytes, P < 0·01; mononuclear/red cells, P < 0·01) were higher in SCD BM. Total CD34(+) cell count was lower in SCD BM (P < 0·05), however CD34(+) count was higher in SCD BM when collected in acid citrate dextrose-A (ACDA) versus heparin (P < 0 05). Greater than 50% of CD34(+) HSPCs from SCD BM are CD34(dim) due to higher erythroid lineage expression (P < 0·01) as single cell CD34(+)CD45(+)GPA(+) (P < 0·01) and CD34(+)CD45(‒)GPA(+) (P < 0·01) HSPCs. SCD BM is characterized by increased inflammation, aggregation and contamination contributing to significant differences in HSPC quality and yield compared to non-SCD BM.

Elevated fetal hemoglobin (HbF) is associated with reduced severity of sickle cell disease. Therefore, γ-globin protein levels and F-cell (HbF-positive red blood cell) percentages are used for estimation of clinical benefit. Here, we monitored transplantation-related changes in HbF and F-cell percentages for rhesus macaques (Macaca mulatta) following total body irradiation or busulfan conditioning prior to CD34(+) cell transplantation. HbF protein expression peaked in the first 4–9 weeks posttransplant (0.99%–2.53%), and F-cells increased in the first 6–17 weeks posttransplant (8.7%–45.3%). HbF and F-cell ratios gradually decreased and stabilized to levels similar to those of control animals (1.96 ± 1.97% for F cells and 0.49 ± 0.19% γ-globin expression) 4–7 months post-transplant. These findings confirm and expand on previous reports of transient induction in HbF and F-cell percentages in rhesus macaques following CD34(+) cell transplantation, an observation that must be taken into consideration when evaluating therapeutic strategies that aim to specifically elevate HbF expression, which are currently in clinical development. Published by Elsevier Inc. on behalf of ISEH – Society for Hematology and Stem Cells.

Sickle cell disease (SCD) is an inherited monogenic disorder resulting in serious mortality and morbidity worldwide. Although the disease was characterized more than a century ago, there are only two FDA approved medications to lessen disease severity, and a definitive cure available to all patients with SCD is lacking. Rapid and substantial progress in genome editing approaches have proven valuable as a curative option given plausibility to either correct the underlying mutation in patient-derived hematopoietic stem/progenitor cells (HSPCs), induce fetal hemoglobin expression to circumvent sickling of red blood cells (RBCs), or create corrected induced pluripotent stem cells (iPSCs) among other approaches. Recent discovery of CRISPR/Cas9 has not only revolutionized genome engineering but has also brought the possibility of translating these concepts into a clinically meaningful reality. Here we summarize genome engineering applications using CRISPR/Cas9, addressing challenges and future perspectives of CRISPR/Cas9 as a curative option for SCD.

Sickle cell disease (SCD) is an inherited monogenic disorder resulting in serious mortality and morbidity worldwide. Although the disease was characterized more than a century ago, there are only two FDA approved medications to lessen disease severity, and a definitive cure available to all patients with SCD is lacking. Rapid and substantial progress in genome editing approaches have proven valuable as a curative option given plausibility to either correct the underlying mutation in patient-derived hematopoietic stem/progenitor cells (HSPCs), induce fetal hemoglobin expression to circumvent sickling of red blood cells (RBCs), or create corrected induced pluripotent stem cells (iPSCs) among other approaches. Recent discovery of CRISPR/Cas9 has not only revolutionized genome engineering but has also brought the possibility of translating these concepts into a clinically meaningful reality. Here we summarize genome engineering applications using CRISPR/Cas9, addressing challenges and future perspectives of CRISPR/Cas9 as a curative option for SCD.

Hematopoietic stem cell (HSC) gene therapy is being evaluated for hemoglobin disorders including sickle cell disease (SCD). Therapeutic globin vectors have demanding requirements including high-efficiency transduction at the HSC level and high-level, erythroid-specific expression with long-term persistence. The requirement of intron 2 for high-level β-globin expression dictates a reverse-oriented globin-expression cassette to prevent its loss from RNA splicing. Current reverse-oriented globin vectors can drive phenotypic correction, but they are limited by low vector titers and low transduction efficiencies. Here we report a clinically relevant forward-oriented β-globin-expressing vector, which has sixfold higher vector titers and four to tenfold higher transduction efficiency for long-term hematopoietic repopulating cells in humanized mice and rhesus macaques. Insertion of Rev response element (RRE) allows intron 2 to be retained, and β-globin production is observed in transplanted macaques and human SCD CD34+ cells. These findings bring us closer to a widely applicable gene therapy for hemoglobin disorders., Vectors used in gene therapy for hemoglobin disorders carry globin in a reverse-orientation to prevent the loss of key regulatory elements by RNA splicing, but this limits their efficiency. Here, the authors develop a vector carrying β-globin in a forward orientation and show that it has improved titers and transduction efficiency in humanized mice and nonhuman primates.

Hematopoietic stem cell gene therapy for hemoglobin disorders, such as sickle cell disease, requires high-level gene marking and robust therapeutic globin expression in erythroid cells (>20% of γ- or β-globin production) for widespread successful clinical application. We previously demonstrated that lentiviral transduction of a truncated human erythropoietin receptor (thEpoR) gene allows for erythropoietin-dependent selective proliferation of gene-modified human erythroid cells during in vitro differentiation (ASH 2017). In this study, we sought to evaluate whether thEpoR can enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous non-human primate transplantation models. To investigate this hypothesis, we designed lentiviral vectors encoding both thEpoR and BCL11A-targeting micro RNA-adapted short hairpin RNA (shmiBCL11A), driven off an erythroid specific ankyrin 1 (ANK1) promoter. Both selective proliferation and high-level fetal hemoglobin (HbF) induction were observed in in vitro erythroid differentiation cultures using transduced human CD34+ cells. Healthy donor CD34+ cells were transduced with shmiBCL11A vector, thEpoR-shmiBCL11A vector, and GFP vector (control). Transduced cells were transplanted into immunodeficient NBSGW mice. Five months post-transplant, xenograft bone marrow cells were evaluated for human cell engraftment (human CD45+) and vector copy number (VCN) in both human CD34+ progenitor cells and glycophorin A+ (GPA+) erythroid cells. HbF production was also measured in GPA+ erythroid cells by reverse phase HPLC. We observed efficient transduction in transduced CD34+ cells in vitro (VCN 2.1-5.1) and similar human cell engraftment among all groups (84-89%). The VCN with thEpoR-shmiBCL11A transduction was 3-fold higher in human erythroid cells when compared to CD34+ cells (p We then performed autologous rhesus CD34+ cell transplantation using either shmiBCL11A vector (142562 and RA0706, n=2, compared to a GPA promoter-derived shmiBCL11A vector) or thEpoR-shmiBCL11A vector (ZL50 and ZM24, n=2, compared to a Venus-encoding vector). Transduced CD34+ cells were transplanted into autologous rhesus macaques following 2x5Gy total body irradiation. Efficient transduction was observed in CD34+ cells in vitro among all 4 macaques (VCN 3.8-8.7) using a high-density culture protocol (Uchida N, Mol Ther Methods Clin Dev. 2019). In shmiBCL11A transduction animals, engraftment of gene-modified cells (VCN 0.2-1.0) and robust HbF induction (14-16%) were observed 1 month post-transplant. However, VCN and HbF levels were reduced down to VCN ~0.1 and HbF ~0.4% in both animals 6 months post-transplant. In contrast, a thEpoR-shmiBCL11A transduction animal (ZL50) resulted in engraftment of gene-modified cells (VCN 0.8-1.0) and robust HbF induction (~18%) 1 month post-transplant, with both gene marking and HbF levels remaining high at VCN 0.6-0.7 and HbF ~15% 4 months post-transplant. These data suggest that shmiBCL11A transduction results in transient HbF induction in gene-modified erythroid cells, while thEpoR-based selective advantage allows for sustained HbF induction with shmiBCL11A. In summary, we developed erythroid-specific thEpoR-shmiBCL11A expressing vectors, enhancing HbF induction in gene-modified erythroid cells in xenograft mice and rhesus macaques. While further in vivo studies are desirable, the use of thEpoR appears to provide a selective advantage for gene-modified erythroid cells in gene therapy strategies for hemoglobin disorders. Disclosures No relevant conflicts of interest to declare.

Elevated fetal hemoglobin (HbF, α2γ2) levels are clinically beneficial for patients with β-hemoglobinopathies. Editing of the erythroid-specific BCL11A enhancer induces HbF, inhibiting sickling and restoring globin chain balance in erythroid cells derived from hematopoietic stem and progenitor cells (HSPCs) from SCD and β-thalassemia patients respectively, without detectable genotoxicity or adverse effects on hematopoietic stem cell (HSC) function (Wu Y, Nat Med, 2019). Here, we sought to evaluate engraftment and HbF induction potential of erythroid-specific BCL11A enhancer edited CD34+ HSPCs in a non-human primate transplantation model in which hemoglobin switching is conserved. We targeted the erythroid-specific +58 DNAse I hypersensitive site of BCL11A, which has identical human and rhesus sequences at the spacer and protospacer adjacent motif (PAM) of the potent #1617 sgRNA. Ribonucleoprotein complex (RNP) composed of 3x-NLS SpCas9 protein and either BCL11A enhancer targeting (#1617) or AAVS1 targeting sgRNA was electroporated into rhesus CD34+ HSPCs (n=3). Following erythroid differentiation, substantial γ-globin expression (54-77%, p We tested BCL11A enhancer editing with autologous HSC transplant in two cohorts, with two macaques per cohort. For cohort 1, we performed competitive engraftment of BCL11A enhancer and AAVS1 edited HSPCs to test long-term reconstitution. For cohort 2, we evaluated BCL11A enhancer editing alone to evaluate HbF induction and hematopoietic reconstitution. For each cohort, purified CD34+ HSPCs were electroporated with RNP one day after G-CSF and plerixafor mobilization and cultured for two days prior to cryopreservation. HSPCs were thawed and infused following 2×5 Gy total body irradiation. For cohort 1 (n=2, ZL25 and ZL22, 1.34-1.39×106 CD34+ HSPCs/kg), we observed reduced indel frequencies (8-41%) at early post-infusion time points compared to cell products (18-49%), suggesting indels in unfractionated HSPCs may overestimate those in engrafting cells and/or hematopoietic ablation was incomplete. From weeks 6 to 83, stable indel frequencies were detected in both BCL11A (~3-18%) and AAVS1 (~10-45%), suggesting no selective advantage for BCL11A enhancer edited, AAVS1 edited, or non-edited HSCs. For cohort 2 (BCL11A enhancer editing alone (n=2, ZM17 and ZM26, 1.78-6.06×106 CD34+ cells/kg), cell products showed improved editing with ~95% indels and ~65-78% γ-globin protein after in vitro erythroid culture. Animals engrafted with typical kinetics and displayed stable indel ratios up to 28 weeks post-transplantation. A significant correlation was detected between γ-globin level and indel frequency comparing all 4 transplanted animals and unedited controls (R2=0.76, p In summary, we evaluated the clinical potential of autologous BCL11A erythroid enhancer editing in rhesus macaques. BCL11A enhancer edited HSCs can persist for at least 83 weeks post-transplant and provide therapeutic levels of HbF in peripheral RBCs without anemia or other apparent hematologic toxicity. Furthermore, these results emphasize input CD34+ HSPC dose and conditioning intensity as critical variables that influence gene editing following autologous HSCT. Overall, these findings support BCL11A erythroid enhancer genome editing as a promising strategy for therapeutic HbF induction. Disclosures Weiss: GlaxoSmithKline: Consultancy; Cellarity INC: Consultancy; Esperian: Consultancy; Beam Therapeutics: Consultancy; Rubius INC: Consultancy.

Sickle cell disease (SCD) is caused by a 20A>T mutation in the β-globin gene. State-of-the-art genome editing technologies have the potential to correct the SCD mutation in hematopoietic stem cells (HSCs), producing adult hemoglobin (Hb) while simultaneously eliminating sickle Hb. We have demonstrated efficient gene correction in SCD CD34+ cells with SCD mutation-specific guide RNA, Cas9 mRNA/protein, and single strand donor DNA, resulting in ~30% gene correction and ~50% indels at the DNA level, and ~60% normal β-globin production at the protein level in in vitro erythroid differentiation (ASH 2018). Gene correction by homology directed repair is thought to be enhanced by cell proliferation; however, cell proliferation might reduce stemness of HSCs. To investigate this hypothesis, we sought to evaluate engraftment of gene-edited CD34+ HSCs in a non-human primate model. To model SCD gene correction, a β-to-βs globin conversion was designed in rhesus macaques. Mobilized rhesus CD34+ cells (n=2) were electroporated using the GMP-compliant, FDA Master File-supported, and scalable MaxCyte GT System to deliver rhesus β-globin-targeting guide RNA (the same target site as the SCD mutation-specific guide RNA), SpCas9 protein, and single strand donor DNA including a SCD mutation (20A>T). We also added an adjuvant to improve gene conversion efficiencies. Following erythroid differentiation, gene correction efficiency was evaluated at DNA levels by deep sequencing and at protein levels by reverse-phase HPLC. We observed high-efficiency genome editing without the adjuvant (20-30% gene conversion and 61-64% indels), and further enhanced genome editing with the adjuvant (51-59% gene conversion and 36-39% indels). After erythroid differentiation, we observed production of βs-globin protein (~100%) but not normal β-globin in gene-edited cells. We then evaluated engraftment of gene-edited rhesus CD34+ cells with β-to-βs globin conversion (n=2, 13U005 and 12U011). Mobilized rhesus CD34+ cells (3.4-3.8e7) were pre-stimulated for 2 days, and edited cells were cryopreserved after electroporation with editing tools. Small aliquots of edited cells (before and after cryopreservation) were differentiated into erythroid cells in vitro, resulting in 17-26% of gene conversion and 57-71% of indels at the DNA level and 50-100% of β-globin production at the protein level, with no difference observed between aliquots taken before and after cryopreservation. Following 9.5 Gy total body irradiation, the frozen edited CD34+ cells (1.6-2.2e7) were injected into autologous macaques. We observed robust recovery of blood counts in 13U005, while peripheral blood recovery was delayed in 12U011, who was supported by serial whole blood transfusion. We observed 7-11% of gene conversion and 44-54% of indels in both granulates and lymphocytes in 13U005 1 month post-transplant. Around 15% sickle Hb production in red blood cells was detected by Hb electrophoresis in 13U005 three months post-transplant and ~7% in 12U011 two months post-transplant. Interestingly, ~10% of fetal Hb production was observed in 12U001, likely due to stress hematopoiesis. In summary, we developed a rhesus β-to-βs globin conversion model with HSC-targeted genome editing strategies. The gene-edited rhesus CD34+ cells are engraftable for at least 3 months post-transplant. Although further follow-up is necessary for transplanted animals, these findings are helpful in designing HSC-targeted gene correction trials. Figure Disclosures Li: MaxCyte, Inc: Employment. Allen:MaxCyte, Inc: Employment. Peshwa:MaxCyte, Inc: Employment.

γ-globin expression can be induced by various gene modification strategies, which could be beneficial for hemoglobin (Hb) disorders. To translate promising ideas into clinics, large animal models have proven valuable to evaluate safety and efficacy of the approaches; however, in vitro erythroid differentiation methods have not been established to determine whether they can be modeled in nonhuman primates.We optimized erythroid differentiation culture to produce high-level adult Hb from rhesus hematopoietic progenitor cells by using low (LC) or high cytokine concentration (HC) protocols with or without feeder cells. In addition, we established rhesus globin protein analysis using reverse-phase high performance liquid chromatography and mass spectrometry.Robust adult Hb production at protein levels was observed in the LC protocol when feeder cells were used, whereas the HC protocol resulted in higher baseline fetal Hb levels (P0.01). We then compared lentiviral transduction of rhesus cells between serum-containing LC media and serum-free StemSpan-based differentiation media, revealing 100-fold more efficient transduction in serum-free differentiation media (P0.01). Finally, rhesus CD34In conclusion, we developed an efficient rhesus erythroid differentiation protocol from hematopoietic progenitor cells with low fetal and high adult Hb production. Further studies are warranted to optimize gene modification and transplantation of rhesus hematopoietic progenitor cells.

We report the case of a 36-year-old woman with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, and corticosteroid replacement therapy since birth. She manifested persistent virilization and high testosterone levels that were attributed to nonadherence to medical treatment. The patient was referred to our gender unit for genitoplastic surgery. We recommended the patient for left oophorectomy after detecting an ovarian mass. Pathologic findings confirmed an ovarian hilus cell tumor. Testosterone levels fell back to normal and masculinization disappeared but ACTH remained elevated. This case represents a very rare type of primary ovarian tumor that must be considered in persistent virilizing symptoms in women with CAH.

Sickle cell disease (SCD) is caused by a 20A>T mutation in the β-globin gene, and can be cured by therapeutic β-globin gene addition into hematopoietic stem cells (HSCs) with lentiviral transduction. However, this method relies upon random integration, leaving the SCD mutation intact and potentially inducing insertional mutagenesis. Genome editing technologies have the potential to correct the SCD mutation without integration, producing adult hemoglobin (Hb) while simultaneously eliminating sickle Hb. In this study, we investigated CRISPR/Cas9-based gene correction for SCD CD34+ cells. Plerixafor-mobilized SCD CD34+ cells were transfected by electroporation using the GMP-compliant, FDA Master File-supported, and scalable MaxCyte GT System to deliver SCD mutation-specific guide RNA at 200mg/ml, SpCas9 mRNA at 200mg/ml or protein at 120mg/ml, and single strand donor DNA with a normal β-globin sequence at 80, 120, or 200mg/ml. We chose Cas9 mRNA and single strand donor DNA due to the ease of clinical grade large-scale production and to avoid the need for viral vector manufacturing. Following erythroid differentiation, gene correction efficiency was evaluated at DNA levels by deep sequencing and at protein levels by reverse-phase HPLC. Cell viability was reduced to 76-87% after electroporation, compared to 90% in the control. We observed high-efficiency genome editing (29-34% gene correction and 49-58% indels) with Cas9 mRNA, showing donor DNA concentration dependence, and editing levels were comparable to Cas9 protein (39% correction and 43% indels). 15-23% Biallelic and 17-26% monoallelic gene correction were detected at the clonal level by colony assay. After erythroid differentiation, up to 54% normal β-globin production was observed with Cas9 mRNA (Figure), comparable to Cas9 protein (67%), while βs-globin amounts were markedly reduced under both conditions (6-10%). Similar correction efficiencies were obtained from two additional SCD patients' CD34+ cells at DNA levels (28-35%) and protein levels (33-56%). These data demonstrate that Cas9 mRNA and single strand donor DNA allow for efficient gene correction in SCD CD34+ cells, exceeding the therapeutic threshold of 20% in SCD. We then evaluated off-target effects on the δ-globin gene, which was reported as a major off-target site in β-globin gene editing due to high homology; however, almost no off-target effects (0.6-1.3% indels) were detected. Interestingly, gene conversion in the 9T>C polymorphism (11bp upstream of SCD mutation) on the β-globin gene was observed, and this conversion always occurred with SCD gene correction (26-33% of SCD gene correction), suggesting that gene conversion is strongly affected by distance from the target site. In addition, we evaluated genome editing among subpopulations of CD34+ cells from 3 healthy donors under the same conditions (normal β-globin to SCD mutation). We observed similar editing efficiencies (conversion and indels) among more immature (CD34+CD133+CD90+) and relatively differentiated populations (CD34+CD133+CD90-, CD34+CD133-, and CD34-) as well as among cells at different phases of the cell cycle (G0/G1, S, and G2/M), suggesting that similar gene correction efficiencies are obtained in all CD34+ cell populations, including the HSC population. We have begun efforts to evaluate gene-corrected SCD CD34+ cell engraftment in the mouse xenograft model, as similarly corrected X-CGD CD34+ cells were engrafted in immunodeficient mice. To examine the effects of indels in the β-globin gene, we next evaluated Hb production from genome-edited SCD CD34+ cells (2 patients) without donor DNA. Editing without donor DNA resulted in 63-70% indels (compared to 26-29% correction and 46-53% indels with donor DNA) and increased non-adult Hb production (small amounts of fetal Hb and significant amounts of a Hb variant), which will require further investigation to characterize. In summary, we observed efficient gene correction in SCD CD34+ cells with a simple Cas9 mRNA, single strand donor DNA, and guide RNA method, resulting in ~30% gene correction and ~50% indels. After erythroid differentiation, the majority of Hb detected was adult Hb; we detected up to 54% normal β-globin production with a marked reduction of βs-globin to ~10%. Evaluation of engraftment potential is required for gene-corrected CD34+ cells, but these methods would be clinically applicable for gene correction in SCD. Figure. Figure. Disclosures Li: MaxCyte, Inc.: Employment. Allen:MaxCyte, Inc.: Employment. Peshwa:MaxCyte, Inc.: Employment.

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent an ideal source for in vitro modeling of erythropoiesis and a potential alternative source for red blood cell transfusions. However, iPS cell-derived erythroid cells predominantly produce ε- and γ-globin without β-globin production. We recently demonstrated that ES cell-derived sacs (ES sacs), known to express hemangioblast markers, allow for efficient erythroid cell generation with β-globin production. In this study, we generated several iPS cell lines derived from bone marrow stromal cells (MSCs) and peripheral blood erythroid progenitors (EPs) from sickle cell disease patients, and evaluated hematopoietic stem/progenitor cell (HSPC) generation after iPS sac induction as well as subsequent erythroid differentiation. MSC-derived iPS sacs yielded greater amounts of immature hematopoietic progenitors (VEGFR2 + GPA−), definitive HSPCs (CD34 + CD45+), and megakaryoerythroid progenitors (GPA + CD41a+), as compared to EP-derived iPS sacs. Erythroid differentiation from MSC-derived iPS sacs resulted in greater amounts of erythroid cells (GPA+) and higher β-globin (and βS-globin) expression, comparable to ES sac-derived cells. These data demonstrate that human MSC-derived iPS sacs allow for more efficient erythroid cell generation with higher β-globin production, likely due to heightened emergence of immature progenitors. Our findings should be important for iPS cell-derived erythroid cell generation.

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell transfusion. However, when using traditional methods with embryoid bodies, ES cell-derived erythroid cells predominantly express embryonic type ε-globin, with lesser fetal type γ-globin and very little adult type β-globin. Furthermore, no β-globin expression is detected in iPS cell-derived erythroid cells. ES cell-derived sacs (ES sacs) have been recently used to generate functional platelets. Due to its unique structure, we hypothesized that ES sacs serve as hemangioblast-like progenitors capable to generate definitive erythroid cells that express β-globin. With our ES sac-derived erythroid differentiation protocol, we obtained ∼120 erythroid cells per single ES cell. Both primitive (ε-globin expressing) and definitive (γ- and β-globin expressing) erythroid cells were generated from not only ES cells but also iPS cells. Primitive erythropoiesis is gradually switched to definitive erythropoiesis during prolonged ES sac maturation, concurrent with the emergence of hematopoietic progenitor cells. Primitive and definitive erythroid progenitor cells were selected on the basis of glycophorin A or CD34 expression from cells within the ES sacs before erythroid differentiation. This selection and differentiation strategy represents an important step toward the development of in vitro erythroid cell production systems from pluripotent stem cells. Further optimization to improve expansion should be required for clinical application.

ObjectiveChanges in eating habits may be influential in the ever-increasing rate of childhood obesity. Our aim was to determine whether those children who consume olive oil have a lower risk of weight gain compared with children who consume other oils.Design and methodsThe study included 18 girls and 74 boys, all aged 13–166 months. A survey was completed for each subject about eating habits and physical activity. A sample of subcutaneous adipose tissue was also obtained for cellular study. Data were recorded on the mean size of the adipocytes, the number of preadipocytes, and the concentration of particular fatty acids. The weight and height of the children were measured 13 months later.ResultsThe likelihood that after 1 year the children would have increased their body mass index (BMI)Z-score above the initial score was less in the children who consumed only olive oil (odds ratio (OR)=0.22; 95% confidence interval (CI): 0.08–0.63;P=0.005). These results remained after adjusting for age, physical activity and BMI (OR=0.19; 95% CI: 0.06–0.61;P=0.005) and after adjusting for age, physical activity and adipocyte volume (OR=0.15; 95% CI: 0.04–0.52;P=0.003).ConclusionsDiets with mono unsaturated fatty acid (MUFA)-rich olive oil could reduce the risk of obesity in childhood.

Background Insulin has several biological functions besides glycaemic control. We investigated and compared the effects of six different commercial insulins on adipocyte cell differentiation, the lipolytic activity of differentiated cells, and the expression levels of genes involved in adipogenesis and associated with insulin activity. Materials and methods 3T3-L1 cells were induced to differentiate with six commercial insulins: glargine, lispro, aspart, detemir, NPH and regular recombinant human insulin (used as control). Cell differentiation, lipolysis and gene expression were measured at day 7 (D7) and day 10 (D10) after induction of differentiation in these cells. Results The highest values of cell differentiation and lipolysis were found at D10 for all the insulins used. Preadipocyte differentiation differed at both times depending on the insulin used, with detemir insulin being the least adipogenic. The PPARγ mRNA level varied according to the insulin and was a good genetic marker of adipogenesis at D7. Cells treated with glargine insulin showed the highest lipolysis and HSL expression on both days. Gene expression levels of InsR, SREBP-1c and SCD-1 differed depending on the insulin studied. Conclusions Detemir insulin was the least adipogenic of the insulins tested, whereas treatment with glargine insulin tended to produce the highest lipolysis levels. Under these experimental conditions, the modifications made in commercial insulins to improve glycaemic control also affect adipocyte differentiation, the lipolysis level of differentiated cells, and the expression of different genes that can modify metabolic pathways independently of glucose metabolism. © 2011 The Authors. European Journal of Clinical Investigation © 2011 Stichting European Society for Clinical Investigation Journal Foundation.

A reliable cell line capable of robust in vitro erythroid differentiation would be useful to investigate red blood cell (RBC) biology and genetic strategies for RBC diseases. K562 cells are widely utilized for erythroid differentiation; however, current differentiation methods are insufficient to analyze globin proteins. In this study, we sought to improve erythroid differentiation from K562 cells to enable protein-level globin analysis. K562 cells were exposed to a variety of reagents, including hemin, rapamycin, imatinib, and/or decitabine (known erythroid inducers), and cultured in a basic culture medium or erythropoietin-based differentiation medium. All single reagents induced observable erythroid differentiation with higher glycophorin A (GPA) expression but were insufficient to produce detectable globin proteins. We then evaluated various combinations of these reagents and developed a method incorporating imatinib preexposure and an erythropoietin-based differentiation culture containing both rapamycin and decitabine capable of efficient erythroid differentiation, high-level GPA expression (>90%), and high-level globin production at protein levels detectable by hemoglobin electrophoresis and high performance liquid chromatography. In addition, β-globin gene transfer resulted in detectable adult hemoglobin. In summary, we developed an in vitro K562 erythroid differentiation model with high-level globin production. This model provides a practical evaluation tool for hemoglobin production in human erythroid cells.

Background and aims:As interleukin-6 (IL-6) has an important role in general metabolism with high circulating levels in obesity and other associated diseases, the factors regulating its synthesis and release have been considered possible therapeutic targets and have recently been studied. We examined the influence of three different diets, each having a different fatty acid compositionsaturated, monounsaturated or polyunsaturated (coconut oil, olive oil and sunflower oil diets), on IL-6 release from rat adipocytes, and the interaction between diet and other regulatory factors of IL-6 release, such as epinephrine.Methods:A group of rats was assigned to one of the three different diets, each with a significantly different concentration of saturated, monounsaturated and polyunsaturated fatty acids. Samples were taken from the omental adipose tissue for measurement of the triacylglycerol fatty acid composition of the tissues and for adipocyte isolation. IL-6 release from adipocytes was measured in vitro, under nonstimulated conditions and also with two concentrations of epinephrine in the medium.Results:Animals fed with the olive oil diet showed lower values of IL-6 release with and without epinephrine stimulation. IL-6 release from adipocytes varied according to the diet, but not according to epinephrine dose. However, a significant interaction was found between the epinephrine dose and the diet in IL-6 release regulation. Conclusions:IL-6 release from adipocytes was markedly regulated by the dietary fatty acid composition, even under epinephrine stimulation, with lower values of IL-6 release in the olive oil diet. The study also showed that epinephrine regulation of IL-6 release was related to the diet. © 2010 Macmillan Publishers Limited All rights reserved.

It has been suggested that hormones released after nutrient absorption, such as glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide 2 (GLP-2), could be responsible for changes in bone resorption. However, information about the role of GLP-1 in this regard is scanty. Diabetes-related bone loss occurs as a consequence of poor control of glucose homeostasis, but the relationship between osteoporosis and type 2 diabetes remains unclear. Since GLP-1 is decreased in the latter condition, we evaluated some bone characteristics in streptozotocin-induced type 2 diabetic (T2D) and fructose-induced insulin-resistant (IR) rat models compared to normal (N) and the effect of GLP-1 or saline (control) treatment (3 days by osmotic pump). Blood was taken before and after treatment for plasma measurements; tibiae and femora were collected for gene expression of bone markers (RT-PCR) and structure (microCT) analysis. Compared to N, plasma glucose and insulin were, respectively, higher and lower in T2D; osteocalcin (OC) and tartrate-resistant alkaline phosphatase 5b were lower; phosphate in IR showed a tendency to be higher; PTH was not different in T2D and IR; all parameters were unchanged after GLP-1 infusion. Bone OC, osteoprotegerin (OPG) and RANKL mRNA were lower in T2D and IR; GLP-1 increased OC and OPG in all groups and RANKL in T2D. Compared to N, trabecular bone parameters showed an increased degree of anisotropy in T2D and IR, which was reduced after GLP-1. These findings show an insulin-independent anabolic effect of GLP-1 and suggest that GLP-1 could be a useful therapeutic agent for improving the deficient bone formation and bone structure associated with glucose intolerance.

Introduction Irisin activates the thermogenic function in adipose tissues. However, little is known on the association between human irisin and different cardiometabolic risk factors. We analyse the influence of morbid obesity on irisin levels and its relation with leptin and different cardiovascular risk factors. Material and methods We measured the serum irisin level and the fibronectin type III domain containing 5 (FNDC5) expression in adipose tissue from 33 morbidly obese subjects and 12 nonobese subjects. We also studied the effect of leptin on FNDC5 expression. Results Serum irisin was higher in the nonobese subjects than in morbidly obese subjects, both before (P = 0·043) and after bariatric surgery (P = 0·042). The variable that best explained the serum irisin levels in a multiple linear regression model was the waist-to-hip ratio (WHR) (R2 = 0·201) (Beta = −0·357, P = 0·046). Those morbidly obese subjects with android-type obesity had lower serum irisin levels than those with gynecoid-type obesity, both before (P = 0·027) and after bariatric surgery (P = 0·006). Only the percentage change in WHR was associated with serum irisin levels after bariatric surgery (r = −0·529, P = 0·005). FNDC5 expression levels in subcutaneous adipose tissue (SAT) were higher in the nonobese than in the morbidly obese subjects (P = 0·042). In SAT explants from nonobese subjects, leptin (20 and 150 ng/mL) produced a decrease in FNDC5 expression (P = 0·009 and P = 0·037, respectively). Conclusions We showed decreased serum irisin levels in morbidly obese subjects, related mainly to WHR. FNDC5 expression could be regulated by leptin.

Background Childhood obesity has grown very fast over recent decades and now it represents a serious public health problem. The number of adipocytes is set in childhood and adolescence and then, an effective understanding of the development of adipose tissue during these periods will help in the prevention of this pathology. Objectives The current study aimed to determine which adipose tissue characteristics are related to a high weight Z-score in childhood. Patients and Methods The current study included 82 children aged 5-130 months who underwent inguinal hernia surgery. Anthropometric variables were measured, and a nutritional and physical activity questionnaire was completed. Subcutaneous adipose tissue samples, taken during the operation, were analyzed for preadipocyte number, adipocyte volume, fatty acid composition (gas chromatography of FAME), and relative gene expression of various genes (real time PCR). Results The results showed that children with a higher weight Z-score spend more time in sedentary activities and less time running or involved in active games. SCD-1 activity index, arachidonic/linoleic index, and adipocyte volume were significantly higher in children with a weight Z-score greater than 0. The preadipocyte number and the genetic expression of the studied genes did not differ between the groups. A multiple regression analysis was done to determine which variables were related to the weight Z-score. R2 values indicated that the model which included adipocyte volume, SREBP-1c, SCD-1 expression, and activity index, predicted 59% of the variability in the weight Z-score among the children. The main variables associated with adipocyte volume were PPARγ, Adiponectin, CB1R expressions, as well as the SCD-1 activity and normalized weight. Conclusions It was concluded that in childhood, the weight Z-score is related to adipocyte volume and adipose tissue gene expression.

Clinical Trial; Journal Article; Research Support, Non-U.S. Gov't; BACKGROUND. Transsexual persons afford a very suitable model to study the effect of sex steroids on uric acid metabolism. DESIGN. This was a prospective study to evaluate the uric acid levels and fractional excretion of uric acid (FEUA) in a cohort of 69 healthy transsexual persons, 22 male-to-female transsexuals (MFTs) and 47 female-to-male transsexuals (FMTs).The subjects were studied at baseline and 1 and 2 yr after starting cross-sex hormone treatment. RESULTS. The baseline levels of uric acid were higher in the MFT group.Compared with baseline, uric acid levels had fallen significantly after 1 yr of hormone therapy in the MFT group and had risen significantly in the FMT group. The baseline FEUA was greater in the FMT group. After 2 yr of cross-sex hormone therapy, the FEUA had increased in MFTs (P = 0.001) and fallen in FMTs (P = 0.004).In MFTs, the levels of uric acid at 2 yr were lower in those who had received higher doses of estrogens (P = 0.03),and the FEUA was higher (P = 0.04).The FEUA at 2 yr was associated with both the estrogen dose (P = 0.02) and the serum levels of estradiol-17beta (P =0.03).In MFTs, a correlation was found after 2 yr of therapy between the homeostasis model assessment of insulin resistance and the serum uric acid (r = 0.59; P = 0.01). CONCLUSIONS. Serum levels of uric acid and the FEUA are altered in transsexuals as a result of cross-sex hormone therapy.The results concerning the MFT group support the hypothesis that the lower levels of uric acid in women are due to estrogen-induced increases in FEUA. Yes