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Pyruvate kinase deficiency (PKD), an autosomal-recessive disorder, is the main cause of chronic non-spherocytic hemolytic anemia. PKD is caused by mutations in the pyruvate kinase, liver and red blood cell (PKLR) gene, which encodes for the erythroid pyruvate kinase protein (RPK). RPK is implicated in the last step of anaerobic glycolysis in red blood cells (RBCs), responsible for the maintenance of normal erythrocyte ATP levels. The only curative treatment for PKD is allogeneic hematopoietic stem and progenitor cell (HSPC) transplant, associated with a significant morbidity and mortality, especially relevant in PKD patients. Here, we address the correction of PKD through precise gene editing at the PKLR endogenous locus to keep the tight regulation of RPK enzyme during erythropoiesis. We combined CRISPR-Cas9 system and donor recombinant adeno-associated vector (rAAV) delivery to build an efficient, safe, and clinically applicable system to knock in therapeutic sequences at the translation start site of the RPK isoform in human hematopoietic progenitors. Edited human hematopoietic progenitors efficiently reconstituted human hematopoiesis in primary and secondary immunodeficient mice. Erythroid cells derived from edited PKD-HSPCs recovered normal ATP levels, demonstrating the restoration of RPK function in PKD erythropoiesis after gene editing. Our gene-editing strategy may represent a lifelong therapy to correct RPK functionality in RBCs for PKD patients., Graphical abstract, Combination of CRISPR-Cas9 ribonucleoprotein electroporation and donor AGAVE transduction in human hematopoietic stem and progenitor cells from pyruvate kinase deficiency patients allows efficient gene editing with restoration of erythrocyte energetic balance. Maintenance of stem cell properties without detectable off-targets indicates clinical applicability of gene editing for this inherited hemolytic anemia.

Hematopoietic Stem and Progenitor Cells are crucial in the maintenance of lifelong production of all blood cells. These Stem Cells are highly regulated to maintain homeostasis through a delicate balance between quiescence, self-renewal and differentiation. However, this balance is altered during the hematopoietic recovery after Hematopoietic Stem and Progenitor Cell Transplantation. Transplantation efficacy can be limited by inadequate Hematopoietic Stem Cells number, poor homing, low level of engraftment, or limited self-renewal. As recent evidences indicate that estrogens are involved in regulating the hematopoiesis, we sought to examine whether natural estrogens (estrone or E1, estradiol or E2, estriol or E3 and estetrol or E4) modulate human Hematopoietic Stem and Progenitor Cells. Our results show that human Hematopoietic Stem and Progenitor Cell subsets express estrogen receptors, and whose signaling is activated by E2 and E4 on these cells. Additionally, these natural estrogens cause different effects on human Progenitors in vitro. We found that both E2 and E4 expand human Hematopoietic Stem and Progenitor Cells. However, E4 was the best tolerated estrogen and promoted cell cycle of human Hematopoietic Progenitors. Furthermore, we identified that E2 and, more significantly, E4 doubled human hematopoietic engraftment in immunodeficient mice without altering other Hematopoietic Stem and Progenitor Cells properties. Finally, the impact of E4 on promoting human hematopoietic engraftment in immunodeficient mice might be mediated through the regulation of mesenchymal stromal cells in the bone marrow niche. Together, our data demonstrate that E4 is well tolerated and enhances human reconstitution in immunodeficient mice, directly by modulating human Hematopoietic Progenitor properties and indirectly by interacting with the bone marrow niche. This application might have particular relevance to ameliorate the hematopoietic recovery after myeloablative conditioning, especially when limiting numbers of Hematopoietic Stem and Progenitor Cells are available.

Congenital dyserythropoietic anemias (CDAs) are a group of inherited anemias that affect the development of the erythroid lineage. CDA type II is the most common one: it accounts for around 60% of all cases, and more than 600 cases have been reported so far. CDA II is caused by biallelic mutations in the SEC23B gene and is characterized by ineffective erythropoiesis with morphologic abnormalities of erythroblasts, hemolysis, and secondary iron overload, which is the most frequent complication. Patients usually suffer from variable degrees of jaundice, splenomegaly, and absolute reticulocyte count inadequate depending on the degree of anemia. Hydrops fetalis, aplastic crisis and gallstones are other associated clinical signs. CDA II bone marrow is characterized by the presence of more than 10% mature binucleated erythroblasts. Another distinctive feature of CDA II erythrocytes is hypoglycosylation of membrane proteins. The management of CDA II is generally limited to blood transfusion and iron chelation. Splenectomy has proved to reduce the number of transfusions in CDA II patients. However, allogenic hematopoietic stem cell transplant (HSCT) represents the only curative option for this disease. Autologous HSCT of genetically corrected cells will mean a definitive treatment for CDA II, overcoming the limitations of allogeneic HSCT, such as limited availability of HLA-matched donors, infections linked to immunosuppression or development of graft versus host disease. This strategy has been used to treat many inherited hematological diseases, including red blood cell diseases such as β-thalassemia, sickle cell disease or pyruvate kinase deficiency. Therefore, we have addressed a similar strategy to be applied to CDAII patients. Two different lentiviral vectors carrying either wild type or codon optimized versions of SEC23B cDNA (wtSEC23B LV or coSEC23B LV, respectively) under the control of human phosphoglycerate kinase promoter (PGK) have been developed. Taking advantage of a CDA II model, in which SEC23B knock-out was done in human hematopoietic progenitors through gene editing, we have determined the most effective SEC23B LV version and the most suitable multiplicity of infection (MOI) to compensate protein deficiency. SEC23B knock out human hematopoietic progenitors (CD34 + cells; 80% frame shift mutations; SEC23BKO) showed a sharp reduction in SEC23B protein level. Those SEC23BKO hematopoietic progenitors were transduced with both lentiviral vectors at MOIs ranged from 3 to 25. We observed that SEC23B protein reached physiological or even supraphysiological levels. In addition, the reduction in the number of erythroid colony forming units (CFUs) identified in SEC23BKO CD34 + cells, was partially restored in the LV transduced SEC23BKO progenitors. Significantly, we observed a clear correlation between the used MOI and the vector copy number (VCN) in the CFUs derived from transduced SEC23BKO CD34 + cells. Furthermore, SEC23BKO hematopoietic progenitors were subjected to an in vitro erythroid differentiation protocol. A sharp decrease in the cell growth throughout erythroid differentiation was observed in SEC23BKO condition. However, the transduction with any of SEC23B LVs at MOIs above 10 was able to recover cell expansion to values equal to wild type cells. Interestingly, total level of protein glycosylation during erythroid differentiation was enhanced after SEC23B LV transduction. Glycosylation level in wtSEC23B LV transduced SEC23BKO cells was most similar to the level in wild type cells. Then, we transduced peripheral blood-derived hematopoietic progenitors (PB-CD34 + cells) from a CDA II patient with wtSEC23B LV at MOI 25 and differentiated in vitro to erythroid cells. A complete restauration of SEC23B protein expression and a cell growth increase of wtSEC23B transduced CDAII was observed with vector copy numbers of 0.3 after 14 days under erythroid conditions. More importantly, we could find a decrease in the percentage of bi-/multinucleated erythroid cells generated in vitro after wtSEC23B LV transduction. In summary, SEC23B LV compensate the SEC23B deficiency in SEC23BKO and in CDAII hematopoietic progenitor cells, paving the way for gene therapy of autologous hematopoietic stem and progenitor cell as an alternative and feasible treatment for CDA II. Disclosures Bianchi: Agios pharmaceutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sanchez: Bloodgenetics: Other: Co-Founder and promoter; UIC: Current Employment. Ramirez: VIVEBiotech: Current Employment. Segovia: Rocket Pharmaceuticals, Inc.: Consultancy, Research Funding. Quintana Bustamante: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company.

Pyruvate kinase deficiency (PKD) is an autosomal recessive disorder caused by mutations in the PKLR gene that lead to a reduction of the erythroid pyruvate kinase (RPK) protein activity, which causes an energetic imbalance in PKD erythroid cells. This disease is associated with reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected patients. In selected cases, allogeneic Hematopoietic Stem Cell Transplantation has been shown to correct the disorder. Therefore, autologous HSCT of genetically corrected cells will offer a durable and curative therapeutic option. In fact, a global Phase I clinical trial (clinicaltrials.gov #NCT04105166) is underway to evaluate the feasibility and safety of lentiviral mediated gene therapy with severe PKD. Looking for a guided integration of the exogenous therapeutic DNA sequences, herein we conducted a gene editing approach to correct PKD in human Hematopoietic Stem Cells (HSCs). We developed a knock-in approach to insert either a TurboGFP expression cassette or a promotor-less therapeutic codon optimized RPK cDNA (coRPK) at the genomic starting site of the PKLR gene, a gene editing strategy that will correct most of the mutations present in PKD patients. This gene editing approach combines RNP nucleofection and adeno-associated viral vector (AAV6) mediated delivery of homologous donors. In order to assess the safety of the proposed gene editing approach, we performed GUIDE-Seq and rhAmpSeqTM analyses of different single guide RNAs targeting the PKLR starting site. We found PKLR sgRNAs that showed a high targeting efficiency, up to 40% targeted hematopoietic progenitors (in vitro semisolid colony forming units) and a safety profile, with no off-targets detected above threshold values (0.1%) and in the absence of cellular toxicity, when applied to healthy cord blood CD34+ (CB-CD34+) cells. These gene-edited CB-CD34+ cells engrafted efficiently in both primary and secondary immunodeficient NSG recipient mice, demonstrating the gene editing in long-term hematopoietic repopulating HSCs. Furthermore, we evaluated the therapeutic potential of this gene editing strategy to restore the energetic imbalance in erythroid cells derived from PKD patients. CD34+ cells from 4 different PKD patients, were purified, nucleofected with PKLR sgRNA and transduced with AAV-coRPK donor. In vitro differentiated erythroid cells derived from edited PKD CD34+ expressed coRPK mRNA and restored their energetic defect up to normal values obtained in in vitro differentiated erythroid cells from healthy donor cells. Moreover, gene edited mobilized Peripheral Blood CD34+ (mPB-CD34+) cells from a PKD patient engrafted efficiently in immunodeficient NBSGW mice, having human cells that showed the specific integration of coRPK donor 2 months post-transplant. Overall, these results demonstrate the feasibility and safety of PKLR gene editing in human HSPCs and, therefore, its potential clinical application for the treatment of PKD patients. Disclosures Dever: Integral Medicines: Current Employment. Turk:Integrated DNA Technologies, Inc. (IDT): Current Employment, Current equity holder in publicly-traded company. Bianchi:Agios Pharmaceuticals: Other: Scientific Advisor. Behlke:Integrated DNA Technologies, Inc. (IDT): Current Employment, Current equity holder in publicly-traded company. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from this company., Patents & Royalties, Research Funding. Segovia:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from the Company., Patents & Royalties, Research Funding.

Congenital dyserythropoietic anemias (CDAs) are a group of inherited anemias that affect the development of the erythoid lineage. They are characterized by ineffective erythropoiesis with distinct morphologic abnormalities of erythroblasts, a degree of hemolysis, and secondary hemochromatosis. Patients usually present with congenital anemia, jaundice, splenomegaly, and an absolute reticulocyte count inadequate for the degree of anemia. CDA type II (CDAII) is the most frequent type. CDAII patients show anemia of variable degrees, and 20% are transfusion dependent. The most specific finding of CDAII marrow is the presence of more than 10% mature binucleated erythroblasts with two nuclei at the same erythroid maturation stage. Treatment of CDAII patients may involve blood transfusions, iron chelation therapy and splenectomy. The only described definitive therapy is allogeneic bone marrow transplantation, which implies additional side effects for these patients. Therefore, new therapeutic approaches are needed. CDA II is caused by mutations in the SEC23B gene. SEC23B is part of coat protein complex II (COPII). COPII is involved in protein processing and Golgi-reticulum trafficking. However, how mutations in SEC23B cause CDA II is not known yet. Therefore, studying the role of SEC23B in the erythropoiesis will help to elucidate the underlying mechanism of CDA II and to develop new therapeutic approaches for the disease. We have developed a CDA II model in human cells through the introduction of genomic mutations in the gene using the CRISPR/Cas9 gene editing system. Different single guides RNAs (sgRNA) targeting the start of the coding sequence of human SEC23B gene were designed and tested in human erythroleukemia K562 cell line and in healthy human cord blood hematopoietic stem and progenitors (hCB-CD34+). The gene editing outcome at SEC23B gene was assessed at: i) genomic level through Sanger sequencing, Inference of CRISPR Edits (ICE) and Next-Generation Sequencing (NGS). ii) Protein level through Western-blot analysis. iii) Functional level through morphological analysis and erythroid differentiation either in vitro or in vivo in human hematopoietic chimeras in NOD.Cg-KitW-41JTyr+PrkdcscidIl2rgtm1Wjl/ThomJ (NBSGW) mice. K562 cells were nucleofected with three different sgRNAs, as ribonucleoprotein (RNP), independently or in combination. Afterwards, seventy five K562 clones were established from the cells nucleofected with the most efficient sgRNA or with the combination of the three sgRNAs. Forty per cent of them showed a high efficiency of knock-out (higher than 50% of alleles). Eight SEC23BKO clones were selected for further analysis. All of those eight clones showed a reduction in SEC23B protein and six of them had a lower proliferation than control cells and morphological abnormalities, such as presence of bi/multinucleated cells. Moreover, when CB-CD34+ cells were nucleofected with the most efficient sgRNA or with the combination of the three sgRNAs, up to 80% of knock-out efficiency and close to 90% reduction of SEC23B protein were obtained. Interestingly, when those gene edited hematopoietic progenitors were differentiated in vitro to erythroid cells, their terminal differentiation was hampered, with a reduce percentage of enucleated cells and the presence of high number of bi/multinucleated cells. Similarly, the in vivo erythroid differentiation of these gene edited progenitors two months after HSPC transplant into NBSGW mice showed again an impairment of terminal erythroid differentiation with an increment in the percentage of erythroid bi/multinucleated cells without altering other human hematopoietic lineages. In summary, CRISPR/Cas9 system has been used to model CDA II in a human cell line and in human hematopoietic progenitors through the knock-out of SEC23B gene. Our system reproduced the most relevant feature characteristic of CDA II pathology. This gene editing based CDA II model will allow the study of how mutations in SEC23B cause CDA II and the development of new therapeutic strategies to cure this disease. Disclosures Tornador: Bloodgenetics: Current Employment. Sanchez:Bloodgenetics: Current Employment. Segovia:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from the Company., Patents & Royalties, Research Funding.

Hematopoietic Stem Cells (HSCs) is a rare cell population that sits atop a hierarchy of progenitors that become progressively restricted to several or a single blood lineage. HSCs are capable of self-renewal and multipotent differentiation to all blood cell lineages. HSCs are crucial in the maintenance of lifelong production of all blood cells. HSCs are highly regulated to maintain homeostasis through a delicate balance between quiescence, self-renewal and differentiation. However, this balance is altered during the hematopoietic recovery after Hematopoietic Stem Cell Transplantation (HSCT). HSCT is routinely used to reconstitute hematopoiesis after myeloablation, being the most commonly-used cell therapy. HSCT efficacy and multilineage reconstitution can be limited by inadequate HSC number, poor homing, engraftment, or limited self-renewal. Recent evidence indicates that estrogens are involved in regulating the hematopoietic system homeostasis. Estrogens are the primary female sex hormones and are responsible for controlling many cellular processes including growth, differentiation and function of the reproductive system. However, estrogens have also been proposed to regulate HSCs. b-Estradiol (E2) was shown to promote the cell cycle of HSCs and multipotent progenitors (MPPs) and increase erythroid differentiation in females (1). On the other hand, tamoxifen reduces the number of MPPs and short-term HSCs but activates proliferation of long-term HSCs (2). The potential clinical application of estrogens in HSCT mainly derives from the possibility that these drugs may enhance the engraftment of transplanted HSCs, thus reducing side effects associated to myeloablative conditioning. Here, we show that a short-term treatment of immunodeficient mice transplanted with hCD34+ cells with estrogens such as E2 and estetrol (E4) improves human hematopoietic engraftment. Fifty-thousand cord blood CD34+ cells (CB-CD34+) were transplanted into sublethally irradiated immunodeficient NSG mice. Three days after transplantation, mice were treated for four days with daily subcutaneous doses of E2, E4 or vehicle. Human hematopoietic engraftment was evaluated in the BM of transplanted mice at four months later. E2 and E4 estrogens increased the proportion of hCD45+ cells 1.8-fold and 2.4-fold as compared to values determined in control mice, without modifying the proportion of myeloid and lymphoid lineages. Significantly, animals treated with either estrogen had significantly higher levels of human hematopoietic progenitors (hCD45+CD34+). To study the engraftment of long-term engraftment HSCs in transplanted mice, human CD45+ cells from primary recipients were sorted and transplanted in secondary NSG recipients. Three months after transplants, the proportion of human hematopoietic cells in secondary recipients was also higher when primary recipients were treated with E2 or E4 than in vehicle-treated animals. Improved engraftments associated to the administration of E2 or E4 estrogens were confirmed when very low doses of CB-CD34+ cells were transplanted (5x103 hCD34+/mouse) in recipients of either sex. Collectively, our data support a new application of estrogens to improve the hematopoietic recovery after HSCT. This application may have particular relevance to enhance the hematopoietic recovery after myeloablative conditioning and when limiting numbers of HSCs are available. Disclosures Bueren: Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Segovia:Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Pyruvate kinase deficiency (PKD) is the most common erythroid inherited enzymatic defect causing chronic nonspherocytic hemolytic anemia. PKD is an autosomal recessive disorder caused by mutations in the PKLR gene, which led in a total or partial reduction of the activity of the erythroid pyruvate kinase (RPK) protein. To date, more than 200 different mutations in the PKLR gene have been related with PKD. The disease is associated with reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected patients. Treatments for PKD are mainly palliative, including regular red blood cell transfusion, splenectomy and iron chelation therapy. Allogeneic hematopoietic stem cell transplant (HSCT) represents the only curative treatment for severely affected patients, so far. Autologous HSCT of genetically corrected cells would offer a durable and curative clinical option. Over the last years, gene editing has emerged as a promising gene therapy approach for blood cell disorders, where genetic alterations can be accurately corrected. The high level of correction got in hematopoietic progenitors and stem cells without remarkable off-target effects suggests that the clinical use of gene editing therapy to correct genetic hematopoietic diseases is highly likely in a short term. Here, we present two gene editing approaches to correct PKD in human hematopoietic cells based of specific point mutation correction and in cDNA knock-in of a codon optimized version the RPK cDNA. We have designed both strategies to maintain the endogenous regulation of RPK once the gene editing has been carried out. First, we designed specific sgRNA targeting NM_000298.5(PKLR):c.359C>T mutation reported as pathogenic in PKD patients, and a single-stranded donor oligonucleotide (ssODN) to correct this mutation. When both sgRNA/Cas9 ribonucleoprotein (RNP) and ssODN were nucleofected together in a heterozygous PKD patient-lymphoblastic cell line (PKD LCL), around 5% of total alleles were correctly edited. In a second strategy, we developed a knock-in gene editing strategy at the genomic starting site of the PKLR gene by combining RNP electroporation and the adeno-associated viral vector (AAV) delivery of the recombination matrix. Specific gRNAs generating up to 60% indels at the RPK starting site were generated. Two different AAV constructs flanked by specific homologous arms were generated to delivery either a TurboGFP expression cassette or a promotor-less therapeutic coRPK. Up to 60% donor integration and stable expression of turbo EGFP and of coRPK driven by PKLR endogenous promoter was obtained in K562 erythroleukemia cells. Similar gene editing efficacies were obtained in human CB-CD34+. Specific integration and stable expression of the transgenes were detected in up to 30% colony forming units (CFUs). Moreover, gene edited cells engrafted efficiently in NSG mice. These results demonstrate the feasibility of editing the PKLR locus in hematopoietic progenitors and hematopoietic stem cells at efficiencies that could be clinically applicable to treat Pyruvate Kinase deficiency. Disclosures Bueren: Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Porteus:CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Segovia:Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.