ALBERT

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

Description: All currently marketed Factor VIII (FVIII) molecules are administered intravenously (IV) for the treatment of hemophilia A (HemA). Conventional FVIII prophylaxis requires a dosing interval of three times per week to every other day. This frequent dosing regimen necessitates repeated venous access and is associated with complications such as secondary infection in children with a venous port/catheter. More recently, extended half-life variants of FVIII have been shown in clinical trials to decrease the dosing interval to twice weekly or less frequent IV dosing, which reduces, but does not eliminate, the burden of treatment. A FVIII molecule with further prolonged half-life and subcutaneous (SQ) delivery potential could significantly relieve the treatment burden for HemA patients and improve the adherence rate to FVIII prophylaxis. Recombinant FVIIIFc-VWF-XTEN has been shown to not bind endogenous VWF, and is able to achieve a 4-fold extension of half-life in hemophilia A mice compared to conventional FVIII, well beyond the approximately 2-fold half-life extension limit demonstrated by other long-acting FVIII variants that bind endogenous VWF. It comprises of two polypeptide chains: 1) a single chain B-domain deleted FVIIIFc-XTEN chain with a XTEN polypeptide inserted at the B-domain region of native FVIII sequence, and 2) a VWF D'D3-XTEN-Fc chain xtend one that n TEN fragemnt o FVIII prophylaxis.ntial rity of the patients depending on the half-life of the FVIII molecule. with a second XTEN polypeptide inserted between D'D3 domain and Fc. The rFVIIIFc-VWF-XTEN protein was produced in HEK293 cells and affinity purified using VIIISelect resin. The pharmacokinetic (PK) profiles of intravenously (IV) and subcutaneously (SQ) administered rFVIIIFc-VWF-XTEN were compared to those of rFVIII in HemA mice. The duration of the in vivo efficacy of rFVIIIFc-VWF-XTEN post-SQ delivery was assessed in a HemA mouse tail vein transection (TVT) bleeding model. After intravenous dosing in HemA mice, we observed a linear PK profile for rFVIIIFc-VWF-XTEN within the therapeutic dose range (25, 50, 100 IU/kg). The half-life of IV-administered rFVIIIFc-VWF-XTEN was about 37 h, which is more than 4-fold longer than that of rFVIII. In addition, animals that received 25 IU/kg of rFVIIIFc-VWF-XTEN treatment retained 5% of normal FVIII activity at 120 h post-dosing, which suggests the potential for full protection from spontaneous bleeding in this animal model. When delivered subcutaneously, the bioavailability of rFVIIIFc-VWF-XTEN was 20%, a significant increase compared to the bioavailability of rFVIII (less than 1%). Starting at 24 h post-dosing, subcutaneous administration of rFVIIIFc-VWF-XTEN achieved plasma FVIII levels that were equal to or greater than those attained with rFVIII delivered intravenously at the same dose. In addition, greater than 5% of normal circulating FVIII level was observed at 96 h post SQ administration of rFVIIIFc-VWF-XTEN with a 100 IU/kg dose, which provided 80% protection on survival in mice subjected to tail vein transection injury. These results suggest that rFVIIIFc-VWF-XTEN could enable less frequent FVIII replacement treatment compared to rFVIII even when administered subcutaneously. The VWF independence of rFVIIIFc-VWF-XTEN enables a 4-fold increase in circulating half-life compared to that of rFVIII. Also, the addition of D'D3 domains and the two XTEN insertions dramatically increases subcutaneous bioavailability to 20%, compared to less than 1% with conventional FVIII. These unique properties of rFVIIIFc-VWF-XTEN make it a potential candidate for both IV and SQ treatments for hemophilia A. Disclosures Drager: Biogen: Employment, Equity Ownership. Patarroyo-White:Biogen: Employment, Equity Ownership. Chao:Biogen: Employment, Equity Ownership. Ismail:Biogen: Employment. Liu:Biogen: Employment, Equity Ownership. Holthaus:Biogen: Employment. Chhabra:Biogen: Employment, Equity Ownership. Kulman:Biogen: Employment. Schellenberger:Amunix Operating Inc: Employment. Liu:Biogen: Employment, Equity Ownership. Peters:Biogen: Employment.