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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.

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.