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Dieffenbach, Carl (NIH/NIAID) [E] Cc: Tabak, Lawrence (NIH/OD) [E] Subject: Seeing Bill Gates tomorrow nih.gov>; niaid.nih.gov> nih.gov> nih.gov>; Panepinto, Julie (NIH/NHLBI) [E] niaid.nih.gov>; Fauci, Anthony (NIH/NIAID) [E] nih.gov>; Schwetz, Tara (NIH/OD) [E] Hi all, Bill Gates is at the White House tomorrow and asked to see me at 9 AM. Among the topics we will likely discuss is the HIV-SCD joint NIH-BMGF effort to develop cures that will be applicable in low income settings. Are there any new developments since the Sunnylands meeting back in May? Bill will likely ask about the future of this partnership. Thanks, Francis In vivo base editing by a single intravenous vector injection for treatment of hemoglobinopathies Chang Li, ... , Evangelia Yannaki, André Lieber JCI Insight. 2022. https://doi.org/10.1172/jci.insight.162939. In-Press Preview Individuals with beta-thalassemia or Sickle Cell Disease and hereditary persistence of fetal hemoglobin (HPFH) possessing 30% HbF appear to be symptom-free. Here, we used a non-integrating HDAd5/35++ vector expressing a highly efficient and accurate version of an adenine base editor (ABE8e) to install, in vivo, a -113A>G HPFH mutation in the gamma-globin promoters in "healthy" CD46/β-YAC mice carrying the human β-globin locus. Our in vivo hematopoietic stem cell (HSC) editing/selection strategy involves only subcutaneous and intravenous injections and does not require myeloablation and HSC transplantation. In vivo HSC base editing in CD46/β-YAC mice resulted in >60% -113A>G conversion with 30% γ-globin of human beta globin expressed in 70% of erythrocytes. Importantly, no off-target editing at sites predicted by CIRCLE-Seq or in silico was detected. Furthermore, no critical alterations in the transcriptome of in vivo edited mice were found by RNA-seq. In vitro, in HSCs from beta-thalassemia and Sickle Cell Disease patients, transduction with the base editor vector mediated efficient -113 A>G conversion and reactivation of γ-globin expression with subsequent phenotypic correction of erythroid cells. Because our in vivo base editing strategy is safe and technically simple, it has the potential for clinical application in developing countries where hemoglobinopathies are prevalent. Research Hematology Stem cells Find the latest version: https://jci.me/162939/pdf In vivo base editing by a single intravenous vector injection for treatment of hemoglobinopathies Chang Li1, Aphrodite Georgakopoulou2, Gregory A. Newby3, 4, 5, Kelcee A. Everette3, 4, 5, Evangelos Nizamis6, Kiriaki Paschoudi 2,7, Efthymia Vlachaki8, Sucheol Gil1, Anna K. Anderson1, Theodore Koob1, Lishan Huang1, Hongjie Wang1, Hans-Peter Kiem9, David R. Liu3, 4, 5, Evangelia Yannaki2, André Lieber1,10 1University of Washington, Department of Medicine, Division of Medical Genetics, Seattle, WA 98195 2Gene and Cell Therapy Center, Hematology Department, George Papanicolaou Hospital, Thessaloniki, Greece 3Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA 4Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA 5Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA 6University of Thessaly, Department of Computer Science and Biomedical Informatics, Lamia, Greece 7Aristotle University of Thessaloniki, School of Biology, Thessaloniki, Greece 8Hematological Laboratory, Second Department of Internal Medicine, Faculty of Health Sciences, School of Medicine, Aristotle University of Thessaloniki, Hippokration General Hospital, Thessaloniki, Greece 9Stem and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109 10University of Washington, Department of Pathology, Seattle, WA 98195 Conflict of interest: HPK is a paid advisor for Ensoma Bio. Corresponding authors: André Lieber: University of Washington, Box 357720, Seattle, WA 98195, phone: email: @uw.edu, phone: Chang Li: University of Washington, Box 357720, Seattle, WA 98195, phone: email: @uw.edu Abstract Individuals with -thalassemia or Sickle Cell Disease and hereditary persistence of fetal hemoglobin (HPFH) possessing 30% HbF appear to be symptom-free. Here, we used a non-integrating HDAd5/35++ vector expressing a highly efficient and accurate version of an adenine base editor (ABE8e) to install, in vivo, a -113A>G HPFH mutation in the -globin promoters in "healthy" CD46/-YAC mice carrying the human -globin locus. Our in vivo hematopoietic stem cell (HSC) editing/selection strategy involves only subcutaneous and intravenous injections and does not require myeloablation and HSC transplantation. In vivo HSC base editing in CD46/-YAC mice resulted in >60% -113A>G conversion with 30% -globin of - globin expressed in 70% of erythrocytes. Importantly, no off-target editing at sites predicted by CIRCLESeq or in silico was detected. Furthermore, no critical alterations in the transcriptome of in vivo edited mice were found by RNA-seq. In vitro, in HSCs from -thalassemia and Sickle Cell Disease patients, transduction with the base editor vector mediated efficient -113 A>G conversion and reactivation of −globin expression with subsequent phenotypic correction of erythroid cells. Because our in vivo base editing strategy is safe and technically simple, it has the potential for clinical application in developing countries where hemoglobinopathies are prevalent. Key points - In vivo HSC transduction of -YAC mice with an ABE8e expressing HDAd5/35++ vector is safe and results in efficient and stable -globin reactivation in ~70% of erythrocytes at levels of 30% of human -globin. - Therapeutic editing is achieved in thalassemia and Sickle Cell Disease patient CD34+ cells with HDAdEF1.ABE8e, leading to phenotypic correction in in vitro differentiated erythroid cells. Introduction Autologous HSC gene therapy for hemoglobinopathies has shown promising clinical efficacy 1-4. However, current protocols involving isolation of patient HSCs, their in vitro genetic modification with integrating vectors, and reinfusion of the modified HSCs following myelotoxic bone marrow conditioning, are technically complex and expensive. We are attempting to develop an in vivo HSC gene therapy approach that does not require myeloablation and integrating vectors, and that is technically easier. In this approach, we are using capsid-modified, helper-dependent HDAd5/35++ vectors (1, 2). These vectors target CD46, a receptor that is expressed on primitive HSCs (2, 3). HDAd5/35++ vectors are injected intravenously after mobilization of HSCs from the bone marrow by agents routinely used for HSC mobilization/harvesting. Mobilized HSCs are transduced while they are in the periphery. A large fraction of HSCs returns to the bone marrow. Mobilization of HSCs is critical for in vivo transduction because in the bone marrow they are surrounded by extracellular stroma proteins (4), and are not accessible to gene transfer vectors (2). To expand in vivo transduced HSCs, we currently use an in vivo selection mechanism based on a mutant O6-methylguanine-DNA methyltransferase (mgmtP140K) gene that confers resistance to O6-BG/BCNU (O6-Benzylguanine/Carmustine) given at doses that are 20-30-fold lower than used for cancer chemotherapy (5-7). We have demonstrated the safety (including the absence of clonal dominance (8-10)) and efficacy of the in vivo approach in mice (9-15) and rhesus macaques (3, 8, 16). One of our goals is to broadly apply this approach for the treatment of hemoglobinopathies, i.e. -thalassemia and Sickle Cell Disease (SCD). Because of its simplicity and lower costs, this method could be applicable in developing countries where the burden from these diseases is high (17). -thalassemia and Sickle Cell Disease (SCD) are the most common inherited diseases in humans worldwide. -thalassemia is caused by mutations in the -globin gene which result in absent (β0/β0) or deficient (β+/β+, β+/β0) β-globin chain synthesis. -thalassemia patients have a multitude of pathological symptoms and die prematurely. Most SCD patients are homozygous for a Glu6Val substitution resulting in the production of βS-globin. Clinically, SCD is characterized by recurrent episodes of severe bone pain, multi-organ failure, and early mortality. Individuals with Hereditary Persistence of Fetal Hemoglobin (HPFH) and thalassemic -globin mutations exhibit few or no pathological effects because HbF inhibits hemoglobin precipitation by reversing the globin chain imbalance and improving erythropoiesis (18). This led to a number of hematopoietic stem cell (HSC) gene therapy approaches aimed toward the reactivation of fetal/-globin, e.g. by targeting two transcriptional repressors, ZBTB7A and BCL11A, that participate in the developmental silencing of the two -globin genes (HBG1/G and HBG2/A) through binding to their respective promoter cis-regulatory elements (19). An approach based on RNA interference to silence bcl11a expression (19) or reactivation of -globin expression by CRISPR-Cas9 editing of the erythroid bcl11a enhancer (20) have delivered promising clinical data. Furthermore, preclinical studies to reactivate -globin by CRISPR/Cas9 editing of the BCL11A and ZBTB7A binding motifs within the HBG1/2 promoters have shown efficient HbF reactivation (21). Along this line, we used an in vivo HSC transduction approach to express a CRISPR/Cas9 specific to the BCL11A binding site within the HBG1/2 promoters and showed efficient −globin reactivation in transgenic mice carrying the wild-type 248-kb b-globin locus yeast artificial chromosome (-YAC mice) (22). However, we also detected an undesired deletion of the HBG1 gene due to CRISPR/Cas9 mediated double strand DNA breaks (DSBs) in