-Author name in bold denotes the presenting author
-Asterisk * with author name denotes a Non-ASH member
Clinically Relevant Abstract denotes an abstract that is clinically relevant.

PhD Trainee denotes that this is a recommended PHD Trainee Session.

Ticketed Session denotes that this is a ticketed session.

3368 In Vivo HSC Gene Therapy with Base Editors Allows for Efficient Reactivation of Fetal Globin in Beta-Yac Mice

Program: Oral and Poster Abstracts
Session: 801. Gene Editing, Therapy and Transfer: Poster III
Hematology Disease Topics & Pathways:
sickle cell disease, Biological, Diseases, thalassemia, Therapies, Hemoglobinopathies, gene therapy, Technology and Procedures, Clinically relevant
Monday, December 7, 2020, 7:00 AM-3:30 PM

Chang Li1*, Afrodite Georgakopoulou, PhD student2*, Sucheol Gil3* and Andre Lieber, MD, PhD3

1Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, WA
2Gene and Cell Therapy Center, Hematology-HCT Unit, G. Papanikolaou Hospital, Thessaloniki, Greece
3University of Washington, Seattle, WA

Base editors are capable of installing precise nucleotide mutations at targeted genomic loci and present the advantage of avoiding double-stranded DNA breaks. Here, we aimed to target critical motifs regulating gamma-globin reactivation with base editors delivered via HDAd5/35++ vectors. Through optimized design, we successfully rescued a panel of cytidine and adenine base editors (CBE and ABE) targeting the BCL11A enhancer or recreating naturally occurring Hereditary Persistence of Fetal Hemoglobin (HPFH) mutations in the HBG1/2 promoter. In HUDEP-2 cells, all five tested vectors efficiently installed target base conversion and led to gamma-globin reactivation. We observed significant gamma-globin protein production (~23% over β-globin) by using an ABE vector HDAd-ABE-sgHBG#2 specific to the -113A to G HPFH mutation in HBG1/2 promoter. This vector was therefore chosen for downstream in vivo hematopoietic progenitor/stem cell (HSPC) transduction studies in mice that carry 248kb of the human β-globin locus (β-YAC mice) and thus accurately reflect globin switching. An EF1a-mgmtP140K expression cassette flanked by frt and transposon sites was included in the vector for allowing in vivo selection of transduced cells. After in vivo HSPC transduction with HDAd-ABE-HBG#2 + HDAd-SB and low doses of chemoselection, an average of over 40% HbF-positive cells in peripheral red blood cells was measured. This corresponded to ~21% gamma-globin production over human β-globin. The -113 A to G conversion in total bone marrow cells was on average 20%. Compared to untransduced mice, no alterations in hematological parameters, erythropoiesis and bone marrow cellular composition were observed after treatment, demonstrating a good safety profile of our approach. No detectable editing was found at top-scored potential off-target genomic sites. Bone marrow lineage-negative cells, isolated from primary mice at week 16 after transduction, were capable of reconstituting secondary transplanted mice with stable HbF expression. Importantly, the advantage of base editing over CRISPR/Cas9 was reflected by the markedly lower rates of intergenic 4.9kb deletion and no detectable toxicity in human CD34+ stem cells. Our observations demonstrate that base editors delivered by HDAd5/35++ vectors represent a promising strategy for precise in vivo genome engineering for the treatment of hemoglobinopathies.

Disclosures: Lieber: Ensoma, Inc: Consultancy, Research Funding.

*signifies non-member of ASH