-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.

535 CRISPR/Cas9 Gene-Edited Hematopoietic Stem Cell Therapy for Sickle Cell Disease

Hemoglobinopathies, Excluding Thalassemia—Basic and Translational Science
Program: Oral and Poster Abstracts
Type: Oral
Session: 113. Hemoglobinopathies, Excluding Thalassemia—Basic and Translational Science: Sickle Cell Disease—Hematopoiesis and Fetal Hemoglobin Augmentation
Monday, December 11, 2017: 7:00 AM
Bldg B, Lvl 3, B302-B303 (Georgia World Congress Center)

Vionnie W. C. Yu, PhD1*, Yi Liu, PhD1*, Matthew Curran2*, Pu Zhang, MD3*, Jennifer Snead, PhD4, Christian Schmedt5*, Yi Yang, PhD1*, Victor Guosheng Lin2*, William R Tschantz, PhD1*, Lisa Quinn1*, Carsten Russ2*, Scott Clarkson2*, Amy Janiak2*, Morag Stewart, PhD6*, Yanick Mulumba7*, Reynald Lescarbeau7*, Brad Murray7*, Jessica Seitzer7*, Walter Strapps7*, Hon-Ren Huang7*, Kevin Sloan7*, Craig S Mickanin, BS1*, Lloyd Klickstein, MD8* and Susan Stevenson1*

1Novartis Institutes for BioMedical Research, Cambridge, MA
2Novartis Institutes for BioMedical Research, Cambridge
3Beth Israel Deaconess Med. Ctr. Harvard Inst. of Med. 951, Boston, MA
4The Genomics Institute of the Novartis Research Foundation, San Diego, CA
5The Genomics Institute of the Novartis Research Foundation, Cambridge
6Intellia Therapeutics, Cambridge, MA
7Intellia Therapeutics, Cambridge
8Novartis Institutes for BioMedical Research, Cambrdige

Sickle cell disease (SCD) is a life-threatening hereditary disorder that affects thirty million individuals worldwide. The disease results from a single amino acid change in the β-globin gene, which causes polymerization of hemoglobin and deformation of red blood cells, leading to vaso-occlusion, severe pain crisis, multi-organ dysfunction, and ultimately reduction of lifespan in affected individuals. Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative therapy for SCD, yet its success is limited by donor availability and high risk of graft-versus-host disease. Increasing expression of fetal γ-globin in adults is an accepted therapeutic strategy for treating SCD. Supporting this approach are GWAS studies that have identified genetic loci, including within the BCL11A gene, that are associated with elevated fetal hemoglobin (HbF) and mitigated disease severity. Leveraging CRISPR/Cas9 genome editing technology, we aim to develop a safe and curative therapy by autologous, genome-modified HSCT leading to sustained generation of HbF-containing, functional red blood cells. Guide RNAs (gRNAs) targeting the erythroid-specific enhancer region of the BCL11A gene were assayed in CD34+ hematopoietic stem and progenitor cells (HSPCs), and one gRNA was selected for in vivo evaluation based on editing efficiency and functional potency. We achieved approximately 80% target site editing in human CD34+ HSPCs and 65% in CD34+CD38-CD90+CD45RA-CD49f+ defined LT-HSCs. Potential off-target editing sites were identified by two approaches: in silico prediction and unbiased oligo insertion method. These sites were then directly interrogated in HSPCs by targeted PCR amplification and NGS analysis, and to date our selected gRNA had zero validated off-target sites. In addition, HSPC composition was not altered following electroporation of ribonucleoprotein complex, and cells were capable of in vitro expansion and multi-lineage differentiation as measured by CFU assays. Upon in vitro differentiation into the erythroid lineage, edited cells demonstrated ~40% reduction in BCL11A mRNA with a corresponding two-fold increase in γ-globin transcript, translating to a 30-40% increase in HbF positive cells. Most importantly, when CD34+ cells derived from SCD patients were gene-edited and differentiated into erythroid cells, a similar editing efficiency and increase in HbF positive cells was observed. To evaluate whether gene editing alters the self-renewal, engraftment or lineage differentiation capacity of HSCs, gene-edited HSPCs were transplanted into NOD-scid IL2rγnull (NSG) mice to assess reconstitution of the hematopoietic system. Mice displayed 30-40% human CD45+ cell engraftment and multi-lineage reconstitution at 16 weeks following transplantation. When long-term engrafted bone marrow cells were harvested from the recipients and differentiated into erythroid cells in vitro, gene-edited bone marrow cells produced significantly higher numbers of HbF positive cells compared to mock-edited bone marrow cells. These data support the development of autologous genome-modified HSPC transplantation as a novel cell therapy for SCD.

Disclosures: Yu: Novartis Institutes for BioMedical Research: Employment. Liu: Novartis Institutes for BioMedical Research: Employment. Curran: Novartis Institutes for BioMedical Research: Employment. Zhang: Novartis: Employment. Snead: Novartis: Employment. Schmedt: Novartis: Employment. Yang: Novartis Institutes for BioMedical Research: Employment. Lin: Novartis: Employment. Tschantz: Novartis Institutes for BioMedical Research: Employment. Quinn: Novartis Institutes for BioMedical Research: Employment. Russ: Novartis: Employment. Clarkson: Novartis: Employment. Janiak: Novartis: Employment. Stewart: Intellia Therapeutics: Employment. Mulumba: Intellia Therapeutics: Employment. Lescarbeau: Intellia Therapeutics: Employment. Murray: Intellia Therapeutics: Employment. Seitzer: Intellia Therapeutics: Employment. Strapps: Intellia Therapeutics: Employment. Huang: Intellia Therapeutics: Employment. Sloan: Intellia Therapeutics: Employment. Mickanin: Novartis: Employment. Klickstein: Novartis: Employment. Stevenson: Novartis: Employment.

Previous Abstract | Next Abstract >>
*signifies non-member of ASH