RE-CREATING HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN (HPFH) WITH CRISPR/CAS9 TO TREAT SICKLE CELL DISEASE (SCD) AND BETA-THALASSEMIA (BETA-THAL)
Author(s): ,
Michelle Lin
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Elizabeth Paik
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Bibhu Mishra
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Song Chou
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
David Burkhardt
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Andrew Kernytsky
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Michael Pettiglio
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Sean Corcoran
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Yi-Shan Chen
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Kaleigh Tomkinson
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Andrew Sanginario
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Amanda Woo
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Ying Zhang
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Min Jin Lee
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Melanie Allen
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Thomas Cradick
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Siyuan Tan
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Jason West
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Michael Weinstein
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Mauricio Cortes
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Todd Borland
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Lawrence Klein
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
William Fodor
Affiliations:
Cell Therapy Group,Madison, CT,United States
,
Angela Yen
Affiliations:
Vertex Pharmaceuticals Incorporated,Boston,United States
,
Sudipta Mahajan
Affiliations:
Vertex Pharmaceuticals Incorporated,Boston,United States
,
Mark Wood
Affiliations:
Vertex Pharmaceuticals Incorporated,Boston,United States
,
Eric Chan
Affiliations:
Vertex Pharmaceuticals Incorporated,Boston,United States
,
Brenda Eustace
Affiliations:
Vertex Pharmaceuticals Incorporated,Boston,United States
,
Matthew Porteus
Affiliations:
Stanford University,Stanford,United States
,
Ciaran Lee
Affiliations:
Rice University,Houston,United States
,
Gang Bao
Affiliations:
Rice University,Houston,United States
,
Annarita Miccio
Affiliations:
Genethon,Evry,France;INSERM,Paris,France;Imagine Institue,Paris,France
,
Annalisa Lattanzi
Affiliations:
Genethon,Evry,France
,
Fulvio Mavilio
Affiliations:
Genethon,Evry,France
,
Tirtha Chakraborty
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Chad Cowan
Affiliations:
CRISPR Therapeutics,Cambridge,United States
,
Rodger Novak
Affiliations:
CRISPR Therapeutics,Cambridge,United States
Ante Lundberg
Affiliations:
CRISPR Therapeutics,Cambridge,United States
EHA Learning Center. Lundberg B. Jun 23, 2017; 181434
Topic: 4Bj Indication for specific and gene-modified cell therapy
Bill Lundberg
Bill Lundberg

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Abstract
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Abstract: S147

Type: Presidential Symposium

Presentation during EHA22: On Friday, June 23, 2017 from 16:00 - 16:15

Location: Hall A

Background
Extensive human genetic and epidemiological data demonstrate that the genetic condition Hereditary Persistence of Fetal Hemoglobin (HPFH) substantially ameliorates the pathology of Sickle Cell Disease (SCD) and β-thalassemia (β-Thal). This condition is associated with several genetic variants at the β-globin locus that lead to transcriptional reactivation of γ-globin genes, resulting in upregulation of fetal hemoglobin (HbF).

Aims
CRISPR/Cas9 is a revolutionary technology that allows for precise, directed changes to genomic DNA. Our strategy is to use CRISPR/Cas9 in human primary CD34+ hematopoietic stem and progenitor cells (HSPCs) to re-create specific HPFH genetic variants as well as other variants associated with elevated HbF and demonstrate their causal relationship to elevated HbF as a potential therapeutic strategy to treat SCD and β-Thal.

Methods
Using CRISPR/Cas9 gene editing, we have successfully re-created genetic variants linked to high HbF levels in HSPCs from healthy donors and SCD and β-Thal patient samples, and determined the relationship of different genetic variants to upregulation of γ-globin in bulk and clonal populations of differentiated erythrocytes. Off-target editing was assessed, and on-target editing in long-term repopulating subsets of HSPCs was measured in vitro and by engraftment in immunocompromised mice. Finally, editing rates at clinical scale in a GMP-capable manufacturing facility were demonstrated.

Results
We first optimized cell culture and electroporation conditions that led to high rates of genomic editing across multiple loci, achieving 84.9±6.2% (Mean±SD) editing efficiency at key regions of interest in CD34+ HSPCs from mobilized peripheral blood of healthy donors (n=16). Similar rates of editing were attained using CD34+ HSPCs derived from healthy-donor bone marrow (n=6). Cas9 delivery as recombinant protein improved cell viability when compared to mRNA-based delivery (88.8±3.7% compared to 75.5±9.3%, Mean±SD, n=56 for each) with no observed reduction in editing efficiencies. To investigate gene editing impact on HbF, edited cells were erythroid differentiated from healthy donors as well as from SCD and β-Thal patients. Specific gene edits significantly increased γ-globin mRNA expression to therapeutically-relevant levels (increased expression to 29-37% as a ratio of γ/α in one β-Thal patient sample and to 25-45% as a ratio of γ/(γ+β) in six SCD patient samples). We demonstrated similarly high rates of editing in the CD34+CD38-CD90+CD45RA- long-term repopulating HSPCs and bulk CD34+ HSPCs (87.9±6.4% compared to 89.7±3.6%, Mean±SD, n=4). We confirmed that engraftment levels of edited cells in immunocompromised mice were similar to control cells (% human CD45 in peripheral blood = 28.6±6.9% in controls versus 27.1±6.6% and 26.3±7.9% for two guide targets, Mean±SD, n=48 for each). In-depth off-target analysis for a selected guide RNA confirmed no detectable genomic cleavage at over 5000 predicted off-target sites with a detection sensitivity of 0.2%, supporting its safety for clinical use. Finally, we have demonstrated editing rates of >85% at clinical scale in a GMP-capable manufacturing facility to enable clinical development for SCD and β-Thal. Required safety toxicology studies are ongoing.

Conclusion
Using CRISPR/Cas9 we successfully created gene edits that upregulate HbF in both healthy donor and patient samples. We have also dissected the genotype-phenotype relationship for specific genetic modifications, identifying the editing strategies which are most promising for re-expressing HbF. We have optimized the conditions for modifying HSPCs, including at clinical scale in a GMP-compliant setting, and demonstrated potential safety with no detectable off-target editing. These experiments support the further development of specific CRISPR/Cas9 editing strategies of HSPCs to treat SCD and β-Thal patients.

Session topic: 25. Sickle cell disease

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