486. Efficient Site-Specific Integration and In Situ Gene Correction of Human Long-Term Repopulating Hematopoietic Stem Cells by Zinc Finger Nucleases

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. High frequencies of reproducible targeted integration of a wild-type cDNA into the endogenous start codon of a gene in LT-HSCs could provide a robust genome editing approach to cure genetic diseases in which patients have different mutations throughout the gene. We describe a clinically relevant method for correcting X-linked severe combined immunodeficiency (SCID-X1). By using a highly specific and active CRISPR/Cas9-AAV6 based strategy and selection-free approach, we achieve up to 20% genome integration frequencies in LT-HSCs of a full-length IL2RG cDNA at the endogenous start site as demonstrated by serial transplantation and analysis of genome edited human cells eight months following initial transplantation. In addition to high frequencies of functional gene correction in LT-HSCs we observed no evidence of abnormal hematopoiesis following transplantation, a functional measure of the lack of genotoxicity. Deep analysis of potential off-target activity detected two sites with low frequency (<0.3%) of off-target mutations. The level of off-target mutations was reduced to below the limit of detection using a high fidelity Cas9. Moreover, karyotype evaluation identified no genomic instability events. We achieved high levels of genome targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect of patient derived cells both in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl.

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Beta-Globin Gene Targeting in Human Hematopoietic Stem Cells Using Cas9 Ribonucleoprotein and rAAV6

Blood ◽

10.1182/blood.v128.22.2310.2310 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2310-2310

Author(s):

Daniel Patrick Dever ◽

Matthew Porteus

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Reporter Gene ◽

Ex Vivo ◽

Globin Gene ◽

Gene Correction ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Nucleotide Mutation

Abstract The b -hemoglobinopathies including sickle cell disease (SCD) and b -thalassemia ( b -thal) affect millions of people worldwide . SCD and b -thal are caused by mutations in the b -globin gene (HBB) resulting in either abnormal sickling or severely reduced protein production, respectively. A curative strategy for the b -hemoglobinopathies would be ex vivo HBB gene correction in patient-derived hematopoietic stem and progenitor cells (HSPCs) followed by autologous hematopoietic stem cell transplantation (auto-HSCT). We report the first CRISPR/Cas9 gene-editing platform for achieving homologous recombination (HR) at the HBB gene in long-term repopulating HSCs derived from mobilized peripheral blood. We combine electroporation of Cas9 protein complexed with chemically modified sgRNAs and delivery of a HR donor by recombinant adeno-associated viral vectors, serotype 6 (rAAV6). Notably, by including a reporter gene in the HR donor, we are able to identify and purify a population of HSPCs with >90% of cells having targeted integration at the HBB gene. These cells can be identified because HR-mediated integration causes the reporter gene to be expressed at log-fold higher levels than the non-integrated reporter. When transplanted into immunodeficient mice, the purified population gives rise to engraftment of HBB-edited human cells in primary and secondary recipients, confirming the presence of long-term repopulating hematopoietic stem cells (LT-HSCs). Importantly, we show efficient correction of the SCD-causing E6V mutation in SCD patient-derived CD34+ HSPCs by either editing the nucleotide mutation or knocking in an anti-sickling b -globin cDNA. Edited SCD CD34+ cells were shown to express adult b -globin (HbA) mRNA after HSPCs were differentiated into erythrocytes in vitro, confirming intact transcriptional regulation of the edited HBB allele. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting HSCs for HR at the HBB locus to advance the development of next generation therapies for b -hemoglobinopathies. Disclosures Porteus: CRISPR Therapeutics: Consultancy, Equity Ownership.

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