Re-Creating Hereditary Persistence of Fetal Hemoglobin (HPFH) to Treat Sickle Cell Disease (SCD) and β-Thalassemia
Authors: Lin Michelle I , Paik Elizabeth J , Mishra Bibhu P , Chou Song , Zhang Ying , Tomkinson Kaleigh , Pettiglio Michael A , Sanginario Andrew , Woo Amanda , Lee Min Jin , Allen Melanie , Cradick Thomas J , Chakraborty Tirtha , Porteus Matthew , Lee Ciaran M , Bao Gang , Miccio Annarita , Lattanzi Annalisa , Mavilio Fulvio , Cowan Chad A , Novak Rodger , Lundberg Ante S 
Published: October 2019

Abstract
Hereditary persistence of fetal hemoglobin, a naturally occurring condition that substantially ameliorates disease in SCD and β-thalassemia, is associated with genetic variation at the β-globin locus. Our strategy is to use the CRISPR-Cas9 system to re-create specific genetic variants associated with HPFH in CD34+ hematopoietic stem and progenitor cells (HSPCs) and demonstrate their causal relationship to elevated fetal hemoglobin (HbF) levels as a potential therapeutic strategy to treat SCD and β-thalassemia.
The CRISPR-Cas9 system has been adapted to achieve site-directed DNA cleavage by guide RNAs (gRNAs) and the Cas9 endonuclease. We have identified highly potent single gRNAs (sgRNAs) that can target regions within the β-globin locus associated with HPFH. The sgRNAs, when combined and delivered as dual sgRNAs, resulted in deletions that mimic naturally occurring deletions associated with HPFH. We have also optimized transfection dose and ratio of these sgRNAs with either Cas9 mRNA or Cas9 protein into primary human CD34+ HSPCs from mobilized peripheral blood of healthy donors and can achieve greater than 85% transfection rate with minimal cell loss (85-95% viability). We found that sgRNAs delivered with Cas9 protein resulted in better cell viability than when delivered with Cas9 mRNA especially at higher doses, while attaining the same rates of editing efficiency. Lastly, erythroid differentiation of CD34+ HSPCs demonstrated significant increase in γ-globin gene expression level by qRT-PCR as well as HbF protein levels, measured by FACS and LC/MS.
In conclusion, we have optimized sgRNA and Cas9 transfection into primary human CD34+ HSPCs. We have identified sgRNAs that are highly effective in targeting the β-globin locus and employed forward genetics to re-create genetic variants associated with HPFH in HPSCs. We have demonstrated the causal relationship of different HPFH genetic variants to elevation of HbF, and obtained comparative data on upregulation of HbF in erythroid cells differentiated from edited CD34+ HSPCs. Our findings provide a viable therapeutic strategy using CRISPR-Cas9 for the treatment of β-hemoglobinopathies.

Disclosures
Lin: CRISPR Therapeutics: Employment. Paik:CRISPR Therapeutics: Employment. Mishra:CRISPR Therapeutics: Employment. Chou:CRISPR Therapeutics: Employment. Zhang:CRISPR Therapeutics: Employment, Equity Ownership. Tomkinson:CRISPR Therapeutics: Employment. Pettiglio:CRISPR Therapeutics: Employment. Sanginario:CRISPR Therapeutics: Employment. Woo:CRISPR Therapeutics: Employment. Lee:CRISPR Therapeutics: Employment. Allen:CRISPR Therapeutics: Employment. Cradick:CRISPR Therapeutics: Employment. Chakraborty:CRISPR Therapeutics: Employment. Porteus:CRISPR Therapeutics: Consultancy, Equity Ownership. Mavilio:Adverum Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Consultancy, Research Funding. Cowan:CRISPR Therapeutics: Employment, Equity Ownership. Novak:CRISPR Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Lundberg:CRISPR Therapeutics: Employment, Equity Ownership.