C6 - Gene Targeting and Gene Correction New Technologies
712: Highly Efficient Gene-Editing of S. aureus Cas9 in Patient Hematopoietic Stem and Progenitor Cells for Treating Sickle Cell Disease
Type: Poster Session
Poster Board Number: 712
Presentation Details
Session Title: Wednesday Posters: Gene Targeting and Gene Correction New Technologies
Introduction: Sickle cell disease (SCD) is a hemoglobinopathy caused by a single nucleotide mutation (A>T) in the sixth codon of the β-globin gene (HBB). Technologies based on CRISPR/Cas9 mediated gene-editing have been developed to provide a permanent cure for SCD by editing the HBB gene in hematopoietic stem and progenitor cells (HSPCs). Recently, Casgevy and Lyfgenia, ex vivo CRISPR/Cas9 gene-editing based therapies for SCD, have received FDA approval. However, the high cost of ~$2-3 million per treatment and the high-quality infrastructure needed for ex vivo editing limit access to these therapies, especially for patients in low-resource countries, such as Africa. Therefore, there is an unmet need for in vivo editing-based therapy that can significantly reduce the cost and eliminate the requirement of highly sophisticated infrastructure. One potential approach for in vivo gene-editing based therapy is to use adeno-associated virus (AAV) vector for delivery of the gene editing machinery to HSPCs in patients. However, the most widely used S. pyogenes Cas9 (SpCas9) cannot be packaged into a single AAV. Therefore in this study we systematically quantified the gene-editing outcomes of S. aureus Cas9 (SaCas9) which can be packaged into AAV to treat SCD, and compared the results with that of SpCas9. Methods: We edited two genes, HBB to correct the sickle mutation and BCL11A enhancer to induce fetal hemoglobin (HbF) with biological replicates in SCD HSPCs from different 2 donors. We delivered sgRNA with SpCas9 or sgRNA with SaCas9 as RNP, along with a corrective ssODN donor template to correct the sickle mutation in HBB and RNP of SpCas9 or SaCas9 to disrupt BCL11A enhancer by electroporation. HBB has a mutually permissive PAM sequence for both Cas9, and we screened 6 targetable sites for SaCas9 around the target site of SpCas9 and chose one with the highest editing efficiency. We analyzed the editing rate and small insertions and deletions (INDELs) profile by Next Generation Sequencing (NGS) and measured large deletions (> 61 bp) by ddPCR. We erythroid differentiated the edited SCD HSPCs for 14 days and measured % HbF by intracellular staining followed by flow cytometry. To evaluate the transduction efficiency of AAV in SCD HSPCs, we generated AAV6-GFP and transduced the SCD HSPCs, and measured % GFP+ cells. Results: We found that SaCas9 is as highly efficient as SpCas9 in the sickle mutation correction (HDR rate by SpCas9: 42%, by SaCas9: 43%). With RNP only, the cutting efficiency of SaCas9 at HBB was comparable to that of SpCas9 (INDEL rate by SpCas9: 78%, by SaCas9: 80%). Large deletions, one of the unintended gene editing outcomes, were not significantly different (33% by SpCas9 RNP, 19% by SpCas9 RNP with the ssODN, 38% by SaCas9 RNP, and 18% by SaCas9 with the ssODN). SaCas9 was also as highly efficient as SpCas9 in BCL11A enhancer disruption (INDEL rate by SpCas9: 86%, by SaCas9: 88%). After erythroid differentiation, cells with BCL11A enhancer disruption by SaCas9 showed comparable HbF induction to SpCas9 (% HbF+: 54% by SpCas9, 50% by SaCas9). The in vitro transduction efficiency of AAV6-GFP was 49% in SCD HSPCs. Discussions: In this work, we showed highly efficient gene-editing by SaCas9 and compared it with the highly optimized and widely used SpCas9 in sickle mutation correction and BCL11A enhancer disruption in SCD HSPCs. The results demonstrated the therapeutic potential of SaCas9 for treating SCD. We confirmed the high transduction efficiency of AAV6 in SCD HSPCs. Based on these results, we will generate AAV6-CRISPR with SaCas9 targeting BCL11A enhancer to investigate its potential for treating SCD by in vivo gene-editing.
ByoungYong Yoo1, So Hyun Park1, Vivien Sheehan2, Gang Bao1
1Rice University, Houston, TX,2Emory University School of Medicine, Atlanta, GA"
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