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B - Gene Targeting and Gene Correction -> B1 – Gene Targeting and Gene Correction – In Vivo Studies (Basic development of novel technologies for genome editing, with or without site-specific endonuclease.

310: Optimization and Characterization of Genome Editing of Human Hematopoietic Stem Cells to Induce Fetal Hemoglobin towards SAGES-1 Clinical Trial

Type: Oral Abstract Session

Presentation Details
Session Title: Gene Targeting and Gene Correction: Hemoglobin, Muscle, and Eye
Location: Room 515 AB
Start Time: 5/19/2023 17:00
End Time: 5/19/2023 17:15

Sickle cell disease (SCD) is a severe blood disorder caused by a point mutation in the HBB gene. One promising genome editing strategy to treat SCD is to reactivate anti-sickling fetal hemoglobin (HbF). Results of ongoing clinical trials targeting the erythroid specific enhancer of BCL11A to induce durable levels of HbF are encouraging. We targeted alternative downstream repressor-binding sites in the γ-globin gene promoters and identified -115 site as more potent inducer of HbF (32% ± 1.5) compared to -196 ZBTB7A repressor binding site (16.8% ± 0.9) in erythroid progeny of CD34+ transplanted NBSGW mice. Here we describe IND-enabling studies to advance a planned St. Jude Autologous Genome Edited Stem Cell (SAGES-1) clinical trial.
To evaluate the efficacy of -115 γ-globin promoter editing, we edited plerixafor-mobilized CD34+ HSPCs from one healthy donor and three individuals with SCD and transplanted them into NBSGW mice. We attained high-frequency editing in CD34+ HSPCs before (85.6% ± 0.9) and after 17 weeks of transplantation (74.2% ± 2.9) that resulted in strong HbF induction (27.6% ± 1.7) in red cell progeny derived from mice bone marrow. Single-cell western blots revealed that 49.3% to 58.2% of edited erythroblasts exhibited γ-globin expression compared to unedited controls (<6%). By single-cell RNA-sequencing, we noticed similar upregulation of γ-globin transcripts in edited erythroblasts. Importantly, functional effects of broad HbF induction assessed where edited cells exhibited three-fold reduction in sickling under hypoxia.
To assess associated genotoxicities, we applied CHANGE-seq to discover and rhAMP-seq to validate genome wide off-target activity. We observed no detectable off-target mutations above background at 194 candidate nominated off-target sites. We further characterized genomic re-arrangements by Uni-Directional Targeted Sequencing (UDiTaS) and noted no recurrent translocations at day 5 and 14. By long-range PacBio sequencing analysis, we observed a dominant 4.9kb deletion resulted from simultaneous double strand beaks in HBG1 and HBG2 promoters. We further confirmed by digital droplet PCR 29.8 % ± 1.3 of 4.9 kb deletions observed in edited CD34+ HSPCs.
To produce genome editing components required for autologous cellular therapy, we optimized a tag-free purification method to generate GMP-grade Cas9-3xNLS with high purity (>99.7% by RPLC) and low endotoxin. Potential guide RNA contaminants were identified by a modified gRNA sequencing assay where we observed 82.2% to 84.2% of the 5' protospacer sequence perfectly matches to the targeted gRNA sequence with no detectable contaminants from unrelated sequences.
We optimized editing of healthy donor CD34+ HSPCs at clinical scale using Maxcyte electroporation system. In our first two pre-GMP engineering runs, using plerixafor mobilized HSPCs, clinical scale CL1.1 cartridge, GMP-like Cas9 protein, and GMP-like sgRNA, we achieve ~92% editing in bulk HSPCs and further xenografted into NBSGW mice. At 17 weeks, we noticed 70-84% editing efficiency with no alterations to lineage or engraftment compared to unedited HSPCs. Furthermore, we observed 23.5-26% HbF in erythroid cells isolated from the bone marrow, well in range of expected therapeutic range.
In summary, we describe a roadmap for rigorous evaluation of -115 γ-globin promoter editing approach to induce therapeutically effective levels of HbF for SCD therapy to support a planned SAGES1 first-in-human clinical trial.

Varun Katta1, Kiera O’Keefe1, Yichao Li1, Rachael K. Wood1, Cicera R. Lazzarotto2, Rachel Levine1, Thiyagaraj Mayuranathan1, Young-In Kim-Hoehamer1, Alicia Powers1, Naoya Uchida3, Timothy Lockey1, Shondra M. Pruett-Miller1, Akshay Sharma1, John F. Tisdale3, Sheng Zhou1, Jonathan Yen1, Mitchell J. Weiss1, Shengdar Q. Tsai1

1St. Jude Children’s Research Hospital, Memphis, TN,2Beam Therapeutics, Cambridge, MA,3NIH, Bethesda, MD
 V. Katta: None.

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