SAGES1: Clinical Translation of CRISPR Genome Editing Strategy to Induce Fetal Hemoglobin to Treat Sickle Cell Disease
Authors: Levine Rachel M , Dempsey Erin A , Nimmagadda Nikitha , Jang Yoonjeong , Mayberry Kalin , Zhang Jingjing , Cullins David , Willis Catherine , Powers Alicia , Kim-Hoehammer Young-In , Lin Jie , Zhang Fei , Das Gupta Neha , Lewis Will , Katta Varun , Li Yichao , Lockey Timothy , Cheng Yong , Lehigh Patti , Fazio Frank , Leonard Alexis Kristine , Sharma Akshay , Zhou Sheng , Tsai Shengdar Q , Weiss Mitchell J , Yen Jonathan S 
Published: December 2024

Background:
Sickle cell disease (SCD) symptoms can be alleviated with elevated expression of fetal hemoglobin (HbF) in red blood cells (RBCs). CRISPR-Cas9 editing to produce indels disrupting DNA regulatory elements that repress g-globin gene expression can induce HbF in adult RBCs. We have previously shown that the disruption of the BCL11A repressor-binding motifs (-115) in the g-globin gene promoters can effectively induce HbF that mimics naturally occurring hereditary persistent fetal hemoglobin variants. Here we present our clinical scale up and optimization, pharmacological, and toxicological studies in support of our recently approved FDA IND application to initiate our St. Jude Autologous Genome Edited Stem Cell (SAGES1) clinical trial (NCT06506461).
Methods:
To translate this editing strategy for clinical application, we optimized RNP electroporation of human donor CD34+ HSPCs with Cas9 and the HBG-115 gRNA. For maximal editing rates and cell recovery, we explored the Cas9 concentration, RNP ratio, and electroporation cell concentration using reagents produced in a Good Manufacturing Practice (GMP) facility. We then scaled up the electroporation protocol to the clinically relevant MaxCyte electroporator. Several other aspects of the culture and electroporation protocol were then investigated to establish optimal conditions and acceptable ranges for an efficient and robust process, including prestimulation cell culture, cryopreservation, and RNP complexation. Pre-GMP engineering runs were conducted using full scale healthy donor plerixafor mobilized HSPCs (n=3) edited with GMP-like Cas9 protein and sgRNA. The Cas9/HBG-115 edited cells were xenotransplanted into NBSGW mice for both in vivo pharmacological and in vivo toxicological studies.
Results:
With our scaled and optimized ex vivo editing protocol, we demonstrated that the Cas9/HBG-115 editing rate of CD34+ HSPCs and HbF expression correlates directly with increasing Cas9 concentration, and that at optimal concentrations, we can achieve &gt;80% editing and ~25% HbF expression in erythroid cells in vitro. In our in vivo pharmacological study, we obtained ~92% editing in the bulk HSPCs before xenotransplantation. Edited and unedited cells engrafted efficiently. We retained high indel rates (70-84%) from bone marrow harvested after 16 weeks. We attained HbF levels of 17.1-25.9% from erythroid cells isolated from the mice bone marrow. We observed no differences in human engraftment or lineages in transplanted mice compared to unedited controls. Indel pattern distribution and HSPC subpopulation frequency of edited CD34+ HSPCs were similar for in vitro and engrafted samples within the same donor. Population frequencies of human hematopoietic lineages were similar between edited and unedited cells and editing rates between human lineages of edited CD34+ HSPCs were also similar to bulk editing rates. From our in vivo toxicological studies, we also did not detect any adverse effects in the mice and there was no detectable off target editing.
Conclusion:
Our IND package demonstrating an optimized, safe, and effective preclinical treatment of Cas9/HBG-115 edited CD34+ HSPCs was approved by the FDA to proceed with the clinical trial that will begin accrual soon.