SAGES1: Clinical Translation of CRISPR Genome Editing Strategy to Induce Fetal Hemoglobin to Treat Sickle Cell Disease
Abstract
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…
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