Functional correction and genome integrity with duplex base editing of ß-thalassemic hematopoietic stem cells

Background: Beta-thalassemia is among the commonest monogenic disorders, posing a major global health challenge. Editing of genetic modifiers of ß-thalassemia, such as BCL11A erythroid enhancer and HBG promoters, enhances fetal hemoglobin (HbF) expression and confers major therapeutic potential. Double-strand-break (DSB)-independent genome editing tools, such as base editors, are potentially safer and better suited for multiplexed application than traditional DSB-dependent CRISPR/Cas technology. However, harmful inadvertent on- and off-target events remain a concern and must be excluded before clinical application, including chromosomal rearrangements, which are invisible to standard detection technologies. Results: Using primary patient-derived CD34+ cells from three donors, we investigated simplex and duplex BE-based disruption of the BCL11A erythroid enhancer and the BCL11A binding site (-115bp) on the HBG promoter for DNA-level events and functional studies at the RNA, protein, and morphological level. Analyses included direct comparison to DSB-based editing, as the current clinically applied standard, and analysis of DNA recombination events by CAST-seq to allow wider inferences for relative safety of DSB-, BE- and duplex BE-based editing. Our study reveals the effectiveness of duplex base editing, with robust ?-globin and HbF induction and significantly improved functional correction over simplex editing. Moreover, duplex editing resulted in low incidence of simple and complex genomic alterations in both therapeutically relevant target loci. Conclusions: Here we display simultaneous duplex base editing by targeting both BCL11A erythroid enhancer and HBG promoter for functional correction and genome integrity. Our study highlights the efficacy, safety, and therapeutic potential of the present duplex BE approach Overall design: After the collection of BFU-E colonies and the investigation of editing levels, three colonies from each treatment were selected for RNA sequencing by Genewiz (Germany). For RNA sequencing workflow, the ultra-low Input RNA-seq assay was performed. The RNA was enriched for full-length transcripts by poly(A) selection and was then processed for library preparation including cDNA synthesis and adapter ligation. Samples were sequenced on the Illumina HiSeq or NovaSeq platforms with 2× 150 bp read length. Raw RNA-Seq data in FASTQ format were trimmed to remove adapter sequences and low-quality nucleotides using Trimmomatic v0.36. The trimmed reads were mapped to the Homo sapiens GRCh38 reference genome available on ENSEMBL using the STAR aligner v2.5.2b, a splice-aware aligner that detects splice junctions and incorporates them into the alignment to improve mapping accuracy, generating BAM files. Unique gene hit counts were then obtained for each sample, and using DESeq2, a comparison of gene expression between the CONTROL vs. DSB-Cas9, CONTROL vs. BE-BCL11A, CONTROL vs. HBG and CONTROL vs. 2xBE groups of samples was performed. The Wald test was applied to generate p-values and log2 fold change values, producing tables of differential expression for all genes as well as significantly differentially expressed genes.