Direct correction of haemoglobin E β-thalassaemia using base editors

Haemoglobin E (HbE) β-thalassaemia causes approximately 50% of all severe thalassaemia worldwide; equating to around 30,000 births per year. HbE β-thalassaemia is due to a point mutation in codon 26 of the human HBB gene on one allele (GAG; glutamatic acid → AAG; lysine, E26K), and any mutation causing severe β-thalassaemia on the other. When inherited together in compound heterozygosity these mutations can cause a severe thalassaemic phenotype. However, if only one allele is mutated individuals are carriers for the respective mutation and have an asymptomatic phenotype (β-thalassaemia trait). Here we describe a base editing strategy which corrects the HbE mutation either to wildtype (WT) or a normal variant haemoglobin (E26G) known as Hb Aubenas and thereby recreates the asymptomatic trait phenotype. We have achieved editing efficiencies in excess of 90% in primary human CD34 + cells. We demonstrate editing of long-term repopulating haematopoietic stem cells (LT-HSCs) using serial xenotransplantation in NSG mice. We have profiled the off-target effects using a combination of circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq) and deep targeted capture and have developed machine-learning based methods to predict functional effects of candidate off-target mutations

Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for β-thalassemia

β-thalassemia is characterised by the presence of an excess of α-globin chains, which contribute to erythrocyte pathology. Here the authors use CRISP/Cas9 to reduce α-globin expression in hematopoietic precursors, and show effectiveness in xenograft assays in mice

The evolving genetic landscape of telomere biology disorder dyskeratosis congenita.

Acknowledgements: The authors would like to thank all the affected probands and their families for their participation in the study. The authors acknowledge financial support provided by UKRI/MRC (MR/P018440/1) and Blood Cancer UK (14032). The authors would like to thank Torben Heick Jensen from Aarhus University for kind donation of HeLa-TIR1 and HeLa-ZCCHC8-3F-mAID cells.Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome, caused by genetic mutations that principally affect telomere biology. Approximately 35% of cases remain uncharacterised at the genetic level. To explore the genetic landscape, we conducted genetic studies on a large collection of clinically diagnosed cases of DC as well as cases exhibiting features resembling DC, referred to as 'DC-like' (DCL). This led us to identify several novel pathogenic variants within known genetic loci and in the novel X-linked gene, POLA1. In addition, we have also identified several novel variants in POT1 and ZCCHC8 in multiple cases from different families expanding the allelic series of DC and DCL phenotypes. Functional characterisation of novel POLA1 and POT1 variants, revealed pathogenic effects on protein-protein interactions with primase, CTC1-STN1-TEN1 (CST) and shelterin subunit complexes, that are critical for telomere maintenance. ZCCHC8 variants demonstrated ZCCHC8 deficiency and signs of pervasive transcription, triggering inflammation in patients' blood. In conclusion, our studies expand the current genetic architecture and broaden our understanding of disease mechanisms underlying DC and DCL disorders

The evolving genetic landscape of telomere biology disorder dyskeratosis congenita

Dyskeratosis congenita (DC) is a rare inherited bone marrow fail-ure syndrome, caused by genetic mutations that principally affecttelomere biology. Approximately 35% of cases remain unchar-acterised at the genetic level. To explore the genetic landscape, weconducted genetic studies on a large collection of clinically diag-nosed cases of DC as well as cases exhibiting features resemblingDC, referred to as ‘DC-like’ (DCL). This led us to identify severalnovel pathogenic variants within known genetic loci and in thenovel X-linked gene, POLA1. In addition, we have also identifiedseveral novel variants in POT1 and ZCCHC8 in multiple cases fromdifferent families expanding the allelic series of DC and DCL phe-notypes. Functional characterisation of novel POLA1 and POT1variants, revealed pathogenic effects on protein-protein interac-tions with primase, CTC1-STN1-TEN1 (CST) and shelterin subunitcomplexes, that are critical for telomere maintenance. ZCCHC8variants demonstrated ZCCHC8 deficiency and signs of pervasivetranscription, triggering inflammation in patients’ blood. In con-clusion, our studies expand the current genetic architecture andbroaden our understanding of disease mechanisms underlying DCand DCL disorders