A Universal Approach to Correct Various HBB Gene Mutations in Human Stem Cells for Gene Therapy of Beta-Thalassemia and Sickle Cell Disease

Abstract

Beta‐thalassemia is one of the most common recessive genetic diseases, caused by mutations in the HBB gene. Over 200 different types of mutations in the HBB gene containing three exons have been identified in patients with β‐thalassemia (β‐thal) whereas a homozygous mutation in exon 1 causes sickle cell disease (SCD). Novel therapeutic strategies to permanently correct the HBB mutation in stem cells that are able to expand and differentiate into erythrocytes producing corrected HBB proteins are highly desirable. Genome editing aided by CRISPR/Cas9 and other site‐specific engineered nucleases offers promise to precisely correct a genetic mutation in the native genome without alterations in other parts of the human genome. Although making a sequence‐specific nuclease to enhance correction of a specific HBB mutation by homology‐directed repair (HDR) is becoming straightforward, targeting various HBB mutations of β‐thal is still challenging because individual guide RNA as well as a donor DNA template for HDR of each type of HBB gene mutation have to be selected and validated. Using human induced pluripotent stem cells (iPSCs) from two β‐thal patients with different HBB gene mutations, we devised and tested a universal strategy to achieve targeted insertion of the HBB cDNA in exon 1 of HBB gene using Cas9 and two validated guide RNAs. We observed that HBB protein production was restored in erythrocytes derived from iPSCs of two patients. This strategy of restoring functional HBB gene expression will be able to correct most types of HBB gene mutations in β‐thal and SCD. stem cells translational medicine 2018;7:87–97

Development of Gene Editing Strategies for Humanβ-Globin (HBB) Gene Mutations

“…Various studies targeted HBB gene with different gene editing methodologies in various types of cell and cell lines (Antony et al, 2018;Cai et al, 2018;Chattong et al, 2017;Dever et al, 2016;Hoban et al, 2016a;Vakulskas et al, 2018;Wattanapanitch et al, 2018;Ye et al, 2016). However, our study is the first study testing long gRNA including SCD specific mutation and its effects in HBB gene editing.…”

Gene editing and CRISPR in the clinic: current and future perspectives

“…Gene editing provides an alternative to viral delivery of intact genes for the treatment of β-thalassemia and sickle cell anemia. While a number of studies have focused on utilizing genome editors to facilitate the correction of mutations in HBB, or to aid in site specific incorporation of an intact copy of HBB [74][75][76][77][78], others have focused on the editing ability alone. It has long been known that mutations resulting in persistence of fetal γ-globin expression (usually silenced at birth) reduce the debilitating effects of mutations in β-globin, including β-thalassemia and sickle cell disease [79].…”

“…This function is critical for correcting disease-causing mutations, but the low efficiency of HDR compared with non-homologous end-joining (NHEJ) in mammalian cells complicates using this method for repairing mutations [209]. Efforts to enhance usage of HDR, while significant and impactful, have only yielded moderate increases in efficiency [74,210,211]. An elegant solution to this problem was developed by David Liu of Harvard University, who demonstrating that fusing a cytidine deaminase (enzyme which deaminates a cytosine into a uracil) with catalytically inactive Cas9 (dCas9) could result in sgRNA-guided correction of a range of C to T mutations that are relevant to human disease [212].…”

CRISPR/Cas9 system: A promising technology for the treatment of inherited and neoplastic hematological diseases

“…Moreover, a study had tried to correct a common β‐thalassemia splicing variant IVS2‐654 ( HBB :c.316‐197C>T) . In addition to the mutation‐specific correction, various studies had explored the possibility of universal gene correction by adding a HBB transgene at the endogenous locus by TTI strategy . Studies have used different Cas9 delivery systems such as pDNA, mRNA, and RNP, and surprisingly the efficiency of gene correction varies dramatically even for the same delivery system irrespective of the cell line tested and donor template used.…”