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

Beta-thalassemia and sickle cell disease (SCD), two of the most common genetic diseases, are caused by mutations in the HBB gene encoding the postnatal form of the beta subunit of hemoglobin. Over 200 different types of beta-thalassemia mutations that diminish beta-globin protein production have been identified throughout the HBB gene containing three exons. In contrast, sickle cell disease is due to a HBB missense mutation at codon 6 located in exon 1. Novel therapeutic strategies to permanently correct the HBB mutation in stem cells and to persistently restore the normal HBB protein production at a high level in differentiated erythrocytes are highly desirable. Although making a sequence-specific nuclease to enhance correction of a specific HBB mutation by homology-directed repair (HDR) is becoming straightforward, treating various HBB mutations of β-thalassemia 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-thalassemia patients with different HBB gene mutations, we devised and tested a universal strategy to achieve HBB gene targeting in exon 1 and 3'-UTR using spCas9 with two validated guide RNAs and a DNA template providing all the HBB coding cDNA sequence. In this way, a HDR event near the guide RNA will provide a functional correction of HBB mutations not only in exon 1, but also downstream sites. To provide a simple readout, we linked a GFP reporter gene downstream to the HBB coding cDNA via the 2A self-cleaving peptide so that the GFP reporter expression is indicative of the HBB expression from the same transcript and pro-peptide, which provides an experimental system to screen bioactive molecules to improve HBB protein expression. We observed that HBB protein production was restored in erythrocytes derived from iPSCs of two β-thalassemia patients and of a patient with sickle cell disease. This strategy of restoring functional HBB gene expression will be able to correct most types of HBB gene mutations in both β-thalassemia and sickle cell disease, whether primary hematopoietic stem cells (HSCs) or iPSC-derived HSCs will be used for gene therapy.