Toward life without sickle cell disease

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In this issue of Molecular Therapy, Katta et al. report on the development of a new and efficient Cas9-mediated therapy for sickle cell disease (SCD).¹ SCD is a devastating genetically inherited disease that affects millions of individuals worldwide.² SCD is caused by a homozygous single-nucleotide missense mutation in the adult β-globin gene substituting a valine for a glutamic acid residue, which induces polymerization of hemoglobin tetramers at low oxygen concentrations. The consequences of sickle hemoglobin (HBS) polymerization are manifold: HBS polymerization induces rigid sickle-like cells, which cause vaso-occlusion and, as a result, tissue hypoxia, leading to inflammation, hemolytic anemia, and chronic pain. Enhanced production of normally silenced fetal hemoglobin in adult individuals ameliorates symptoms associated with SCD. There are different therapeutic strategies to combat the disease, none without risks and side effects. Hydroxyurea, US Food and Drug Administration (FDA) approved since 1998 for adults and since 2017 for children, increases the expression of fetal hemoglobin, thereby reducing pain crises and blood transfusion requirements in patients with SCD.² However, hydroxyurea is variably effective and by no means a cure.

SCD has been the focus of viral gene therapy research for three decades, and since the early 2000s, lentiviruses have proven to confer therapeutic levels of globin or anti-sickling globin variants in animal studies and, ultimately, patients with β-hemoglobinopathies (Figure 1).^3,4 Gene editing using site-specific nucleases and homologous recombination has also been proposed and experimentally developed for a long time. The discovery of the prokaryotic CRISPR-Cas9 defense system and its adaptation for modulating the human genome accelerated the potential for therapeutic gene editing.⁵ The Cas9 system consists of a single guide RNA that targets a Cas9 nuclease to a specific site in the genome. Double-strand cuts occur in DNA complementary to the guide RNA near a PAM (protospacer-adjacent motif) sequence. The DNA lesions are most commonly repaired by non-homologous end joining (NHEJ), leading to the insertion or deletion of short DNA sequences. Genome-wide association studies identified variants associated with elevated fetal hemoglobin (hemoglobin G1 and G2; HBG1 and HBG2) and mapped a quantitative trait locus to BCL11A.⁶ BCL11A (B cell lymphoma/leukemia 11A) acts as transcriptional repressor of fetal globin genes by binding to its recognition DNA motifs 115 bp upstream of HBG1 and HBG2.⁷ Together with LRF (leukemia/lymphoma-related factor), BCL11A efficiently suppresses fetal hemoglobin expression during adult hematopoiesis.⁷ This repression can be alleviated and fetal globin transcription induced by deleting the BCL11A binding sites or an activating DNA element that controls the expression of BCL11A itself.

Figure 1.

Outline of genetic strategies for a permanent cure of sickle cell disease

The human β-globin gene locus consists of an LCR (locus control region) super-enhancer, five developmentally regulated globin genes (HBE, HBG2, HBG1, HBD, and HBB), and a 3′ β-globin-enhancer (3′βE); LCR elements in combination with β-globin-associated DNA regulatory regions have been successfully used in gene therapy experiments (left; ITR, inverted terminal repeat; β∗, anti-sickling β-globin). Katta et al.¹ used CRISPR-Cas9 to eliminate BCL11A binding sites in the HBG2/1 promoters (right), which can lead to deletions of DNA sequences lying in between the two BCL11A sites (far right).

In the current study, Katta et al. used mobilized CD34+ cells from patients with sickle cell disease and demonstrated that Cas9-mediated deletion of the −115 BCL11A binding site is more efficient in activating fetal globin and reducing sickling compared to deleting the BCL11A activating element or the LRF binding site.¹ As expected, in a relatively small percentage of edited clones, the authors detected a 4.9 kb deletion spanning the two duplicated promoter motifs in HBG1 and HBG2 targeted by the guide RNA (Figure 1). However, fetal globin expression was still elevated in clones in which the BCL11A binding site was eliminated in the remaining hybrid HBG promoter driving HBG1. The authors used a number of different assays, including CHANGE-sequencing (CHANGE-seq) and single cell RNA sequencing (RNA-seq), to identify potential off-target sites, copy-number variations (CNVs), and genomic rearrangements. Importantly, the authors did not detect any adverse genomic effects after −115 HBG gene editing. Given previous concerns with respect to Cas9-mediated off-site targeting and induction of genomic rearrangements, the current study is promising and suggests that deleterious effects of gene editing may be locus specific and may also depend on the specific methodology. On a cautionary note, a recent study points to the possibility that Cas9 off targets are frequently associated with nuclear processes that cause DNA super-coiling, including transcription and DNA replication.⁸ Nevertheless, the study by Katta et al. provides a clinically scalable protocol for a new editing strategy that may be safe and more efficient compared to currently FDA-approved gene editing protocols for SCD.¹

Gene addition (gene therapy) and CRISPR-Cas9-mediated genome editing share common limitations, including exorbitant costs ($2 to $4 million per patient), concerns with insertional mutagenesis or off-target nuclease activity, and risks associated with ex vivo treatment of hematopoietic stem and progenitor cells (HSPCs). There are efforts to bring the high price tags for FDA-approved gene therapy or CRISPR editing therapeutics ($2 to $4 million) down to a range that may be affordable for many individuals afflicted with hemoglobinopathies.⁹ Current and future technical advances will likely reduce concerns with Cas9 off-site nuclease activity. Alternative procedures, including prime editing (PE) or base editing (BE), avoid nuclease activity, but a recent study points to serious genotoxic consequences after PE and BE in HSPCs.¹⁰ An alternative to ex vivo gene editing of HSPCs represents the direct delivery of nanoparticles containing the editing components to fetal liver HSPCs in utero.¹¹ Finally, there are also new and promising developments in the generation of small drugs that enhance fetal hemoglobin expression. For example, a recent screen for molecular glue degraders that stimulate fetal globin production identified transcription factor WIZ as a potential target.¹²

In summary, the past decade has produced significant progress in the development of new therapies and clinical protocols for the management of sickle cell anemia. This includes the current study by Katta et al., which outlines a refined protocol for the treatment and permanent cure of SCD.¹

Declaration of interests

The authors declare no competing interests.