Customizing the genome as therapy for the b-hemoglobinopathies

The b-hemoglobinopathies, namely sickle cell disease (SCD) and b-thalassemia, result fromgeneticmutations in theb-globingeneandare among themost commonmonogenic diseases in theworld. SCDresults from a nonsynonymousA toTmutation in codon 6 of theb-globin gene leading to a Glu-Val replacement, whereas b-thalassemias are caused by diverse point mutations or deletions. Treatment options are largely supportive. Transfusion and iron chelation aremainstays in the thalassemiaswhereas painmanagement, hydration, andhydroxyurea are used in SCD. The hemoglobin tetramer is composed of 2 a-like globin chains encoded by any of the 3 genes in thea-globin cluster on chromosome 16 and2b-likeglobinchains encoded fromanyof the5genes in theb-globin locus on chromosome 11. The expression of the 3 genes at the a-globin locus (z,a1,a2) and the 5 genes at theb-globin locus (e, g, g, d,b) are developmentally regulated. It has been appreciated for many years that levels of fetal hemoglobin (HbF; a2g2), subject to developmental silencing in the months after birth, is a modifier of disease severity in patients with b-globin disorders. This protective effect of HbF has motivated the therapeutic strategy to reinduce its expression in adult life. Hydroxyurea, a cytotoxic agent that inhibits ribonucleotide reductase, inducesHbFmodestly through an unknownmechanism.However, it has dose-limiting myelosuppressive effects and some patients are nonresponders to therapy. Although bone marrow transplant (BMT) represents the sole established curative option for patients, its use is limited by donor availability and graft-versus-host disease (GVHD). A clinical trial has demonstrated successful gene addition of an antisickling form of b-globin to a transfusion-dependent bb thalassemia patient who gained transfusion independence as a result of gene transfer. Several additional somatic gene therapy trials for b-thalassemias and SCD are ongoing. Despite a deep understanding of molecular defects and gene control mechanisms, treatment options for the majority of patients remain limited. The emergence of designer nucleases for eukaryotic genome editing has ushered in an era of unprecedented control over the genome. The development of zincfingernucleases (ZFNs), transcriptionactivatorlike (TAL) effector nucleases (TALENs), and meganucleases established genome editing as a valuable laboratory technique. The emergence of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) nuclease system, which utilizes a single guide RNA (sgRNA) to direct the Cas9 nuclease for site-specific cleavage, has engendered tremendous excitement about potential clinical applications. The breakneck speed at which new variations on the general theme are developed is truly remarkable. Other Cas9-like systems include the CRISPR/Cpf1 nuclease platform, dimeric RNA-guided FokI nucleases, and use of Cas9s derived from a variety of prokaryotic species. It is unlikely that the discovery of novel CRISPR-based systems and Cas9-like nucleases capable of eukaryotic genome editing will end soon. The relative benefits of the newly developed CRISPR-based systems, ZFNs, and TALENs are still subject to debate. AlthoughCRISPR-based systems are often cited as themost efficient,ZFNsare theonly editing technology that has been brought thus far to a clinical trial. The CCR5 gene has been targetedbyZFNs in autologousCD4Tcells frompatientswithHIV.The gene-modified cells were subsequently reinfused, which led to a decrease in the blood level of HIV in most patients. Notably, this study demonstrated that reinfusion of autologous genome-edited primary human cells could be achieved,well tolerated, andpossibly lead to clinical benefit. Genome-editing–based therapies rely on gene correction or disruption. Double-strand break (DSB) induction by an engineered nuclease is repaired by the endogenous repair pathways of homology-directed repair (HDR) or nonhomologous end joining (NHEJ). Genetic correction strategies exploit the HDR pathway to insert custom sequences into the genome through codelivery of an extrachromosomal repair template in conjunction with an engineered nuclease. The creation of a DSB improvesHDRfrequency.Assuch,wild-type (or customized) sequences can be provided as an extrachromosomal donor for repair following site-specific cleavage by the nuclease. In contrast, genetic disruption strategies rely on the NHEJ pathway following nuclease-induced