Genetic therapies for the first molecular disease

Abstract

Sickle cell disease (SCD) is a monogenic disorder characterized by recurrent episodes of severe bone pain, multi-organ failure, and early mortality. Although medical progress over the past several decades has improved clinical outcomes and offered cures for many affected individuals living in high-income countries, most SCD patients still experience substantial morbidity and premature death. Emerging technologies to manipulate somatic cell genomes and insights into the mechanisms of developmental globin gene regulation are generating potentially transformative approaches to cure SCD by autologous hematopoietic stem cell (HSC) transplantation. Key components of current approaches include ethical informed consent, isolation of patient HSCs, in vitro genetic modification of HSCs to correct the SCD mutation or circumvent its damaging effects, and reinfusion of the modified HSCs following myelotoxic bone marrow conditioning. Successful integration of these components into effective therapies requires interdisciplinary collaborations between laboratory researchers, clinical caregivers, and patients. Here we summarize current knowledge and research challenges for each key component, emphasizing that the best approaches have yet to be developed.

Figure 1. Four major steps in autologous hematopoietic stem cell (HSC) therapies to treat SCD.

(i) Clinical researchers inform participants about the procedure, associated risks, and alternative treatments, then obtain written consent. (ii) CD34⁺ HSPCs are mobilized with plerixafor and isolated from blood by apheresis. (iii) HSPCs are manipulated ex vivo to correct the SCD mutation or induce HbF expression. (iv) The participant receives bone marrow conditioning with myelotoxic/myeloablative agents, followed by infusion of the modified HSPCs.

Figure 2. Tools for genetic manipulation of patient CD34⁺ HSPCs to treat SCD.

(A) LV gene therapy: An antisickling β-like globin gene or BCL11A shRNA flanked by erythroid regulatory elements is inserted into a replication-deficient LV that is packaged into vector particles. The LV integrates semi-randomly into the host HSPC genome and is expressed in erythroid progeny. The β-like globin forms functional hemoglobin, while the BCL11A shRNA induces γ-globin expression to raise HbF levels. (B) Genome editing: The RNA-guided Cas9 nuclease binds the DNA target site via its associated guide RNA (gRNA) and creates a precise DSB that is repaired either by NHEJ, generating insertion-deletion mutations that induce HbF; or by HDR, which utilizes a donor DNA repair template to correct the SCD codon. (C) Base editing: Catalytically impaired Cas9n fused to either a cytosine or adenosine deaminase introduces precise base pair alterations. Adenosine (A) base editors convert A:T to G:C; cytosine base editors convert C:G to T:A. Base editors are used to induce HbF or convert the SCD codon to a benign variant. (D) Prime editing: Cas9n fused to a reverse transcriptase binds the target site via base pairing with the guide portion of the associated prime editing guide RNA (pegRNA) and creates a single-stranded DNA nick. The reverse transcriptase domain uses the pegRNA template to synthesize the desired edit following the nick. Cellular DNA repair machinery removes the endogenous DNA “flap” and repairs the nick to generate a heteroduplex intermediate that is converted to the edited product by DNA repair.

Figure 3. Genetic manipulations to treat SCD.

(A) The developmentally regulated β-like globin gene cluster is shown. Noncoding transcriptional regulatory regions are shown as DNase I–hypersensitive sites (HSs) at the locus control region (LCR) and 3′ to the HBB gene (3′HS1). The fetal γ-globin genes (HBG1 and HBG2) are expressed during late gestation, resulting in the production of RBC HbF. Around birth, γ-globin expression declines and is replaced by β-globin, resulting in a shift from HbF to HbA (α₂β₂) normally, or HbS (α₂β^S₂) in the case of SCD. Inhibiting the γ- to β-globin switch has been a historical Holy Grail for treating SCD and β-thalassemia. (B) Induction of HbF by interfering with the expression or function of HBG1/HBG2 transcriptional repressors BCL11A or ZBTB7A. Strategies for manipulation of autologous SCD patient HSCs include disruption of an erythroid-specific BCL11A gene enhancer via genome-editing nuclease–mediated NHEJ or base editing, transduction with an LV that drives erythroid-specific expression of a BCL11A shRNA, and disruption of BCL11A or ZBTB7A binding motifs in the HBG1 and HBG2 promoters. (C) Conversion of the mutant SCD codon (valine) to normal glutamic acid can be engineered by genome-editing nuclease–mediated HDR or by prime editing. Alternatively, the SCD mutant valine codon can be converted to alanine by adenosine base editing to generate the nonsickling benign variant Hb G-Makassar.