Diverse Approaches to Gene Therapy of Sickle Cell Disease

Abstract

Sickle cell disease (SCD) results from a single base pair change in the sixth codon of the β-globin chain of hemoglobin, which promotes aggregation of deoxyhemoglobin, increasing rigidity of red blood cells and causing vaso-occlusive and hemolytic complications. Allogeneic transplant of hematopoietic stem cells (HSCs) can eliminate SCD manifestations but is limited by absence of well-matched donors and immune complications. Gene therapy with transplantation of autologous HSCs that are gene-modified may provide similar benefits without the immune complications. Much progress has been made, and patients are realizing significant clinical improvements in multiple trials using different approaches with lentiviral vector-mediated gene addition to inhibit hemoglobin aggregation. Gene editing approaches are under development to provide additional therapeutic opportunities. Gene therapy for SCD has advanced from an attractive concept to clinical reality.

Keywords: CRISPR; gene editing; gene therapy; hematopoietic stem cell transplant; lentiviral vector; sickle cell disease.

Figure 1:. Lentiviral Vector Approaches for Gene Therapy of Sickle Cell Disease.

γ-Globin lentiviral vector containing γ-globin gene coding and non-coding regions, β-globin promoter and modified Locus Control Region (LCR) hypersensitive site (HS) elements HS2, HS3, HS4. γ/β-Globin hybrid lentiviral vector containing γ-globin gene coding regions, β-globin 3’ untranslated region (UTR), β-globin promoter, and modified LCR elements HS2, HS3, HS4. T87Q lentiviral vector containing a modified β-globin gene containing a T87Q amino acid change to promote anti-sickling properties, β-globin promoter, and modified LCR elements HS2, HS3, and HS4. βAS3 lentiviral vector containing three anti-sickling amino acid changes (G16D, E22A, T87Q) in the β-globin gene, β-globin promoter, and modified LCR elements HS2, HS3, and HS4. BCL11A shRNA^miR lentiviral vector containing BCL11A shRNA^miR, synthetic polyadenylation signal (polyA), β-globin promoter, and modified LCR elements HS2, and HS3. All vectors are SIN lentiviral vectors (ΔU3). Ψ, packaging signal; LTR, long terminal repeat; cPPT, central polypurine tract; RRE, rev-response element; HS2, HS3, HS4, DNase hypersensitive sites 2, 3, and 4 from the β-globin locus control region (LCR); WPRE, woodchuck hepatitis virus posttranscriptional regulator element. *Not drawn to scale.

Figure 2:. Gene Editing Approaches for Gene Therapy of Sickle Cell Disease.

Disruption of BCL11A erythroid enhancer. Applying CRISPR to disrupt the erythroid enhancer region in intron 2 of BCL11A on chromosome 2 leads to BCL11A downregulation. Without BCL11A repression of γ-globin, the LCR will interact with HBG2 and HBG1 promoting γ-globin expression. Disruption of γ-globin promoter. Altering the BCL11A binding sequence in the γ-globin promoters of HBG2 and HBG1 will inhibit BCL11A binding and γ-globin repression. The LCR will interact with HBG2 and HBG1 promoting γ-globin expression. Correction of E6V with Homology Directed Repair. Using CRISPR/Cas9 and sgRNA to target the point mutation (T) and suppling a corrected template to incorporate the wildtype nucleotide (A) with homology directed repair. Base Editing to Makassar Variant. Adenine base editing to convert the GTG (Val) to the codon GCG (Ala), to produce a non-pathogenic variant (HBB^G).