Engineering Globin Gene Expression

Abstract

Hemoglobinopathies, including sickle cell disease and thalassemia, are among the most common inherited genetic diseases worldwide. Due to the relative ease of isolating and genetically modifying hematopoietic stem and progenitor cells, recent gene editing and gene therapy strategies have progressed to clinical trials with promising outcomes; however, challenges remain and necessitate the continued exploration of new gene engineering and cell transplantation protocols. Current gene engineering strategies aim at reactivating the expression of the fetal γ-globin genes in adult erythroid cells. The γ-globin proteins exhibit anti-sickling properties and can functionally replace adult β-globin. Here, we describe and compare the current genetic engineering procedures that may develop into safe and efficient therapies for hemoglobinopathies in the near future.

Keywords: CRISPR/Cas9; TALEN; gene editing; gene therapy; globin; hematopoiesis; hemoglobin; locus control region; zinc finger.

Figure 1

Human Hematopoietic Gene Therapy Hematopoietic gene therapy is unique in that progenitor stem cells can be removed from the patient, therapeutically manipulated, and then returned to the patient to establish long-lived self-perpetuating production of modified mature cells, thereby limiting exposure of off-target body systems to the modifying agents. Stem cells must first be mobilized with the aid of colony-stimulating factors and harvested from the patient typically via the use of apheresis. The cells are then modified in vitro. In this example, the cells are exposed to a viral vector capable of delivering a therapeutic gene. The patient must then receive myeloablative chemotherapy to provide physical space and reduced competition for the modified cells to engraft. The modified cells are then infused back to the patient, where they migrate to the bone marrow and engraft with the goal of establishing cell lines that will provide long-term, ongoing benefit to the patient.

Figure 2

Lentivirus Vector Employed in Globin Gene Therapy The diagram on top depicts the human β-globin gene locus, which consists of five genes (green boxes) that are expressed in a developmental stage-specific manner. High-level expression of the adult β-globin gene is mediated by the LCR HSs and the β-globin 3′ enhancer (blue ovals). Functional elements within all lentivirus vector systems include the long-terminal repeats (LTRs; gray boxes), splicing acceptor (SA) and donor (SD) sites, and the rev-responsive element (RRE), a structured RNA required for efficient viral replication. The TNS9 vector contains large segments of LCR elements HS2, HS3, and HS4, a wild-type β-globin gene, and a β-globin 3′enhancer. The HPV569 vector is similar to the TNS9 vector but contains a mutant β-globin gene that encodes a protein with a T to Q substitution at position 87. In addition, this vector contains two copies of the chicken HS4 (cHS4) insulator sequence in the LTRs. The Lenti-βAS3-FB vector is similar to HPV569 but expresses a β-globin protein with three amino acid substitutions (AS3) and contains single FB insulators in the LTRs. The GLOBE vector contains the β-globin gene and two large segments of the LCR (HS2 and HS3).

Figure 3

Elevation of γ-Globin Expression by Genome Editing Nucleases targeted to genomic sites create double-strand breaks (DSBs) that can be repaired by non-homologous end joining (NHEJ) or by homology-directed repair (HDR). Shown on the left are three examples for deletions causing increases in fetal hemoglobin production. The first shows the creation of a 13-kb deletion encompassing the δ- and β-globin gene. This deletion removes negative regulatory elements and positions the β-globin 3′ enhancer in close proximity to the γ-globin genes. The second example shows deletion of a repressor binding site in the γ-globin gene promoters. The third example shows creation of a frameshift in the coding region or a deletion of a positive DNA-regulatory element in one of the BLC11A erythroid-specific enhancers. Shown on the right are two examples for HDR-mediated correction of the sickle cell mutation. The first example shows an adeno-associated vector (AAV) that delivers the homologous arms (HAs), the therapeutic donor DNA, and a selectable marker (ITR: inverted terminal repeats). The second example shows a single stranded oligodeoxynucleotide containing 5′ and 3′ homologous arms (HAs) and the wild-type β-globin sequence. The diagram on the bottom right outlines the major steps involved in genome editing using autologous cell transplantation.

Figure 4

Alternative Approaches Using Synthetic Zinc Fingers (A) Synthetic ZF activator binding to promoter. ZFs fused to an activation domain (AD) such as VP64 and specific for the γ-globin genes brings the activation domain into close proximity of the promoter, resulting in activation of γ-globin transcription. (B) Decoy ZF protein binding to a repressor region. ZFs can be designed to bind target sites for known repressors. The ZF protein provides competitive inhibition of the repressor elements, thereby activating fetal hemoglobin. (C) ZF protein facilitated chromatin looping. ZF protein fused to the dimerization domain of Ldb1 and specific for the γ-globin gene promoter can mediate chromatin looping, which brings the LCR into proximity to the promoter allowing for activation of fetal hemoglobin.