Genetic Manipulation Strategies for β-Thalassemia: A Review

Abstract

Thalassemias are monogenic hematologic diseases that are classified as α- or β-thalassemia according to its quantitative abnormalities of adult α- or β-globin chains. β-thalassemia has widely spread throughout the world especially in Mediterranean countries, the Middle East, Central Asia, India, Southern China, and the Far East as well as countries along the north coast of Africa and in South America. The one and the only cure for β-thalassemia is allogenic hematopoietic stem cell transplantations (HSCT). Nevertheless, the difficulty to find matched donors has hindered the availability of this therapeutic option. Therefore, this present review explored the alternatives for β-thalassemia treatment such as RNA manipulation therapy, splice-switching, genome editing and generation of corrected induced pluripotent stem cells (iPSCs). Manipulation of β-globin RNA is mediated by antisense oligonucleotides (ASOs) or splice-switching oligonucleotides (SSOs), which redirect pre-mRNA splicing to significantly restore correct β-globin pre-mRNA splicing and gene product in cultured erythropoietic cells. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) are designer proteins that can alter the genome precisely by creating specific DNA double-strand breaks. The treatment of β-thalassemia patient-derived iPSCs with TALENs have been found to correct the β-globin gene mutations, implying that TALENs could be used as a therapy option for β-thalassemia. Additionally, CRISPR technologies using Cas9 have been used to fix mutations in the β-globin gene in cultured cells as well as induction of hereditary persistence of fetal hemoglobin (HPFH), and α-globin gene deletions have proposed a possible therapeutic option for β-thalassemia. Overall, the accumulated research evidence demonstrated the potential of ASOs-mediated aberrant splicing correction of β-thalassemia mutations and the advancements of genome therapy approaches using ZFNs, TALENs, and CRISPR/Cas9 that provided insights in finding the permanent cure of β-thalassemia.

Keywords: CRISPR-Cas9; antisense oligonucleotides; splice-switching; transcription activator-like effector nucleases; zinc finger nucleases; β-thalassemia.

Figure 1

Normal and aberrant splicing mechanisms. (A) Normal HBB produce normal pre-mRNA with intact three exons and subsequently translated into normal β-globin. (B) HBB with mutation in exon 1 that activates a de novo splice site may either produce correctly spliced or aberrantly spliced mRNA which later be translated into normal or no β-globin, respectively. (C) HBB with intron 1 mutation may activate correct or aberrant splicing pathways that give rise to normal or no β-globin, respectively. (D) Intron 2 mutation in HBB may induce correct or aberrant splicing mechanisms that yield normal or no β-globin, respectively. Red point marked the mutation location. Dashed line indicates the aberrant splicing mechanisms.

Figure 2

How ASOs work to correct the aberrant splicing. In the presence of ASOs targeted at the mutation on the gene, the correction of aberrant splicing of human β-globin occurs and leads to the production of functional β-globin protein. Boxes indicate exons; lines, introns; red dots, mutation.

Figure 3

Modified U7 snRNP. Complexes of spliceosome proteins (green) combine with U7 snRNA that is incorporated with antisense oligonucleotides targeted to the aberrant splice site of the gene. Boxes represent exons and introns.

Figure 4

Gene editing by designer nucleases. ZFNs, TALENs, and CRISPR/Cas9 mediated the genome modifications through two main double strand break repair pathways. Indel mutations resulted from NHEJ pathway. Gene correction, insertion and replacement using DNA donor template are the outcomes of HDR pathway. FokI, endonuclease from Flavobacterium okeanokoites; PAMs, protospacer adjacent motifs; NHEJ, non-homologous end joining; HDR, homology-directed repair; dsDNA, double-stranded DNA; ssODN, single strand oligodeoxynucleotides.