Transcriptome Analyses of β-Thalassemia −28(A&gt;G) Mutation Using Isogenic Cell Models Generated by CRISPR/Cas9 and Asymmetric Single-Stranded Oligodeoxynucleotides (assODNs)

Li, Jing; Zhou, Ziheng; Sun, Hai‐Xi; Ouyang, Wenjie; Dong, Guoyi; Liu, Tianbin; Liu, Ge; Zhang, Xiuqing; Liu, Chao; Gu, Ying

doi:10.3389/fgene.2020.577053

Cited by 5 publications

(3 citation statements)

References 68 publications

(82 reference statements)

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“…On hemoglobin electrophoresis, he had an elevated HbA2 fraction (5.4%) (normal range 1.7-3.2), normal HbF (1.0%) (normal range 0-1.0), and HbH inclusion bodies were not detected. Thalassemia genotyping showed that he is heterozygous for HBB (NM_000518.5):c.-78A>G, a known prevalent pathogenic variant in the ethnic Chinese population (Li et al, 2020).…”

Section: Case Reportmentioning

confidence: 99%

Clinical application of targeted long read sequencing in prenatal beta‐thalassemia testing and genetic counseling

Chin,

Benton,

Yang

et al. 2023

Molec Gen & Gen Med

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BackgroundBeta thalassemia, related to HBB mutation and associated with elevated hemoglobin A2 (HbA2), is an important genetic hemoglobinopathy with high incidences of disease and carrier rates in Singapore. Carrier screening is essential to facilitate prenatal counseling and testing. However, when individuals with elevated HbA2 do not have an identifiable HBB disease‐associated variant, there is ambiguity on risk to their offspring.MethodsWe describe a case report of a proband with elevated HbA2, no identifiable HBB disease‐associated variant, whose partner was a beta thalassemia carrier. Through clinical HBB gene sequencing, multiplex ligation‐dependent probe amplification (MLPA) analysis, as well as targeted Nanopore long read sequencing of selected genes, we performed a complete analysis of HBB including the promoter region, 5′UTR and coding gene sequence, as well as evaluation for potential modifier variants and other rare structural variants.ResultsThis process identified that the proband was heterozygous for KLF1:c.544T>C (p.Phe182Leu), a potential functional polymorphism previously known to be associated with benign elevated HbA2 levels. The presence of disease variants in the HBB locus was excluded.ConclusionThis finding provided clarity and enabled family planning for the proband and her family.

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Section: Case Reportmentioning

confidence: 99%

Clinical application of targeted long read sequencing in prenatal beta‐thalassemia testing and genetic counseling

Chin,

Benton,

Yang

et al. 2023

Molec Gen & Gen Med

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“…Interestingly, the abnormalities due to the −28(A>G) mutation were corrected by CRISPR/Cas9 and asymmetric assODNs in K562 −28( A > G ) . This study is the first to report on the whole-transcriptome analysis based on isogenic cell lines, thus it provides a platform to conduct future investigation of the mechanism of −28(A>G) β-thalassemia ( 50 ).…”

Section: Genome Manipulation Strategiesmentioning

confidence: 99%

Genetic Manipulation Strategies for β-Thalassemia: A Review

Zakaria

Bahar²,

Abdullah³

et al. 2022

Front. Pediatr.

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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.

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“…In vivo editing of the promoter in β-YAC/CD46tg mice resulted in a 20% conversion rate in HSPCs and >40% γ-globin expression in peripheral RBCs [ 73 ]. The HBB -28A>G mutation is one of the most frequently detected mutations in β-thalassemia patients in China and East Asia preventing the transcription of the HBB gene [ 74 ]. Two base editor variants, the eA3A-BE and eA3A(N57Q)-BE3, were employed in erythroid precursor cells derived from a compound heterozygous thalassemia patient (4bp-deletion in exon 1 of one HBB allele and HBB -28 A>G mutation in the other), and they effectively generated the C>G substitution in HBB-28.…”

Section: Double-strand Break-free Gene Editingmentioning

confidence: 99%

In Vivo Hematopoietic Stem Cell Genome Editing: Perspectives and Limitations

Psatha

Paschoudi

Παπαδοπούλου

et al. 2022

Genes

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The tremendous evolution of genome-editing tools in the last two decades has provided innovative and effective approaches for gene therapy of congenital and acquired diseases. Zinc-finger nucleases (ZFNs), transcription activator- like effector nucleases (TALENs) and CRISPR-Cas9 have been already applied by ex vivo hematopoietic stem cell (HSC) gene therapy in genetic diseases (i.e., Hemoglobinopathies, Fanconi anemia and hereditary Immunodeficiencies) as well as infectious diseases (i.e., HIV), and the recent development of CRISPR-Cas9-based systems using base and prime editors as well as epigenome editors has provided safer tools for gene therapy. The ex vivo approach for gene addition or editing of HSCs, however, is complex, invasive, technically challenging, costly and not free of toxicity. In vivo gene addition or editing promise to transform gene therapy from a highly sophisticated strategy to a “user-friendly’ approach to eventually become a broadly available, highly accessible and potentially affordable treatment modality. In the present review article, based on the lessons gained by more than 3 decades of ex vivo HSC gene therapy, we discuss the concept, the tools, the progress made and the challenges to clinical translation of in vivo HSC gene editing.

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Transcriptome Analyses of β-Thalassemia −28(A>G) Mutation Using Isogenic Cell Models Generated by CRISPR/Cas9 and Asymmetric Single-Stranded Oligodeoxynucleotides (assODNs)

Abstract: Analyses of β-Thalassemia −28(A>G) Mutation Using Isogenic Cell Models Generated by CRISPR/Cas9 and Asymmetric Single-Stranded Oligodeoxynucleotides (assODNs).

Cited by 5 publications

References 68 publications

Clinical application of targeted long read sequencing in prenatal beta‐thalassemia testing and genetic counseling

Clinical application of targeted long read sequencing in prenatal beta‐thalassemia testing and genetic counseling

Genetic Manipulation Strategies for β-Thalassemia: A Review

In Vivo Hematopoietic Stem Cell Genome Editing: Perspectives and Limitations

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