CRISPR AND Thalassemia

CRISPR therapy for hematological disease has proven effective for transplant dependent beta thalassemia and sickle cell anemia, with additional disease targets in sight. The success of these therapies relies on high rates of CRISPR-induced double strand DNA breaks in hematopoietic stem and progenitor cells (HSPC). To achieve these levels, CRISPR complexes are typically delivered by electroporation ex vivo which is toxic to HSPCs. HSPCs are then cultured in stimulating conditions that promote error-prone DNA repair, requiring conditioning with chemotherapy to facilitate engraftment after reinfusion...

Stop codon readthrough (SCR) is the process where translation continues beyond a stop codon on an mRNA. Here, we describe a strategy to enhance or induce SCR in a transcript-selective manner using a CRISPR-dCas13 system. Using specific guide RNAs, we target dCas13 to the region downstream of canonical stop codons of mammalian AGO1 and VEGFA mRNAs, known to exhibit natural SCR. Readthrough assays reveal enhanced SCR of these mRNAs (both exogenous and endogenous) caused by the dCas13-gRNA complexes. This effect is associated with ribosomal pausing, which has been reported for several SCR events...

In recent years, a growing number of clinical trials have been initiated to evaluate gene therapy approaches for the treatment of patients with transfusion-dependent β-thalassemia (TDT) and sickle cell disease (SCD). Therapeutic modalities being assessed in these trials utilize different molecular techniques, including lentiviral vectors to add functional copies of the gene encoding the hemoglobin β subunit in defective cells and CRISPR/Cas9, TALE nuclease (TALEN), and zinc finger nuclease (ZFN) gene editing strategies to either directly address the underlying genetic cause of disease or induce fetal hemoglobin production by gene disruption...

No abstract text is available yet for this article.

In 2020, Emmanuelle Charpentier and Jennifer Doudna were awarded the Nobel Prize in Chemistry for their research on the endonuclease, clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein 9 (CRISPR-Cas9) method for DNA editing. On 16 November 2023, the UK Medicines and Healthcare Products Regulatory Agency (MHRA) was the first to approve the CRISPR-Cas9 gene editing therapy, Casgevy (exagamglogene autotemcel), for the treatment of patients with transfusion-dependent b-thalassemia and the treatment of sickle cell disease in patients aged ≥12 years with recurrent vaso-occlusive crises...

Ex vivo cellular system that accurately replicates sickle cell disease and β-thalassemia characteristics is a highly sought-after goal in the field of erythroid biology. In this study, we present the generation of erythroid progenitor lines with sickle cell disease and β-thalassemia mutation using CRISPR/Cas9. The disease cellular models exhibit similar differentiation profiles, globin expression and proteome dynamics as patient-derived hematopoietic stem/progenitor cells. Additionally, these cellular models recapitulate pathological conditions associated with both the diseases...

Exagamglogene autotemcel (Casgevy™) is a genetically modified autologous CD34+ cell enriched population. It contains human haematopoietic stem and progenitor cells edited ex vivo by CRISPR/Cas9 (a DNA double strand break-inducing nuclease system) to differentiate into erythroid cells that produce high levels of foetal hemoglobin. Developed by Vertex Pharmaceuticals and CRISPR Therapeutics, exagamglogene autotemcel received its first approval on 16 November 2023 in the UK for the treatment of transfusion-dependent β-thalassemia (TDT) in patients aged ≥ 12 years for whom haematopoietic stem cell (HSC) transplantation is appropriate and a human leukocyte antigen matched related HSC donor is not available...

β-thalassemia is an inherited blood disease caused by reduced or inadequate β-globin synthesis due to β-globin gene mutation. Our previous study developed a gene-edited mice model (β654-ER mice) by CRISPR/Cas9-mediated genome editing, targeting both the βIVS2-654 (C>T) mutation site and the 3' splicing acceptor site at 579 and corrected abnormal β-globin mRNA splicing in the β654-thalassemia mice. Herein, we further explored the therapeutic effect of the hematopoietic stem cells (HSCs) from β654-ER mice on β-thalassemia by consecutive HSC transplantation...

PURPOSE: Fetal hemoglobin (HbF) upregulation is a mitigating factor in β-hemoglobinopathies therapy like β-thalassemia and sickle cell diseases. Finding molecular mechanisms and the key regulators responsible for globin switching could be helpful to develop effective ways to HbF upregulation. In our prior in silico report, we identified a few factors that are likely to be responsible for globin switching. The goal of this study is to experimentally validate the factors. METHODS: We established K562 cell line with BCL11A knock down leading to increase in HBG1/2 using CRISPR/Cas9 system...

In humans, specific aberrations in β-globin results in sickle cell disease and β -thalassemia, symptoms of which can be ameliorated by increased expression of fetal globin (HbF). Two recent CRISPR-Cas9 screens, centered on ∼1500 annotated sequence-specific DNA binding proteins and performed in a human erythroid cell line that expresses adult hemoglobin, uncovered four groups of candidate regulators of HbF gene expression. They are (1) members of the nucleosome remodeling and deacetylase (NuRD) complex proteins that are already known for HbF control; (2) seven C2H2 zinc finger (ZF) proteins, including some (ZBTB7A and BCL11A) already known for directly silencing the fetal γ-globin genes in adult human erythroid cells; (3) a few other transcription factors of different structural classes that might indirectly influence HbF gene expression; and (4) DNA methyltransferase 1 (DNMT1) that maintains the DNA methylation marks that attract the MBD2-associated NuRD complex to DNA as well as associated histone H3 lysine 9 methylation...

Beta-thalassemia (β-thalassemia) is an autosomal recessive disorder caused by point mutations, insertions, and deletions in the HBB gene cluster, resulting in the underproduction of β-globin chains. The most severe type may demonstrate complications including massive hepatosplenomegaly, bone deformities, and severe growth retardation in children. Treatments for β-thalassemia include blood transfusion, splenectomy, and allogeneic hematopoietic stem cell transplantation (HSCT). However, long-term blood transfusions require regular iron removal therapy...

With the development and wide usage of CRISPR technology, the presence of R-loop structures, which consist of an RNA:DNA hybrid and a displaced single strand (ss) DNA, has become well accepted. R-loop structures have been implicated in a variety of circumstances and play critical roles in the metabolism of nucleic acid and relevant biological processes, including transcription, DNA repair, and telomere maintenance. Helicases are enzymes that use an ATP-driven motor force to unwind double-strand (ds) DNA, dsRNA, or RNA:DNA hybrids...

Gene therapy represents a significant potential to revolutionize the field of hematology with applications in correcting genetic mutations, generating cell lines and animal models, and improving the feasibility and efficacy of cancer immunotherapy. Compared to different genetic engineering tools, clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated protein 9 (Cas9) emerged as an effective and versatile genetic editor with the ability to precisely modify the genome. The applications of genetic engineering in various hematological disorders have shown encouraging results...

The rapid advancements in gene therapy have opened up new possibilities for treating genetic disorders, including Duchenne muscular dystrophy, thalassemia, cystic fibrosis, hemophilia, and familial hypercholesterolemia. The utilization of the clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system has revolutionized the field of gene therapy by enabling precise targeting of genes. In recent years, CRISPR/Cas9 has demonstrated remarkable efficacy in treating cancer and genetic diseases...

No abstract text is available yet for this article.

Genome editing (GE) is one of the most efficient and useful molecular approaches to correct the effects of gene mutations in hereditary monogenetic diseases, including β-thalassemia. CRISPR-Cas9 gene editing has been proposed for effective correction of the β-thalassemia mutation, obtaining high-level " de novo " production of adult hemoglobin (HbA). In addition to the correction of the primary gene mutations causing β-thalassemia, several reports demonstrate that gene editing can be employed to increase fetal hemoglobin (HbF), obtaining important clinical benefits in treated β-thalassemia patients...

No abstract text is available yet for this article.

Reactivation of fetal hemoglobin (HbF) is a critical goal for the treatment of patients with hemoglobinopathies. β-globin disorders can trigger stress erythropoiesis in red blood cells (RBCs). Cell-intrinsic erythroid stress signals promote erythroid precursors to express high levels of fetal hemoglobin, which is also known as γ-globin. However, the molecular mechanism underlying γ-globin production during cell-intrinsic erythroid stress remains to be elucidated. Here, we utilized CRISPR-Cas9 to model a stressed state caused by reduced levels of adult β-globin in HUDEP2 human erythroid progenitor cells...

Clustered regularly interspaced short palindromic repeat (CRISPR)-based biosensors have been developed to facilitate the rapid and sensitive detection of nucleic acids. However, most approaches using CRISPR-based detection have disadvantages associated with the limitations of CRISPR RNA (crRNA), protospacer adjacent motif (PAM) or protospacer flanking sequence restriction, single channel detection, and difficulty in quantitative detection resulting in only some target sites being detected qualitatively. Here, we aimed to develop a barcode-based Cas12a-mediated DNA detection (BCDetection) strategy, which overcomes the aforementioned drawbacks and enables (1) detection with a universal PAM and crRNA without PAM or crRNA restriction, (2) simultaneous detection of multiple targets in a single reaction, and (3) quantitative detection, which can significantly distinguish copy number differences up to as low as a two-fold limit...

The thalassemia issue is a growing worldwide health concern that anticipates the number of patients suffering from the disease will soon increase significantly. Patients with β-thalassemia intermedia (β-TI) manifest mild to intermediate levels of anemia, which is a reason for it to be clinically located between thalassemia minor and β-thalassemia major (β-TM). Notably, the determination of the actual rate of β-TI is more complicated than β-TM. The leading cause of this illness could be partial repression of β-globin protein production; accordingly, the rate of β-globin gene repression is different in patients, and the gene repression intensity creates a different clinical status...