Therapeutic Applications of the CRISPR-Cas9 System in β‐thalassemia    

β-thalassemia is a genetic disease caused by human hemoglobin beta (HBB) gene mutation. β‐Thalassemia is one of the most common types of autosomal recessive disorders, affecting millions of people all over the world. Homozygous patients with β-thalassemia mutations have severe anemia and usually require frequent transfusions and iron chelation. To date, hematopoietic stem cell transplantation is the only cure available when histocompatible donors are available. A patient with HBE1-b-thalassemia treated with lentiviral delivery of a normal HBB gene into his hematopoietic stem and progenitor cells (HSPC) do not need further blood transfusions. Nevertheless, gene therapy using viral vectors that are randomly integrated into multiple sites of the host genome may cause harm, as found in other genetic diseases.

CRISPR/Cas9-mediated Gene Editing in iPSCs

The ideal way to curing a genetic disease such as β-thalassemia is to correct the mutations that cause the disease. The production of induced pluripotent stem cells (iPSCs) from the patients' own somatic cells could provide a rich source of cells for correction of the β-thalassemia mutation. The mutation-corrected iPSCs can be differentiated into haematopoietic stem and progenitor cells (HSPCs) for autologous transplantation. This method can avoid the problems of immune responses to allogeneic transplantation and the possibility of insertional mutations associated with viral gene delivery.

CRISPR-Cas9 system simulates a defense mechanism of the adaptive immune response in archaea and bacteria to destroy foreign genetic material. This mechanism can be used to target the mammalian genome engineering, such as knockout/knock-in genes, imaging location of genomic locus, epigenetic changes and adding a suppressive or additive agent to specific sites on genomic DNA. CRISPR-Cas9-mediated genome editing is used to correct HBB gene mutations in patients through HDR, resulting in normal erythropoiesis. In the past two years, some research groups have successively applied CRISPR-Cas9 technology to correct β-thalassaemia mutations in patient-derived iPSCs. Gene-corrected iPSCs can restore HBB expression with a minimal off-target effect. In a recent study, the fibroblasts from a patient with β-thalassaemia were reprogrammed to become transgene-free naïve-state iPSCs, which indicated significantly higher targeting efficiencies in the CRISPR-Cas9 genome editing system compared with primed iPSCs.

Currently Human CRISPR Trials

An alternative and universal method currently tested in the first human CRISPR trials in Europe and USA, is the reactivation of the γ‐globin genes HBG1 and HBG2, which, together with α‐globin constitute fetal haemoglobin usually expressed during embryonic development and early infancy, but later replaced by adult haemoglobin. A benign condition known as hereditary persistence of fetal haemoglobin (HPFH) has been shown to improve both β‐thalassaemia and sickle cell disease. The expression of HBG1 and HBG2 is controlled by the transcriptional repressor BCL11A, and CRISPR/Cas9 has been used to disrupt the BCL11A gene or its binding sites. Studies have shown 15-30% allelic editing frequencies in CD34+ HSPCs with a concomitant increase in γ‐globin levels in differentiated erythrocytes, which reflected the phenotype seen in humans with HPFH.

Our CRISPR/Cas9 System Services

CRISPR/Cas9 PlatformCB is committed to providing the most professional and comprehensive genetic editing technology solutions for our clients. To support your projects, we offer a comprehensive custom CRISPR/Cas9 gene editing service from strategy design to final β‐thalassemia model generation.

➢ Establishment of gene-edited cells

➢ Animal models generation of β‐thalassemia by CRISPR/Cas9 system

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Animal Models

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Cell Lines

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Products

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