Sickle cell disease is an autosomal recessive disease caused by a single point mutation in the HBB gene . It is among the best understood genetic diseases, but currently the only curative therapy is allogeneic HSCT. However, the deep understanding of the pathophysiology of the disease has led to multiple exciting genome editing strategies to cure the disease. All currently involve the ex vivo manipulation of the patient’s own HSPCs which are then trans planted back into the patient following myeloablative conditioning.

Fetal Hemoglobin Derepression

 It is well documented that the severity of sickle cell disease is ameliorated by increasing the level of fetal Hb (HbF) in red blood cells because HbF acts as an antisickling Hb.[1] Upregulation of HbF is one of the major mechanisms of action for hydroxyurea, a disease modifying small molecule.[2] Decades of research have uncovered the pathways by which HbF is downregulated at birth. One of the central proteins in this downregulation pathway is BCL11A, which is a transcription factor that suppresses gamma-globin expression.[3] There are several different genome editing approaches to inhibit the BCL11A repression (Fig. 1). The first is to inactivate an erythroid-specific enhancer for the BCL11A gene such that the protein is not expressed in the red cell lineage.[4,5] The inactivation is done by delivering a CRISPR/Cas9 RNP complex to HSPCs that makes a break in the enhancer resulting in indels that inactivate the genetic element. Early clinical trial results show exciting promise in this approach. The small number of patients who have been treated are showing an elimination of vaso-occlusive pain crises and 50% to 60% HbF with 40% to 50% HbS in the red cell lineage in the first months following treatment.[6]This amount of HbF is predicted to prevent almost all sickling in the pancellular state, as the early results suggest it is. The long-term safety and efficacy are being evaluated.

Fig1. DIFFERENT GENOME EDITING STRATEGIES FOR SICKLE CELL DISEASE.

The second is to inactivate BCL11A-binding sites in the gamma globin gene cluster (mimicking naturally occurring hereditary persistence of fetal hemoglobin [HPFH] variants). In this strategy the CRISPR/Cas9 RNP is targeted to the BCL11A-binding sites in the gamma-globin cluster, thereby creating indels that prevent BCL11A from binding and repressing the gamma-globin genes.[7-9] Preclinical results show encouraging results in deprepressing HbF using this direct globin cluster editing.

Direct Gene Correction

 An alternative to the indirect approach of HbF derepression is to use HDR to directly convert the pathologic HbS allele to the nonpathologic HbA allele. Preclinical success using CRISPR/Cas9 RNP com bined with either ssODN or AAV6 to template the correction has been achieved.[10,11,12] The frequency of allele correction has been 30% to 50% with ssODN and 40% to 80% with AAV6, resulting in 50% to 90% of cells having at least one allele corrected. The predicted threshold for clinical benefit based on mixed chimerism following allogeneic HSCT is that 5% to 20% cellular correction would have substantial, if not curative, clinical benefit. This increase in effect is because of the selective advantage of nonsickling red blood cells both in red blood cell development within the bone marrow and in the periphery.[13,14] Because CRISPR/Cas9 RNP is so active, the predominant genotype following the gene correction process is β thalassemiac trait. Thus patients with homozygous sickle cell disease (SS disease) may be converted to β thalassemia trait, a condition of mild anemia that millions of people around the world live healthy lives with and a condition that may even be more healthy than sickle cell trait. Clinical trials using both ssODNs and AAV6 began in 2022.

References

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[1] Platt OS, et al. Mortality in sickle cell disease. Life expectancy and risk factors for early death. N Engl J Med. 1994;330(23):1639–1644.

[2] Charache S, et al. Hydroxyurea: effects on hemoglobin F production in patients with sickle cell anemia. Blood. 1992;79(10):2555–2565.

[3] Bauer DE, Orkin SH. Hemoglobin switching’s surprise: the versatile transcription factor BCL11A is a master repressor of fetal hemoglobin. Curr Opin Genet Dev. 2015;33:62–70.

[4] Canver MC, et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature. 2015;527(7577):192–197.

[5] Wu Y, et al. Highly efficient therapeutic gene editing of human hematopoietic stem cells. Nat Med. 2019;25(5):776–783.

[6] Frangoul H, et al. CRISPR-Cas9 Gene Editing for Sickle Cell Disease and beta-Thalassemia. N Engl J Med. 2021;384(3):252–260.

[7] Traxler EA, et al. A genome-editing strategy to treat beta hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition. Nat Med. 2016;22(9):987–990.

[8] Ikawa Y, et al. Gene therapy of hemoglobinopathies: progress and future challenges. Hum Mol Genet. 2019;28(R1):R24–R30.

[9] Hoban MD, Bauer DE. A genome editing primer for the hematologist. Blood. 2016;127(21):2525–2535.

[10] DeWitt MA, et al. Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Sci Transl Med. 2016;8(360):360ra134.

[11] Dever DP, et al. CRISPR/Cas9 beta-globin gene targeting in human haematopoietic stem cells. Nature. 2016;539(7629):384–389.

[12] Lattanzi A, et al. Development of β-globin gene correction in human hematopoietic stem cells as a potential durable treatment for sickle cell disease. Sci Transl Med. 2021;13(598):eabf2444.

[13] Iannone R, et al. Results of minimally toxic nonmyeloablative transplantation in patients with sickle cell anemia and beta-thalassemia. Biol Blood Marrow Transplant. 2003;9(8):519–528.

[14] Fitzhugh CD, et al. At least 20% donor myeloid chimerism is necessary to reverse the sickle phenotype after allogeneic HSCT. Blood. 2017;130(17):1946–1948.

اخر الاخبار

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عَبرَ المجالس الحسينية.. المجمَع العلمي يقدّم محاضرات دينية وتلاوات قرآنية في بغداد والنجف وميسان

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راتبه عشرات ملايين الدولارات.. زوكربرغ يخطف "عقل أبل"

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مواضيع ذات صلة

Genome Editing of Immune Effector Cells for Safer and More Potent Cancer Therapies (CAR-T Cells)

Therapeutic Genome Editing for Hematologic Diseases: β-Thalassemia

TFIIIA Binding to the Middle of its Gene as Well as to RNA

Biology of 5S RNA Synthesis in Xenopus

Entropy, a Basis for Lambda Repressor Inactivation

Induction from the Lysogenic State

The Need for and the Realization of Hair-Trigger Induction

Site for Cro Repression and CI Activation

Chronology of Becoming a Lysogen

N Protein and Antitermination of Early Gene Transcription

Spontaneous Mutations in bacteria

Mechanisms of Gene Transfer in Bacteria