PRAGYA SANTRA, AMITY UNIVERSITY KOLKATA
Sickle cell anaemia is an autosomal red blood cell disorder where the shape of the RBCs become distorted and crescent sickle-shaped. Such structural modifications to the RBCs disables them if their ability to transfer oxygen to the whole body. Deoxygenated Sickle cells have an affinity to bind mutually forming polymers. Due to abnormal shapes and the uneven surface area, they block the blood vessels and capillaries, causing immense pain in the areas with obstructed insufficient blood flow. The state of inadequate oxygen, or hypoxia that is thus created, causes cell death in the tissues of various regions, even potentially leading to failing internal organs which can be fatal.
The disorder is primarily caused because of a mutation occurring in a single base pair (a point mutation) in the DNA sequence of a gene encoding the β-globin subunit of haemoglobin (HbF;α2β2). The mutation occurs in the 6th position of the amino acid sequence, where the originally occurring Glutamic acid (E) is replaced with a Valine (V). This results in the formation of haemoglobin S (Hgb S).
Bone marrow transplantation was formerly considered as the best course of treatment for the affected individuals, but this only promised effective outcomes for patients that were under the age of 16 years. Even so, the treatment came with some big challenges– being able to find a suitable donor, which is often cumbersome. This included the risk of Graft-versus-Host disease, along with other potentially life-threatening complications. Rather than conducting the bone marrow transplantation process, another more effective and permanent solution is a therapeutic approach. This approach for the treatment of the disorder would include the reactivation of expression of the haemoglobin-F (HbF;α2γ2) gene in the foetal stage or at the infantile stage of the individual. Activation of the BCL11A gene after birth represses the activity of haemoglobin-F. Haemoglobin-F remains unaffected to the mutation as it lacks the β-globin subunit but possesses the γ-subunit. Also, it interferes with the polymerization of haemoglobin S.
Treatment of Sickle Cell Anaemia by CRISPR-Cas9 Gene Therapy:
Disease and disorders caused by mutations are prime candidates for gene editing. The diseased stem cells are extracted from the patients, followed by performing an edit in the mutant gene using a normal gene, then reintroducing it back into the patient. CRISPR-Cas9 is used to edit the hematopoietic stem progenitor cells (HSPCs) and re-implant the edited cells in the patient’s bloodstream. CRISPR-Cas9 utilizes two methods to treat Sickle Cell Anaemia:
- Repairing haemoglobin-S to further proliferate into normal forms:
CRISPR-Cas9 is used to introduce a break in the double-strand of the DNA, for editing out the β-globin gene. Besides, a donor template with the normal version of the gene is introduced to incorporate it at the edited portion during repair of the double-strand break. The edited strand of DNA would then have the normal β-globin subunit rather than the mutated form. It would then be re-introduced in the patient’s bloodstream which further would produce normal and healthy RBCs.
- Replacing haemoglobin-S with haemoglobin-F:
CRISPR-Cas9 can be used in more such similar ways as the one mentioned above to perform a double-strand DNA break, editing out the β-subunits and replacing with γ-genes to further proliferate into haemoglobin-F. Also, the BCL11A gene suppresses haemoglobin-F in adults. CRISPR-Cas9 therapy is used to mutate the BCL11A gene to put down its activation promoting haemoglobin-F expression. CRISPR-Cas9 guided RNA libraries to screen and validate BCL11A erythroid enhancer for producing haemoglobin-F. CTX001 is such a drug which introduces a double-strand break in BCL11A, restricting its functionality to suppress haemoglobin-F. Also, CRISPR based screening method identifies other enzymes, heme-regulated inhibitor (HRI), suppressing haemoglobin-F and systematically introduced mutations to deactivate them.
Using CRISPR-Cas9, sickle cell disorder has finally been noticed to be brought under significant control. It even holds potential in completely curing the disease. The general public just awaits to be convinced about the benefits of this therapy. CRISPR-Cas9, with such precise and efficient gene editing capabilities, once again depicts its sought-after potentials in revolutionalizing the treatment of various such genetic disorders that are difficult to treat using conventional means.
Also read: CR3022 antibody: fight against SARS-CoV-2
SOURCES:
- Selami Demirci, Alexis Leonard, Juan J. Haro-Mora, Naoya Uchida, and John F. Tisdale; CRISPR/Cas9 for Sickle Cell Disease: Applications, Future Possibilities, and Challenges; 5 February 2019; Cell Biology and Translational Medicine; Volume 5; Pg.; 37-52; doi: https://doi.org/10.1007/5584_2018_331
- Daniel P. Dever et al; CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells; 07 November 2016; Nature; Volume: 539, Pg.-384–389; doi: https://doi.org/10.1038/nature20134
- So Hyun Park et al; Highly efficient editing of the β-globin gene in patient-derived hematopoietic stem and progenitor cells to treat sickle cell disease; 05 September 2019; Nucleic Acids Research; Volume-47; Issue-15; Pg,-7955–7972, doi: https://doi.org/10.1093/nar/gkz475
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