By Marcus Yang
The gene editing capabilities of CRISPR technology introduce exciting new developments in curing many diseases and conditions. One such disease is Sickle Cell Anemia (SCA). This debilitating genetic disorder is caused by a single-point missense mutation in the β-globin gene (HBB). This means that in sickle-cell anemia, the Glutamic Acid (GAG codon), is converted into Valine (GUG codon), creating an abnormal hemoglobin. The normal HBB gene provides the instructions for making beta-globin. A typical hemoglobin protein consists of four subunits: two beta-globin and two alpha-globin. In individuals with SCA, hemoglobin S (HbS) replaces at least one of the beta-globin subunits in the hemoglobin protein. HbS is the product of a mutated HBB gene, producing an abnormal hemoglobin. This mutation/abnormality leads to the crescent-shaped and rigid red blood cells in anemic patients.
So, with this in mind, how can CRISPR gene-editing technology be engaged to cure SCA? Two methods are currently being tested. CRISPR-Cas9 can either correct the HBB gene missense mutation directly, or it can be used to reactivate the production of fetal hemoglobin (HbF), a form of hemoglobin produced in the fetus in utero, that once switched on again, can compensate for HbS.
The first method has lots of potential but also has limitations. To correct the missense mutation, scientists create a guide RNA (gRNA), which is an RNA sequence that will guide the Cas9 protein to the faulty HBB gene. The Cas9 protein will then cut out a short stretch of double-stranded DNA. Once this mutated section has been excised, scientists will provide a DNA template with the correct sequence, allowing the body’s natural homology-directed repair (HDR) mechanism to repair the mutation, restoring the production of normal hemoglobin. The limitation of this method, however, is that HDR in adult cells tends to be less efficient.
The second innovative approach utilizes the capabilities of HbF, which is exclusively expressed during fetal development but is silenced after birth. Scientists plan to reactivate HbF production by turning off the BCLA11 gene - a key repressor of HbF expression in adult cells - using CRISPR-Cas9 and, more recently, Cas12 proteins. By excising the repressor that prevents the expression of HbF, the production of fetal hemoglobin resumes, allowing for the production of normal hemoglobin in patients with sickle cell disease and beta thalassemia. This has had significant success in clinical trials, with patients who have undergone this procedure experiencing restored hemoglobin production and alleviated symptoms. In December of last year, the FDA approved this therapy, named Casgevy, in treating SCD and transfusion-dependent beta thalassemia.
The actual process of using the CRISPR-Cas9 based treatment to treat sickle cell anemia begins by drawing samples of HSCs (hematopoietic stem cells) from the patient’s bone marrow. The cells are then edited ex vivo (outside of the body) using CRISPR to either directly correct the HBB mutation or to get rid of the BCL11A repressor. Once the modifications have been made, scientists will allow the edited cells to grow and multiply in a culture. In order for these cells to be infused back into the patient, the patient must undergo chemotherapy, more specifically myeloablative conditioning, to clear up room in the bone marrow and allow for the edited cells to occupy the space. Once reintroduced, the modified HSCs should produce healthy hemoglobin proteins and red blood cells, helping to treat SCA.
While the applications of CRISPR are promising and have proven to be extremely successful, gene-based treatments for SCD face several hurdles. The efficiency of HDR in adult cells and the extreme cost of both chemotherapy and CRISPR treatment all pose significant obstacles to widespread implementation.
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