Question

Question 7) If someone with sickle cell disease has 30% of their hemoglobin be fetal hemoglobin, they will be symptom free and their blood cells will not clog their capillaries in low blood oxygen situations. How might CRISPR-CAS9 be used to help sickle cell patients? и подk got lovA Normal adult human erythroid cells or ko -2 BCL11A LCR * TGACCA. TGACCAD y-globin B-globin HPFH or CRISPR edited erythroid cells Edit BCLILA protein Edit B globin-3 Too qeveral -4 to 5 ↑ LCR expression Fix the SNPs - 4 LCR NNNNNNTGACCA 7-globin B-globin

Answer 1

Sickle cell anemia is a genetic blood disorder that affects hemoglobin, the oxygen-transporting molecule in RBC. Sickle cell disease causes the body to produce hemoglobin S, an abnormal form of the molecule that distorts the shape of red blood cells, disrupting their function.

Sickle cell disease is caused by mutation of a single base in the DNA sequence of the gene. The gene is autosomal, meaning that it is not linked to sex, and it is recessive.

The condition is caused by a specific mutation that alters the amino acid sequence of ß-globin, replacing the glutamic acid at position 6 with a valine. This single amino acid alteration results in the formation of hemoglobin S, the disease-causing form of the molecule.

The procedure for gene therapy involves extracting stem cells from the patient, replacing the mutated gene with a normal copy, and reinserting the cells back into the patient.

There are two main ways that CRISPR-Cas9 is being used to treat sickle cell disease. One approach consists of repairing the gene for hemoglobin S in order to cause the normal form to be produced. The second approach involves replacing hemoglobin S with hemoglobin F. Both of these approaches are applied in the clinic by harvesting a patient’s blood, editing the hematopoietic stem progenitor cells (HSPCs) with CRISPR-Cas9 and re-implanting the edited cells into the patient’s bloodstream.

CRISPR-Cas9 is used to introduce a DNA break to the ß-globin gene. A donor template containing the normal version of the gene is also introduced so that the mutation is corrected when the cell repairs the DNA break according to its double-strand break repair mechanisms. The edited cells, now engineered to produce normal hemoglobin, are re-implanted in the patient’s bloodstream.

Another promising CRISPR-Cas9 sickle cell therapy, and one that is closer to a clinical application than ß-globin gene editing, involves eliminating a natural repressor of hemoglobin F. By mutating the BCL11A gene, the expression of hemoglobin F is indirectly promoted. This approach was developed after it was found that sickle cell patients with a natural mutation in their BCL11A gene were resistant to disease symptoms. Initially, researchers used antisense therapy to reduce the expression of BCL11A in cell lines, and expression of hemoglobin F was correspondingly promoted. Antisense therapy involves introducing a DNA strand that binds to a target mRNA transcript and inactivates it, preventing production of the corresponding protein.

CTX001 involves introducing a break to the BCL11A gene, thereby eliminating the repression of hemoglobin F. The drug has successfully corrected sickle cell disease in transgenic mice with clinically relevant efficiency.