Modification of globin gene expression by RNA targeting strategies

Objective Sickle cell anemia is a genetic blood disease resulting from production of mutant β-globin (βS) and has severe clinical consequences. It is known that a higher cellular γ-globin level, e.g., higher ratio of cellular γ-globin to βS-globin (γ/βS ratio), inhibits sickle hemoglobin (HbS) polymerization tendency. Hence, therapeutic treatment of sickle cell anemia has been focused on introducing γ-globin gene into red blood cells to increase the cellular γ/βS ratio. Here, we have introduced ribozymes and small interfering RNAs (siRNAs) against βS-globin mRNA into blood cells as a means to increase the γ/βS ratio. Materials and Methods Single and multiribozymes against βS-globin mRNA have been tested in vitro and in human erythroleukemia K562βS cells that stably express exogenous βS-globin gene. Primary human hematopoietic progenitor cells were also transfected with multiribozyme and the γ/(γ + β) ratio determined and compared with cells transfected with long hairpin β-globin cDNA and synthetic siRNA genes. Results We have found that the multiribozyme zb21A containing two ribozyme units effectively reduces βS-globin mRNA both in vitro and in K562βS cells. The γ-globin mRNA to βS-globin mRNA ratio in the multiribozyme transfected cells is about a factor of 2 more than that in the control cells. We have also found that the γ/(γ + β) ratio in the transfected hematopoietic progenitor cells is increased by more than twofold in cells treated with multiribozyme zb21A or siRNA ib5. Conclusion Our results suggest that introducing multiribozymes or siRNAs into red blood cells is comparable in their effectiveness to increase the ratio of cellular γ-globin mRNA to β- or βS-globin mRNA, providing possible strategies to increase the effectiveness of γ-globin gene transfer as gene therapy for treatment of patients with sickle cell anemia.