Modification of globin gene expression by RNA targeting strategies

Abstract

Objective-Sickle cell anemia is a genetic blood disease resulting from production of mutant β-globin (β S ) and has severe clinical consequences. It is known that a higher cellular γ-globin level, e.g., higher ratio of cellular γ-globin to β S -globin (γ/β S 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/β S ratio. Here, we have introduced ribozymes and small interfering RNAs (siRNAs) against β S -globin mRNA into blood cells as a means to increase the γ/β S ratio.Methods-Single and multi-ribozymes against β S -globin mRNA have been tested in vitro and in human erythroleukemia K562β S cells that stably express exogenous β S -globin gene. Primary human hematopoietic progenitor cells were also transfected with multi-ribozyme 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 multi-ribozyme zb21A containing two ribozyme units effectively reduces β S -globin mRNA both in vitro and in K562β S cells. The γ-globin mRNA to β S -globin mRNA ratio in the multi-ribozyme 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 2-fold in cells treated with multi-ribozyme zb21A or siRNA ib5.Conclusion-Our results suggest that introducing multi-ribozymes or siRNAs into red blood cells are comparable in their effectiveness to increase the ratio of cellular γ-globin mRNA to β-or β Sglobin mRNA, providing possible strategies to increase the effectiveness of γ-globin gene transfer as gene therapy for treatment of patients with sickle cell anemia.Sickle cell anemia is an inheritable blood disease caused by a single nucleotide mutation in the human β-globin gene (codon 6 GAG→GTG). Individuals homozygous for this mutant β-globin gene, designated as β S -globin, have severe clinical symptoms, because the abnormal hemoglobin, sickle cell hemoglobin [HbS (β6 Glu→Val)], produced by this gene can polymerize under low oxygen tension, change red blood cell rheology and shape (sickle), and result in vasoocclusive crisis, infarction, and organ damage [1]. It is known that in some patients with milder sickle cell anemia symptoms, there are higher fetal hemoglobin [HbF (α 2 γ 2 )] levels Offprint request to: Dr. Chien Ho, Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh PA 15213; E-mail address: chienho@andrew.cmu.edu.. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process...

Stem Cell Engineering for the Treatment of Severe Hemoglobinopathies

“…Strategies utilizing ribozymes or RNA interference [112,113] have not yet been investigated in vivo.…”

RNA Interference Technologies and Therapeutics

“…A significant reduction in mouse adult b-globin mRNA by bS-globin-specific siRNA has been reported in murine erythroleukemia cells. [167] …”

Genética das doenças hematológicas: as hemoglobinopatias hereditárias

“…To achieve this, the mechanisms involved in regulation of the globin genes need to be better elucidated. 84,85,[119][120][121] A number of antioxidants have been tested with a view to protecting the cell membrane, including vitamins C and E and certain flavonoids from plants, but the results are not conclusive and the studies need to be amplified in humans. 122,123 Allogeneic transplant of hematopoietic stem cells from related and HLA-compatible donors is a very promising alternative and is the only that offers the possibility of cure to date.…”

“…84 Among the main obstacles are the instability of the vectors and the difficulty in integrating them into this type of cell. Other approaches, such as silencing the β S gene using interference RNA (iRNA), 85 or homologous recombination to substitute the β S gene with the β A gene, 86,87 are promising, but improvements are still needed to the gene transfer methods and efficacy must be demonstrated in animal models.…”