Modification of globin gene expression by RNA targeting strategies.

OBJECTIVE: Sickle cell anemia is a genetic blood disease resulting from production of mutant beta-globin (beta(S)) and has severe clinical consequences. It is known that a higher cellular gamma-globin level, e.g., higher ratio of cellular gamma-globin to beta(S)-globin (gamma/beta(S) ratio), inhibits sickle hemoglobin (HbS) polymerization tendency. Hence, therapeutic treatment of sickle cell anemia has been focused on introducing gamma-globin gene into red blood cells to increase the cellular gamma/beta(S) ratio. Here, we have introduced ribozymes and small interfering RNAs (siRNAs) against beta(S)-globin mRNA into blood cells as a means to increase the gamma/beta(S) ratio. MATERIALS AND METHODS: Single and multiribozymes against beta(S)-globin mRNA have been tested in vitro and in human erythroleukemia K562beta(S) cells that stably express exogenous beta(S)-globin gene. Primary human hematopoietic progenitor cells were also transfected with multiribozyme and the gamma/(gamma + beta) ratio determined and compared with cells transfected with long hairpin beta-globin cDNA and synthetic siRNA genes. RESULTS: We have found that the multiribozyme zb21A containing two ribozyme units effectively reduces beta(S)-globin mRNA both in vitro and in K562beta(S) cells. The gamma-globin mRNA to beta(S)-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 gamma/(gamma + beta) 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 gamma-globin mRNA to beta- or beta(S)-globin mRNA, providing possible strategies to increase the effectiveness of gamma-globin gene transfer as gene therapy for treatment of patients with sickle cell anemia.</p

Effect of nitrite and nitrate supplementation on the levels of nitrite and nitrate in whole blood.

<p>C57BL/6 mice were divided into three groups. Control group received normal water (no treatment), second group received nitrite (0.1 g/L)-containing water and the third group received nitrate (1 g/L)-containing water for a week. All groups were fed standard diets during the treatment period. Nitrite (A) and nitrate (B) in the whole blood from each group were measured using a chemiluminescence NO analyzer (Sievers, Model 280 NO analyzer). Data are means ± SEM (n≥4). * p<0.05.</p

Effect of nitrite and nitrate supplementation on platelet reactivity in whole blood.

<p>Whole blood from the control (no treatment) mice or the mice that were given additional nitrite (0.1 g/L) or nitrate (1 g/L) in the drinking water for a week was taken and diluted with saline (1∶4) for aggregation (A and B) and ATP release (C) measurement. Chronolume® was added to the diluted blood for 2 min at 37°C for ATP measurement and then platelets were activated by collagen (0.5∼3 µg/ml) addition. Aggregation amplitude was shown as maximal impedance (A) and the total response duration was expressed as area under the 5-min aggregation curve (AUC, B). The amount of ATP released from platelets was analyzed by luciferin-luciferase assay using an ATP standard (C). Control (no treatment): open diamond, nitrite (0.1 g/L): red square, nitrate (1 g/L): blue triangle. Data are means ± SEM (n≥5). * p<0.05 compared with control (no treatment) at each concentration of collagen.</p

Influences of different perturbations in NO metabolism on nitrite and nitrate levels in whole blood.

<p>C57BL/6 mice were randomly divided into four groups and treated with antibiotics (bacitracin (4 mg/ml), neomycin (4 mg/ml), tetracycline (1 mg/ml)) or L-NAME (1 g/L) or low nitrite/nitrate (NOx) diet for a week. Age-matched eNOS knock-out mice were used. Nitrite (A) and nitrate (B) were measured using a chemiluminescence NO analyzer (Sievers, Model 280 NO analyzer). Relative values to wild-type control on standard diets were calculated in each treatment group and inserted in both (A) and (B). Data are means ± SEM (n≥7). *p<0.05.</p

Measurement of tail bleeding time.

<p>Mice were divided into three groups for 1-week treatment; control (no treatment, standard diets), nitrite supplementation (0.1 g/L) and low NOx diet group. Nitrite in the whole blood from each group of mice was measured using a chemiluminescence NO analyzer (A) (Sievers, Model 280 NO analyzer). The tail was amputated 0.5 cm from the tip and blood was blotted onto filter paper every 10 seconds (B). Data are means ± SEM (n≥8). *p<0.05.</p