ABSTRACT: One approach to gene therapy for the treatment of hemoglobinopathies has been focused on increasing normal globin gene expression. However, because of the high concentration of hemoglobin in the red blood cell (32-34 g/dl), merely introducing the normal globin gene may not be enough to counteract the effect of an abnormal globin. We propose that in addition to strategies to add normal beta- or gamma-globin production to sickle erythrocytes, a decrease in overall hemoglobin concentration would further decrease the polymerization potential and should be considered with other gene therapy approaches. Ribozymes offer the potential to target a selected gene product. A model system has been set up using the human alpha-globin gene for specific gene suppression by ribozymes by cleaving a-globin mRNA transcripts. Ribozymes, specifically targeted to five different sites in the 5' portion of human alpha-globin mRNA, have been designed and tested in vitro. Cleavage of 32P-labeled a-globin mRNA by these ribozym es has been observed in vitro and the highest level of activity has been found for a multi-ribozyme combining all five ribozymes. The multi-ribozyme gene along with promoters with varying activities in erythroid cells was transfected into human erythroleukemia K562 cells. The multi-ribozyme gene, under the control of human alpha-2-globin promoter alone and combined with the locus control region enhancer, caused a decrease in the level of alpha-globin mRNA of 50-75% compared to the control, determined by RNase protection and by real-time quantitative PCR. The decrease in a-globin transcripts has been found to be correlated with expression of the multi-ribozyme in a dose-dependent manner and does not appear to be mediated by an antisense effect. These results suggest that the multi-ribozyme may be useful in gene therapy as an effective suppressor of a specific globin gene.    © 1999 Academic Press

Keywords: Ribozyme, multi-ribozyme, gene therapy, alpha-globin mRNA, K562 cells.

Reprint requests to: Chien Ho, Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, fax: (412) 268-7083, e-mail: chienho@andrew.cmu.edu.

ABSTRACT: The effect of five different transferrin variants (TFv1, TFv2, TFv3, TFv4, and TFv5) on the hemoglobin level, mean corpuscular volume (MCV), ferritin level, percent transferrin saturation (%TS), and the unsaturated iron binding capacity (UIBC) was investigated in subjects with defined HFE haplotypes, 919 persons undergoing health screening and 113 patients with clinical hemochromatosis. The most common variant is TFv4; the population distribution of this variant was also studied. None of the variants were found to have an effect on any of the parameters of iron metabolism that were investigated. Moreover, the frequency of these variants in patients with clinically significant hemochromatosis was no different from that in the general population. We conclude that these polymorphisms in transferrin do not play a role in the expression of hemochromatosis, nor do they produce any other significant changes in iron metabolism.    © 1999 Academic Press

Keywords: Hemochromatosis, ferritin, HFE.

Reprint requests to: Pauline Lee, Ph.D., The Scripps Research Institute, Department of Molecular and Experimental Medicine, 10550 N. Torrey Pines, La Jolla, CA 92037, phone: (858) 784-2217, e-mail: plee@scripps.edu.