Multi-ribozyme targeting of human alpha-globin gene expression

One approach to gene therapy for the treatment of hemoglobinopathies has be en focused on increasing normal globin gene expression. However, because of the high concentration of hemoglobin in the red blood cell (32-34 g/dl), m erely introducing the normal globin gene may not be enough to counteract, t he effect of an abnormal globin. We propose that in addition to strategies to add normal (beta)- or (gamma)-globin production sickle erythrocytes, a d ecrease in overall hemoglobin concentration would further decrease the poly merization potential and should be considered with other gene therapy appro aches. Ribozymes offer the potential to target a selected gene product. A m odel system has been set up using the human (alpha)-globin gene for specifi c gene suppression by ribozymes by cleaving (alpha)-globin mRNA transcripts . Ribozymes, specifically targeted to five different sites in the 5' portio n of human (alpha)-globin mRNA, have been designed and tested in vitro. Cle avage of P-32-labeled (alpha)-globin mRNA by these ribozymes has been obser ved in vitro and the highest level of activity has been found for a multi-r ibozyme combining all five ribozymes. The multi-ribozyme gene along with pr omoters with varying activities in erythroid cells was transfected into hum an erythroleukemia K562 cells. The multi-ribozyme gene, under the control o f human (alpha)2-globin promoter alone and combined with the locus control region enhancer, caused a decrease in the level of or globin mRNA of 50-75% compared to the control, determined by RNase protection and by real-time q uantitative PCR. The decrease in (alpha)-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 res ults suggest that the multi-ribozyme may be useful in gene therapy as an ef fective suppressor of a specific globin gene.