title: Anti-beta s-ribozyme reduces beta s mRNA levels in transgenic mice: potential application to the gene therapy of sickle cell anemia.
abstract: Our current strategy for gene therapy of sickle cell anemia involves retroviral vectors capable of transducing "designer" globin genes that code for novel anti-sickling globins (while resisting digestion by a ribozyme), coupled with the expression of a hammerhead ribozyme that can selectively cleave the human beta s mRNA. In this report, we have tested in vivo an anti-beta s hammerhead ribozyme embedded within a cDNA coding for the luciferase reporter gene driven by the human beta-globin promoter and hyper-sensitive sites 3 and 4 of the locus control region. We have created mice transgenic for this luciferase-ribozyme construct and bred the ribozyme transgene into mice that were already transgenic for the human beta s gene. We then measured expression of the beta s transgene at the protein and RNA levels by HPLC and primer extension. The presence of the ribozyme was associated with a statistically significant reduction in the level of beta s mRNA in spleen stress reticulocytes (from 60.5 +/- 4.1% to 52.9 +/- 4.2%) and in the percentage of beta s globin chains in very young mice (from 44.5 +/- 0.6% to 40.8 +/- 0.7%). These results demonstrate that it is possible to decrease the concentration of beta s chains and mRNA with the help of a hammerhead ribozyme. While the enormous amount of globin mRNA in reticulocytes is a challenge for ribozyme technology, the exquisite dependence of the delay time for formation of Hb S nuclei on the concentration of Hb S in red blood cells suggests that even a modest reduction in Hb S concentration would have therapeutic value.
authors.affiliation: Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: R
authors.initials: R
authors.lastname: Alami
authors.forename: J G
authors.initials: JG
authors.lastname: Gilman
authors.forename: Y Q
authors.initials: YQ
authors.lastname: Feng
authors.forename: A
authors.initials: A
authors.lastname: Marmorato
authors.forename: I
authors.initials: I
authors.lastname: Rochlin
authors.forename: S M
authors.initials: SM
authors.lastname: Suzuka
authors.forename: M E
authors.initials: ME
authors.lastname: Fabry
authors.forename: R L
authors.initials: RL
authors.lastname: Nagel
authors.forename: E E
authors.initials: EE
authors.lastname: Bouhassira
mesh_terms: D000595:Amino Acid Sequence; D000755:Anemia, Sickle Cell / Q000235:genetics / Q000628:therapy; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D005260:Female; D005818:Genetic Engineering; D015316:Genetic Therapy; D005914:Globins / Q000235:genetics / Q000378:metabolism; D006451:Hemoglobin, Sickle / Q000032:analysis / Q000235:genetics*; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D012313:RNA / Q000235:genetics / Q000378:metabolism; D016372:RNA, Antisense / Q000235:genetics* / Q000502:physiology; D016337:RNA, Catalytic / Q000235:genetics* / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D013997:Time Factors; D019076:Transgenes
chemical_list: D017931:DNA Primers; D006451:Hemoglobin, Sickle; D016372:RNA, Antisense; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012313:RNA; D005914:Globins