Restoration of human beta-globin gene expression in murine and human IVS2-654 thalassemic erythroid cells by free uptake of antisense oligonucleotides.
Correct human beta-globin mRNA has been restored in erythroid cells from transgenic mice carrying the human gene with beta-globin IVS2-654 splice mutation and from thalassemia patients with the IVS2-654/beta(E) genotype. This was accomplished in a dose- and time-dependent manner by free uptake of morpholino oligonucleotide antisense to the aberrant splice site at position 652 of intron 2 in beta-globin pre-mRNA. Under optimal conditions of oligonucleotide uptake, the maximal levels of correct human beta-globin mRNA and hemoglobin A in patients' erythroid cells were 77 and 54%, respectively. These levels of correction were equal to, if not higher than, those obtained by syringe loading of the oligonucleotide into the cells. Comparison of splicing correction results with the cellular uptake of fluorescein-labeled oligonucleotide indicated that the levels of mRNA and hemoglobin A correlate well with the nuclear localization of the oligonucleotide and the degree of erythroid differentiation of cultured cells. Similar but not as pronounced results were obtained after the oligonucleotide treatment of bone marrow cells from IVS2-654 mouse. The effectiveness of the free antisense morpholino oligonucleotide in restoration of correct splicing of IVS2-654 pre-mRNA in cultured erythropoietic cells from transgenic mice and thalassemic patients suggests the applicability of this or similar compounds in in vivo experiments and possibly in treatment of thalassemia.
Suwanmanee
Thipparat
T
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7295, USA.
Sierakowska
Halina
H
Lacerra
Giuseppina
G
Svasti
Saovaros
S
Kirby
Suzanne
S
Walsh
Christopher E
CE
Fucharoen
Suthat
S
Kole
Ryszard
R
D000818:Animals; D001692:Biological Transport; D015672:Erythroid Precursor Cells / Q000187:drug effects* / Q000502:physiology; D015870:Gene Expression / Q000187:drug effects*; D005914:Globins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D006441:Hemoglobin A / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D051379:Mice; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology*; D012322:RNA Precursors / Q000187:drug effects / Q000378:metabolism; D012326:RNA Splicing / Q000187:drug effects; D013789:Thalassemia / Q000235:genetics / Q000473:pathology*
D016376:Oligonucleotides, Antisense; D012322:RNA Precursors; D005914:Globins; D006441:Hemoglobin A