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Vol. 62, Issue 3, 545-553, September 2002
-Globin Gene Expression in Murine and
Human IVS2-654 Thalassemic Erythroid Cells by Free Uptake of Antisense
Oligonucleotides
Lineberger Comprehensive Cancer Center (T.S., H.S., G.L., S.S.,
R.K., S.K.), Departments of Pharmacology (T.S., H.S., G.L., S.S.,
R.K.), Pathology (S.K.), Medicine (S.K., C.E.W.), and Gene Therapy
Center (C.E.W.), University of North Carolina, Chapel Hill, North
Carolina; and Thalassemia Research Center, Institute of Science and
Technology for Research and Development, Mahidol University, Salaya
Campus, Nakornpathom, Thailand (S.F.)
Correct human 
-globin mRNA has been restored in erythroid
cells from transgenic mice carrying the human gene with -globin IVS2-654 splice mutation and from thalassemia patients with the IVS2-654/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
-globin pre-mRNA. Under optimal conditions of oligonucleotide
uptake, the maximal levels of correct human -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.
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