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Prepublished online as a Blood First Edition Paper on May 17, 2002; DOI 10.1182/blood-2002-01-0219.
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Blood, 15 September 2002, Vol. 100, No. 6, pp. 2012-2019
GENE THERAPY
Development of virus vectors for gene therapy of  chain
hemoglobinopathies: flanking with a chromatin insulator reduces
 -globin gene silencing in vivo
David W. Emery,
Evangelia Yannaki,
Julie Tubb,
Tamon Nishino,
Qiliang Li, and
George Stamatoyannopoulos
From the Department of Medicine, Division of Medical
Genetics, University of Washington, Seattle; and the Gene and Cell
Therapy Center, Hematology Department and Bone Marrow Transplantation
Unit, George Papanikolaou General Hospital, Thessaloniki, Greece.
We have previously described the development of oncoretrovirus
vectors for human  -globin using a truncated  -globin promoter, modified -globin cassette, and  -globin enhancer. However, one of
these vectors is genetically unstable, and both vectors exhibit variable expression patterns in cultured cells, common characteristics of oncoretrovirus vectors for globin genes. To address these problems, we identified and removed the vector sequences responsible for genetic
instability and flanked the resultant vector with the chicken
-globin HS4 chromatin insulator to protect expression from
chromosomal position effects. After determining that flanking with the
cHS4 element allowed higher, more uniform levels of -globin expression in MEL cell lines, we tested these vectors using a mouse
bone marrow transduction and transplantation model. When present, the
-globin cassettes from the uninsulated vectors were expressed in
only 2% to 5% of red blood cells (RBCs) long term, indicating they
are highly sensitive to epigenetic silencing. In contrast, when
present the -globin cassette from the insulated vector was
expressed in 49% ± 20% of RBCs long term. RNase protection analysis indicated that the insulated -globin cassette was expressed at 23% ± 16% per copy of mouse -globin in transduced RBCs.
These results demonstrate that flanking a globin vector with the cHS4 insulator increases the likelihood of expression nearly 10-fold, which
in turn allows for -globin expression approaching the therapeutic range for sickle cell anemia and thalassemia.
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