We have previously described the development of oncoretrovirus vectors for human 
gamma-globin using a truncated beta-globin promoter, modified gamma-globin 
cassette, and alpha-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 beta-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 gamma-globin expression in MEL 
cell lines, we tested these vectors using a mouse bone marrow transduction and 
transplantation model. When present, the gamma-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 gamma-globin cassette from the insulated vector was 
expressed in 49% +/- 20% of RBCs long term. RNase protection analysis indicated 
that the insulated gamma-globin cassette was expressed at 23% +/- 16% per copy 
of mouse alpha-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 gamma-globin expression 
approaching the therapeutic range for sickle cell anemia and beta thalassemia.