CONSISTENT, PERSISTENT EXPRESSION FROM MODIFIED RETROVIRAL VECTORS INMURINE HEMATOPOIETIC STEM-CELLS

Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) have shown inconsistent levels and duration of expression as well as a propensity for the acquisition of de novo methylation in vivo. MoMuL V-based vectors are known to contain sequences that are capable of sup pressing or preventing expression from the long terminal repeat, Previ ously, we constructed a series of modified retroviral vectors and show ed that they function significantly better than MuLV-based vectors in vitro. To test the efficacy of the modified vectors in hematopoietic s tem cells in vivo, we examined gene expression and proviral methylatio n in differentiated hematopoietic colonies formed in the spleens of mi ce after serial transplantation with transduced bone marrow (2 degrees CFU-S). We found a significant increase in the frequency of expressio n with our modified vectors (>90% expression in vector DNA containing 2 degrees CFU-S) over the frequency observed with the standard MoMuLV- based vector (28% expression in vector containing 2 degrees CFU-S). Ex pression from the modified vectors was highly consistent, with express ion in >50% of the vector-containing 2 degrees CFU-S from all 20 trans plant recipients analyzed, whereas expression from the standard MoMuLV -based vector was inconsistent, with expression in 0-10% of the vector containing 2 degrees CFU-S from 8 recipients and expression in >50% o f the vector-containing 2 degrees CFU-S from 4 other recipients. In ad dition, we established that the modified vectors had a lower level of DNA methylation than the control vector. These findings represent sign ificant advances in the development and evaluation of effective retrov iral vectors for application in vivo.