RETROVIRAL GENE THERAPY VECTORS INCLUDING INSULATOR ELEMENTS TO PROVIDE HIGH LEVELS OF GENE EXPRESSION

摘要

Silencing of retrovirus vectors poses a significant obstacle to stem cell gene therapy, and may also impact stem cell marking and genetic manipulation studies. Retrovirus silencing is ameliorated but not eliminated by self-inactivating (SIN) mutations. The invention relates to a novel mammalian silencer-blocking assay to overcome residual silencing using insulator elements. A human β-globin transgene regulated by the Locus Control Region is silenced in transgenic mice when linked to retrovirus vector sequences. Dimer cHS4 core insulators flanking the β-globin reporter completely block MSCV retrovirus silencing in transgenic mice. This distinguishes silencer-blocking activity by cHS4 from its enhancer-blocking activity. Retrovirus vectors cannot be created at high titer with two internal dimer cHS4 cores. However, transgenic mouse silencer-blocking assays demonstrate that a combination of internal gypsy and dimer cHS4 cores cooperate to attenuate retrovirus silencing. EGFP retrovirus vectors with gypsy and the dimer cHS4 core increase expression from MSCV transduced F9 Embryonic Carcinoma cells. The highest EGFP expression in F9 cells was produced by a high titer SIN retrovirus with gypsy in the LTRs. The silencer-blocking assay described here complements current enhancer-blocking and position-effect blocking assays. The gypsy insulated SIN retrovirus vector is particularly well suited for gene therapy and for stem cell marking studies.