330. Effect of Different Inducible Minimal Promoters in the RheoSwitch® Therapeutic System That Give Robust and Long-Term In Vivo Gene Expression

Abstract

The RheoSwitch|[reg]| Therapeutic System (RTS) is used to regulate gene expression in a ligand-dependent manner, and may have utility in human gene therapies that require the specific regulation of the level of gene expression, or where it is desirable to terminate therapy either when it is no longer required, or where continuation of the therapy would be unsafe. One version of the RTS is comprised of an ecdysone receptor (EcR) fused to a GAL4 DNA binding domain, a chimeric retinoid X receptor fused to a VP16 activation domain (pVP16-Hs-LmRXR), a reporter gene, such as human secreted alkaline phosphatase (hSEAP) placed under the control of a 6xGAL4 response element, and an inducible minimal promoter (p6xGAL4RE- Inducible Promoter- hSEAP). Studies are described that have evaluated the effects of 10 different inducible promoters on ligand- dependent hSEAP transgene expression in vitro in NIH 3T3 cells and in vivo following quadriceps electroporation in C57BL/6 mice. These data can be interpreted to classify the inducible promoters into three performance groups: 1) low expression and undetectable basal expression, 2) intermediate expression with some basal activity in cells, and 3) robust expression in cells and in vivo. One inducible promoter constructed with a consensus TATA box (TATA3) induced only low levels of expression until three Sp1 binding sites (SP1- TATA) were inserted into the promoter, yielding much higher expression. Additionally, a minimal transthyretin (TTR) promoter showed high levels of expression in response to ligand both in cell culture and in vivo. Of these two minimal promoters, Sp1-TATA3 gave higher expression, but higher basal expression was also observed. Additional evaluation of the Sp1-TATA3 and the TTR minimal promoters showed that insulating these promoters with chicken beta globin insulator further increased the expression levels. The expression cassette containing the TTR minimal promoter was chosen to evaluate the long-term expression of the RTS in vivo due to its high ligand-dependent transgene induction relative to basal expression. To assess long-term induction of in vivo expression, hSEAP protein levels and mRNA levels of hSEAP were analyzed in two groups of C57BL/6 mice electroporated (quadriceps muscle) with the RTS. Group one received a single IP dose of ligand 3 days after electroporation. The second group received a single IP dose of ligand on days 3, 185, and 353 after electroporation. Levels of hSEAP protein in serum were assayed over a series of time points and hSEAP was induced at all times. In addition, animals were sacrificed on day 17 and 365, respectively, and muscle tissue was collected for mRNA analysis by RT-QPCR. Levels of mRNA hSEAP were comparable at both 17 and 365 days after RTS electroporation. These studies establish that RTS containing the TTR minimal promoter both induces high levels of gene expression in vivo as well as sustained performance one year after electroporation.