| Popis: |
The RheoSwitch® Therapeutic System (RTS) is used to regulate gene expression in response to a systemically administered small molecule ligand, or Activator Drug, and may have utility in human gene therapies that require a specifically regulated level of gene expression, as well as in situations where it is desirable to terminate the therapy either when it is no longer needed, or where continuation of the therapy would be unsafe. The version of RTS used in these studies is comprised of an ecdysone receptor (EcR) fused to a GAL4 DNA binding domain, a chimeric of retinoid X receptor fused to a VP16 activation domain (pVP16-Hs-LmRXR), a reporter gene (such as GFP) placed under the control of a 6xGAL4 response element, and a minimal promoter (p6xGAL4-TATA-SEAP), was constructed into Adenovirus (Ad). The Ad-RTS-GFP has been used to evaluate ligand dependent transgene expression in tissues. Following intravenous injection or direct intracerebral injection of Ad-RTS-GFP in the brain, GFP expression was shown to be dependent on systemic administration of ligand. While ligand had been previously shown to partition efficiently to various organs in the thoracic cavity and leg muscles, its ability to cross the blood brain barrier was an important issue for gene or cell therapies targeted to the brain. In the present studies, mice were given a single intracerebral injection of 108-109 infectious virus particles and GFP expression was induced with an intraperitoneal (IP) injection of Activator Drug A (100 mg/ kg) 2-3 days after viral injection. Brain tissue was removed 3 days after ligand administration and cryostat sections were prepared for assessment of GFP expression. A strong fluorescence signal was observed throughout the brain in mice receiving ligand and infected cells were primarily in the ventricle, midline and outer regions of the brain. In other studies, ligand-dependent GFP expression in brain was observed following administration of ligand by either intramuscular or oral routes. These studies demonstrate that Activator Drug crosses the blood brain barrier and that the RTS system is active in both liver and brain when delivered using an Adenoviral vector. |
