Signal sequences can be harnessed and controlled for use in the next phase in gene therapy: controlled targeting and delivery of gene products. To achieve this, the inherent signals present in a protein will be removed and replaced with a switchable mechanism. Optimally, gene products could be directed to a specific subcellular compartment where they are exclusively active, and removed from the compartment in a controlled manner. Localization controllable proteins could be used to correct for mislocalization of endogenous proteins that occurs in some cancers and other disease states. The main goal of our project is to use hormone inducible nuclear import signals and export signals as a bidirectional """"""""on/off switch"""""""" for controlled targeting of proteins to subcellular compartments. The protein delivered by this system should retain its therapeutic effect when it reaches the active compartment, in this case, the nucleus. Such a system would be useful when a controlled response in a specific subcellular compartment is desired, or for removal of the therapeutic protein that is toxic when expressed for prolonged, high levels in a particular subcellular compartment.
The first aim of our project is to determine if a protein can be bidirectionally targeted to the correct subcellular compartment via a hormone inducible import/export switch, and also to determine the mechanism of import and export. The import switch, regulated by hormone, directs the protein to the nucleus, while the export switch, triggered when hormone is removed or absent, directs the protein to the cytoplasm.
The second aim i s to determine if the amount of protein directed to a subcellular compartment can be regulated and tuned. The amount of protein targeted to a specific subcellular compartment can be regulated by altering the strength of the signal(s) and/or by changing the dose of the external hormone added.
The third aim i s to determine if the targeted protein is still active when directed to its working subcellular compartment (and non-toxic in its inactive compartment). The protein selected for delivery to the nucleus is the repression domain of the nuclear corepressor NCoR, which has a therapeutic use for down regulation of progesterone receptor activity (implicated in reproductive cancers).
The fourth aim i s to expand the range of agents that confer inducibility to the nucleus by using alternative ligand binding domains from other nuclear receptors including the estrogen receptor, glucocorticoid receptor, and ecdysone receptor. In summary, the ultimate goal is to regulate the delivery, in terms of the location and amount, of a gene product or therapeutic protein to its subcellular site where it maintains its biological activity.
|Cadwallader, Amy B; Lim, Carol S; Rollins, Douglas E et al. (2011) The androgen receptor and its use in biological assays: looking toward effect-based testing and its applications. J Anal Toxicol 35:594-607|
|Cadwallader, Amy B; Rollins, Douglas E; Lim, Carol S (2010) Effect of anabolic-androgenic steroids and glucocorticoids on the kinetics of hAR and hGR nucleocytoplasmic translocation. Mol Pharm 7:689-98|
|Kakar, Mudit; Davis, James R; Kern, Steve E et al. (2007) Optimizing the protein switch: altering nuclear import and export signals, and ligand binding domain. J Control Release 120:220-32|
|Kakar, Mudit; Cadwallader, Amy B; Davis, James R et al. (2007) Signal sequences for targeting of gene therapy products to subcellular compartments: the role of CRM1 in nucleocytoplasmic shuttling of the protein switch. Pharm Res 24:2146-55|
|Davis, James R; Kakar, Mudit; Lim, Carol S (2007) Controlling protein compartmentalization to overcome disease. Pharm Res 24:17-27|
|Li, Henan; Fidler, Matthew L; Lim, Carol S (2005) Effect of initial subcellular localization of progesterone receptor on import kinetics and transcriptional activity. Mol Pharm 2:509-18|