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Protocols

A summarization of our protocols related to our current research endeavors are detailed.

Inducible AKT System

The inducible AKT system was established at Baylor College of Medicine and adapted to KU Medical Center by Dr. Benyi Li when he established the lab in 2001. It is based on the Chemically Induced Dimerization (CID) approach to bring a pleckstrin homology domain (PH domain) deleted AKT-1 to the cellular plasma membrane for activation. This inducible AKT system functions independently of the AKT upstream kinase PI3K or other environmental factors. The other unique feature of the inducible AKT system (iAKT) is that one can artificially initiate the activation of AKT kinase by adding a chemical inducer (e.g. AP22783) and also control the period of AKT kinase action by removing the inducer.

graphic of AKT protocole, adapted by Dr. Benyi Li, KU School of Medicine

The iAKT system contains three components: a membrane docking protein, an inducible AKT molecule and a chemical inducer. The plasma membrane docking protein was created by fusing a c-Src myristoylation signal (Myr) to two copies of the modified rapamycin binding domain FRB1 to generate Myr-FRBL2 (pt.1). The inducible AKT (iAKT) molecule was created by fusing three tandem rapamycin binding domains of FK506 binding protein (Fpk3) to the N-terminus of the PH domain deleted mutant of AKT1 (DPH.Akt) to generate Fpk3-DPH.Akt (pt.2). The chemical inducer AP22783 is a bivalent, cell permeable rapamycin analog with no bioactivity to the cell. It binds to the FRB1 domain and concomitantly to the Fpk domain. The iAKT system can be activated by AP22783-mediated heterodimerization of the FRBL2 and Fpk3 domains, resulting in plasma membrane association of the Fpk3-DPH.Akt molecule. The plasma membrane associated DPH.AKT is phosphorylated and activated independent of PI3K or growth factor when tested in human Jurkat-TAg and 293T cells. Active iAKT can protect cells from apoptosis induced by various stimuli. The strategy and procedure for plasmid construction and the functional assay protocol were described in our recent paper published in Gene Therapy 2002, 9(4)233-244.

Prostate-Specific Activation of the iAKT System in Mouse Model

Using a Cre/LoxP approach, we developed a transgenic mouse model that expresses the iAKT system in mouse prostate epithelial cells. Unlike other prostate conditional mouse models, there are three advantages over traditional approaches:

  1. the iAKT expression can be expressed in a tissue-specific manner when cross-bred with a conditional CRE transgenic mouse;
  2. the iAKT system is under a strong promoter, CMV/chick b-action (termed as CX promoter) recombination, therefore is not androgen dependent;
  3. Even when the proteins are expressed, the iAKT system is inactive without the presence of the CID, and therefore, it can be controlled by timing and length.

The schema below illustrates the prostate-specific expression of the iAKT system in our transgenic mouse model described in our publication Carcinogenesis 2007.

graphic of IAKT protocol used in the studies of Dr. Benyi Li

Nanoparticle-Based Prostate Cancer-Specific Delivery of AR siRNA

Successful delivery of therapeutic agents efficiently and specifically to the targeted cancer tissue is desirable for the treatment of human cancers. A current hotspot for drug delivery systems is polymeric nanoparticles, which do not have the side effects of traditional viral vectors (e.g. insertional mutagenesis and immunogenesis) or liposome vehicles (e.g. toxicity and inefficiency). Properly designed nanoparticles can be delivered specifically to a target tissue and then the encapsulated drug can be released inside the targeted cells after degradation of the nanoparticle matrix without affecting surrounding tissues. Nanoparticles that are less than 100 nm in size and have a hydrophilic surface can avoid the uptake by the reticuloendothelial system and passively accumulate in tumors due to the enhanced permeability and retention effect in leaky tumor vasculature. Additional coatings (e.g. PEG) can further increase their circulating time by repelling plasma proteins. Two recent studies successfully targeted prostate cancer cells with bioactive nanoparticles (termed as Bio-Nanoparticle hereafter). The prostate tissue specific targeting was achieved by conjugating the nanoparticles with antibodies or Aptamers against human prostate-specific membrane antigen (PSMA). PSMA is a well-known tumor antigen. It is primarily expressed on the surface of prostate cancer epithelial cells and also expressed in the microvasculature of most studied tumors. This highly restricted expression pattern and its elevated expression levels in metastatic prostate cancers warranted it as a promising target for the diagnosis, detection and management of prostate cancer. As such, PSMA has currently been used for molecular imaging, cancer vaccine development and targeted drug delivery in prostate cancers. Particularly, a RNA-based aptamer targeting human PSMA was recently developed (PSMA-A10), which interacts specifically with PSMA extracellular domain.

Nucleic acid ligands, or so-called aptamers, are RNA or DNA molecules that can fold into unique conformational structures and interact with protein targets due to their complementary surface charge and shape. Aptamers are short, single-strand oligonucleotides without immunogenecity or toxicity. A particular aptamer for a given target can be identified by a special approach, called systemic evolution of ligands by exponential enrichment (SELEX). The sequence-defined aptamers can be synthesized in large amounts and can be conjugated onto other particles such as polymer-based nanoparticles to achieve targeted cell-specific delivery.

Currently, we are going to establish a nanoparticle-based approach for targeted delivery of the AR siRNA molecules to prostate cancer cells, as illustrated below.

AR siRNA hairpin-structure and nanoparticle
graphic of AR-siRNA molecules

School of Medicine

University of Kansas Medical Center
Department of Urology
Mail Stop 3016
3901 Rainbow Boulevard
Kansas City, KS 66160
Phone: 913-588-6146