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Overview of Research Activities:

Current research activity in Uludag's group is comprised of two areas of focus. First one deals with the regeneration and remodeling of bone using protein-based growth factors. The second one looks at developing biomaterials as vectors for the delivery of DNA to target cell in non-viral gene delivery system. The avenues taken are described as follows:

Bone Regeneration Several protein-based cytokines and growth factors (such as Fibroblast Growth Factors and Bone Morphogenetic Proteins) have been identified that have significant potential to stimulate bone formation. These proteins were shown to act at bone sites as: (1) mitogens (i.e., induce proliferation of cells), (2) morphogens (i.e., induce differentiation of cells), (3) chemotactic agents (i.e., attract cells to repair sites), and (4) metabolic regulators (i.e., stimulate and/or inhibit cellular activity). Extensive efforts from both academic and industrial groups are currently leading to novel therapeutic agents to stimulate bone repair in specific local sites (e.g., BMPs for fracture healing in long bones). A better understanding of the protein delivery challenges, in particular controlling the local concentration of the proteins, has been paramount in developing these therapeutic agents for clinical use. These proteins, however, have not been effectively used in systemic bone regeneration, again due to protein delivery challenges; i.e., challenges in delivering the protein to the site of action after intravenous injection into the blood stream. We are exploring new ways to deliver the growth factors to bone after systemic administration. Once targeted to the site of activity, the growth factors are expected to stimulate bone formation at a low does and with minimal activity at extraskeletal sites. Ultimately, we are interested in developing clinically useful therapeutic agents for both local and systemic regeneration of the mineralized tissues. A review of protein targeting research to bone can be found in Gitten et al., 2005. Several approaches are underway in the lab Identification of Appropriate Proteins for Therapy. We focused our initial efforts on two proteins, Bone Morphogenetic Protein-2 and basic Fibroblasts Growth Factor. Studies are being conducted to examine the potency of these growth factors to induce new bone formation, as well as other effects in cell culture and in animals. We are currently exploring other useful proteins, such as osteoprotegerin. Our goal is to identify the proteins with the most potent (effective at minimal dose) and lasting effects based on a variety of biological systems. Varkey et al., 2006 Design of Bone-Seeking Ligands. Significant efforts are being directed towards organic synthesis efforts to prepare novel compounds that (i) has bone affinity, and (ii) can carry proteins to bone after systemic injection. The compounds synthesized are based on bisphosphonates and involve their unique arrangement for an effective drug carrier. Bansal et al., 2004 Pharmacokinetics of Bone-Targeted Therapeutics. A better understanding of the pharmacokinetics of our proteins is necessary to fully explore the potential of the bone-targeting approach. It is necessary to characterize the extent of bone targeting, the residence time of the proteins at bone tissue, as well as the non-specific deposition at other tissues. A comprehensive understanding of protein biodistribution in experimental animal models is generated in this way. Gitten et al., 2005.

Non-Viral Gene Therapy Delivering genes of a bioactive protein, in contrast to delivering the actual protein itself, has the potential to induce a more lasting therapeutic effect; upon successful gene delivery, a bioactive protein is continuously produced by the patient at a site where it is able to exert its effect. Gene delivery is effectively achieved by using viral carriers, where therapeutic genes are incorporated into designer viruses. The viruses, however, are associated with significant side-effects; they could be immunogenic and inflammatory, and sometimes cause irreparable damage to the patients cells when they integrate into the genome. Non-viral to transfer genes into a patient’s cells will greatly facilitate therapeutic approaches based on gene delivery. Towards this goal, novel biomaterials are being synthesized in our group that bind to plasmid DNA and transport it into the cells. Our intent is to design 'nano'-engineered vesicles, based on architecterally-controlled polymers, to facilitate cellular uptake of DNA as well as to ensure its nuclear delivery and expression. Avenues currently being pursued in the lab Biomaterials with Cell-Surface Binding Moieties. Synthetic biomaterials that interact with cell surface receptors are expected to facilitate the uptake of exogenous molecules such as a plasmid DNA. As the starting point, we synthesized biomaterials with RGD-mofits that bind to cell surface integrins. Although our first attempt described in Clements et al., (2005) was not successful, we are now pursuing studies to understand the relationship between the way RGD peptide residues are incorporated into a biomaterial and the obtained cell response. In addition, we are exploring other cell surface receptors (such as cadherins and selecting) that will be beneficial in tissue engineering applications Lipophilic Polycations. Cationic polymers are ideal for binding of plasmid DNA and condensing it into nano-sized complexes suitable for cellular uptake. Imparting a lipophilic character to these polymers was considered beneficial to accelerate DNA delivery into the cells, since the plasma membrane is lipophilic in nature. Towards this end, cationic polymers (such as polyethyleneimine and polylysine) are being modified with endogenous fatty acids. Our studies indeed demonstrated an enhanced (10-fold) delivery of extracellular plasmid DNA into the cells. We are now exploring the ideal properties of a lipophilic, cationic polymer that will maximize DNA transfer into the cells and allow its sustained expression. Intracellular Trafficking of Exogenous DNA. Once a plasmid DNA is transported into the cells, it has to find its way into the cells’ nucleus so that it is effectively expressed (i.e., used for mRNA and protein production). This requires the DNA to be protected from degradation inside the cells and effectively transported to the nucleus. We are designing DNA carriers that can effectively allow plasmid DNA to escape degradative enzymes, and can act as substrates for nuclear transport machinery. Peptide-motifs identified in natural molecules are being utilized in our research efforts to achieve successful DNA trafficking inside the cells.