(160m) Directed Evolution of PDGFR-? and PDGF-BB for Promotion of Bone Regeneration.

With over 2 million bone grafts performed annually worldwide, the need for bone tissue is high; however, the supply of autologous and allogeneic donor tissue is unable to meet patient demand, a fact only heightened by factors such as increased life expectancy and rising obesity rates. In an effort to meet the ever-increasing demand for bone tissue, many have turned to tissue engineering approaches that incorporate stem cells into biomaterial scaffolds. These stem cells require signaling proteins, such as platelet-derived growth factor (PDGF) homodimeric ligand PDGF-BB, in order to receive the correct biochemical cues to support bone regeneration. Unfortunately, these signaling proteins are not easily incorporated into engineered scaffolds due to concerns over resulting systemic toxicities upon administration.

PDGF ligands, including PDGF-BB, signal through cognate receptor tyrosine kinases called PDGF receptors (PDGFRs) and are active in many physiological processes such as bone repair and treatment of periodontal defects and diabetic ulcers. Activation of PDGFR-β by PDGF-BB has also been shown to induce robust osteogenic responses in adipose-derived stem cells (ASCs), making it an attractive candidate for use in engineered bone scaffolds. However, both its pleiotropic effects and its implications in diseases such as cancer, vascular disorders, and fibrotic disorders make endogenous PDGF-BB difficult to use in the clinic. Thus, it is advantageous to engineer PDGF-BB in such a way that it only stimulates osteogenic responses in transplanted ASCs.

To overcome the current challenges associated with PDGF in bone regeneration, we are engineering an orthogonal protein system in the form of a new PDGF ligand/receptor pair that is genetically mutated to interact exclusively with one another rather than with any endogenous proteins. The orthogonal PDGF ligand will interact with its engineered PDGF receptor expressed on transplanted ASCs, enabling the targeted activation of regenerative pathways.

In order to remodel the interface between PDGF-BB and PDGFR-β, we are using computational methods to inform mutation of PDGFR-β in order to destroy binding to PDGF-BB. We are also utilizing experimental evolutionary methods to generate and isolate PDGF-BB mutants that bind to the mutated receptor. The binding properties of the resulting orthogonal proteins will be analyzed before their effect is studied in ASCs engineered to express the mutated PDGFR-β. Ultimately, this will create a PDGF ligand that can be administered to patients safely while also promoting bone regeneration in engineered scaffolds.