INTELLECTUAL MERIT: Synthetic gene carriers (or nonviral vectors) that efficiently deliver genes to desired biological targets with high biocompatibility and versatility are a technology in high demand in basic and translational gene therapy research. The extra- and intracellular performance of nonviral vectors remains relatively poor compared to viral vectors, which, though proven to be very efficient, are often immunogenic and difficult to modify in order to vary their structures and functions. The proposed CAREER research will synthesize novel, stimuli-transforming polypeptides that complex nucleic acids with a cationic arm under neutral pH, but which, upon hydrolysis in a cellular compartment, are converted to neutral, naturally occurring polypeptides, thus destabilizing the complex and releasing nucleic acids. Specifically, the project will investigate novel biomaterials that incorporate ketalized serine (kSer) residues, which are analogous to lysine (Lys) but which transform into serine (Ser) upon acid-hydrolysis. The kSer residues will be used along with lysine residues, polyethylene glycol (PEG) and other functional polypeptides to create versatile polymeric non-viral vectors. Preliminary in vitro studies have shown that poly(kSer-Lys)/DNA polyplexes and PEG-poly(kSer)/DNA micelles significantly enhanced gene delivery efficiency via several improved intracellular pathways. The PI proposes to develop these novel acid-transforming polypeptide (ATPP)-based gene carriers by better understanding key extra- and intracellular gene delivery pathways. He will (1) investigate hybridization of ATPPs with fusogenic and nuclear localization signal peptides in order to improve targeting, (2) explore the effects of stimuli-sensitive linkages between ATPPs and functional peptides and/or PEG that can be severed by such stimuli to improve efficacy, and (3) optimize the ATPP-containing polymers to improve DNA complexation, cellular internalization, intracellular targeting, DNA release, and transfection.
BROADER IMPACTS: Efficiently delivering therapeutic genes, especially using nonviral methods, is of great interest not only in the clinic (e.g., gene therapy) but also in the genetic manipulation of biological phenomena (e.g., reprogramming somatic cells to be induced stem cells) in biocompatible and versatile ways. The novel biomaterials developed from this research will be available to undergraduate and high school students for a hands-on research experience and for a summer research program. The proposed project will motivate undergraduate students to think creatively and independently and give them a chance to test their hypotheses with the PI. The outreach program aims to stimulate interest in scientific and engineering careers among participating high school students, particularly those from underrepresented groups in nearby urban areas. The proposed research will be translated to classrooms and beyond through undergraduate students' direct participation in the CAREER research and an outreach research program. A new undergraduate course will introduce basic concepts of gene delivery using stimuli-responsive polymeric biomaterials; it will then challenge students to develop creative ideas on the CAREER research. Students with the most innovative suggestions will be invited to test their hypotheses in the PI's laboratory and will assist high school participants in a summer research program on gene delivery using the acid-transforming polypeptides. This outreach program will grow out of the PI's lectures in combination with hands-on experiments at local high schools in socio-economically disadvantaged areas. The most motivated high school students will participate in a summer research program, followed by developing a science fair project under the mentorship of the undergraduate student mentors. Impacts of these educational activities will be assessed by students' publications and presentations and by their choice of professional careers in science and engineering.
