472846 Sustained Transgene Expression Via Substrate Mediated Gene Transfer Results from Multiple Transfection Events

Tuesday, November 15, 2016: 2:18 PM
Imperial A (Hilton San Francisco Union Square)
Norman Truong and Tatiana Segura, Chemical and Biomolecular Engineering, University of California Los Angeles, Los Angeles, CA

Statement of purpose: Sustained delivery of therapeutic genes in vitro and in vivo has a wide range of applications in studying biology and in developing therapies to treat disease. Non-viral vectors such as cationic polymers still present promising approaches; however, bolus transfection methods with polyethyleneimine (PEI)-based DNA polyplexes suffer from considerable levels of cytotoxicity and short-lived transgene expression levels. Here, we designed and characterized a hyaluronic acid (HA)-based porous hydrogel system for non-viral gene delivery by loading with surface-associated DNA polyplexes (Figure 1A). With this, we observed enhanced and sustained transgene expression over 30 days of cell culture, with better cell viability, both marked improvements over comparable bolus transfection techniques. Finally, we investigated mechanisms thought to be responsible for the sustained expression profile.

Methods:  HA hydrogels were formed via a Michael-addition reaction between acrylated HA and thiolated peptide crosslinkers with the addition of RGD peptide for cell adhesion. The sphere templating method was used to introduce 60-µm diameter pores. Plasmid DNA was complexed with polyethyleneimine to form DNA polyplexes, which were surface coated by electrostatic association to the pore surfaces of the hydrogel by incubating the formed hydrogel in the polyplex solution. DNA loading was assessed with 32P-labelled DNA and scintillation counting. Transfection profiles were obtained by loading DNA encoding for the Gaussia luciferase gene, seeding D1 mouse mesenchymal stem cells in the hydrogels, and detecting the expressed protein with the associated assay.

Results:  32P analysis of DNA-loaded porous hydrogels showed that up to 4 µg of DNA could be loaded into the scaffold. Although the DNA was electrostatically immobilized to the scaffold, minimal polyplex release was observed with only 5% of the initial loaded DNA released over 7 days. Cell culture within this scaffold resulted in sustained transgene expression over a period of more than 30 days at levels higher than those seen using bolus transfection techniques (Fig. 1B). In addition, adjusting the DNA loading concentration resulted in modulation of transgene expression (Fig. 1B). While repeated bolus transfections in 2D culture result in marked toxicity (Fig. 1C), culture in surface-coated hydrogels was significantly less severe (Fig. 1D). To determine if sustained expression was due to re-transfection events, it was observed that levels of internalized plasmid DNA in cells cultured in surface-coated hydrogels was sustained at higher levels over time than in cells cultured in non-coated hydrogels but administered a bolus polyplex transfection (Fig. 1E).

Conclusions: After DNA loading by surface coating, there was minimal basal DNA release over time, suggesting that this may serve as a robust system for long-term DNA availability and expression. Indeed, sustained expression was seen over thirty days of culture and could be enhanced by increasing DNA loading. Although repeated bolus transfections result in severe toxicity, repeated internalization events are possible in surface-coated scaffolds without the same toxic effects and are responsible for the sustained expression observed. This system is a promising means of sustained gene delivery for various biomedical applications without the need for genomic integration via viral methods, which have innate safety concerns.

Description: Macintosh HD:Users:Norman:CloudStation:4. Publications:NT surface coating paper:AIChE submission:AIChE fig NT.tif

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See more of this Session: Biomaterials: Graduate Student Award Session
See more of this Group/Topical: Materials Engineering and Sciences Division