(20g) Sustained Transgene Expression Via Substrate-Mediated Gene Transfer Results from Multiple Transfection Events

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.