(549f) Cell-Controlled and Spatially Localized Gene Delivery with Fibrin-Conjugated VSV-g Pseudotyped Lentivirus: Implications for Lentiviral Microarrays

We have previously reported that fibrin hydrogels can be used for effective plasmid DNA and lentivirus encapsulation and gene delivery. Here we report our recent efforts to generate cell-controlled and spatially arranged live cell arrays with fibrin immobilized lentiviruses for enhanced gene delivery and high throughput applications. Gene transfer in fibrin gels was strongly dependent on matrix degradation by target cells however a significant fraction of lentiviral particles diffused out of these gels over time. To eliminate viral diffusion we engineered lentiviral particles that can bind covalently to fibrin during polymerization. Briefly, we generated fusion proteins between the viral envelope glycoprotein (VSV-G), peptide domains that are recognized by factor XIII and protease cleavage sites that are recognized by plasmin. All modified variants exhibited high transduction efficiencies relative to wild type enveloped viruses and bound to fibrin hydrogels in a factor XIII dose dependent manner. As a result diffusion of virus from the gels decreased dramatically even for fibrin gels with low fibrinogen concentration. When the modified lentivirus preparations were spotted in an array format, gene transfer was strictly confined to virus-containing fibrin spots with no cross-contamination between neighboring sites. Our results suggest this transduction system with a high signal/noise ratio may be ideal for generation of lentiviral microarrays for high throughput studies. We demonstrated this by patterning multiple fibrin-conjugated, lineage-specific lentiviruses on glass slides. As a result, cell-type specific expression was localized to corresponding vector coordinates thereby revealing a high degree of spatial control. These lentiviral arrays are currently employed to examine the dynamics of mesenchymal stem cell differentiation towards the osteogenic lineage in real-time and in a high-throughput manner.