396. Quantitative Analysis of Non-Viral Gene Therapy in a Three-Dimensional Liver Bioreactor
Authors: Nathan Tedford, Jennifer Fang, Karel Domansky, Linda G. Griffith, Douglas A. Lauffenburger
Journal: Molecular Therapy (2006)

Successful delivery of DNA is a crucial first step in attaining effective gene therapy. While vectors based upon recombinant viruses have shown high transfection efficiencies, they may also pose health risks to patients and can be difficult to target to specific cell types. Non-viral (NV) vectors look to offer a safer alternative and can be engineered to more effectively treat a specific cell type or pathology, but these vectors are plagued with low transfection levels. Many barriers exist in the successful trafficking of these NV complexes to the nucleus. Current evaluations of NV gene delivery treatments in more clinical settings often focus on a single barrier at a time and may not lead to an overall improvement in gene delivery. Concurrently, more quantitative or systematic in vitro studies may not correlate well with in vivo data. Our combined approach of quantitative vector trafficking and expression assays coupled with simulation of vector specific mathematical models that describe each step of the delivery process has shown that a systems level approach can reveal the most rate- limiting steps for a given vector. These studies have shown that different vectors have different rate limiting steps and that these results can be counterintuitive when only one step is examined at a time. Established NV vectors have been compared to novel polymer carriers and adenovirus to show how synthetic carriers differ from a viral vector in in vitro culture. Ensuing model analysis has given added insight into how each vector is mechanistically different and how these carriers could be improved to obtain more effective NV therapeutics. These studies have now been extended to primary liver, coupled with device development to attain a more clinically relevant model system and more spatial resolution to study intracellular vector trafficking and localization. A scaled up and improved 3-D, perfused bioreactor has been built that allows for the long-term culture of primary hepatocytes. Within the microfabricated reactor flow channels, cells self assemble over time into tissue structures that more closely mimic hepatic morphology and phenotype than conventional 2-D cultures. This bioreactor allows for multiplexed, high-throughput assays that are not possible in animal models, and provides the micro-scale circulation and tissue structure that are lacking in traditional in vitro culture. By studying NV gene delivery in this system, quantitative experiments and experimentally-driven computational models have been developed that may better describe how a vector will perform in vivo. Gene delivery efficiency, kinetics and transgene expression in this system have been directly compared to 2-D culture systems. A newly constructed density gradient electrophoresis device can separate and collect the vesicular organelles that play an important role in gene delivery. Combined with quantitative assays for the complexed DNA plasmid or viral vector of interest, vector dynamics are tracked at cell entry, through the stages of vesicular trafficking and escape, and at nuclear import, providing data sets to generate more accurate and predictive mathematical models for vector enhancement.