850. Quantitative Analysis of Non-Viral Gene Therapy in a Three-Dimensional Liver Tissue Construct

• Published in: Molecular therapy Vol. 9; no. S1; p. S323
• Main Authors: Tedford, Nathan C, Jackson, David W, Domansky, Karel, Griffith, Linda G, Lauffenburger, Douglas A
• Format: Journal Article
• Language: English
• Published: Milwaukee Elsevier Inc 01.05.2004; Elsevier Limited
• Subjects: Brain cancer; Engineering; Gene therapy; Phosphates; Polymers; Quantitative analysis; Tumors; Vectors (Biology); United States > US; Massachusetts
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1016/j.ymthe.2004.06.751

Successful delivery of DNA lies at the heart of gene therapy, and its feasibility in treating a number of diseases depends on the continued development of more effective gene delivery vectors. While vectors based upon recombinant viruses have shown high transfection efficiencies, they may also pose certain health risks to patients and can be difficult to target to individual cell or tissue types of interest. Non-viral vectors look to offer a safer alternative and can be engineered to more effectively treat a specific cell type, tissue, or pathology, but these vectors are still plagued with low transfection levels. Many barriers exist in the successful trafficking of these non-viral complexes to the nucleus. Current evaluations of non-viral gene delivery treatments in more clinical settings often focus on a single barrier at a time, and as a result, may not lead to an overall improvement in gene delivery. Concurrently, more quantitative or systematic in vitro experiments may not correlate well with in vivo data. A scaled up and improved three-dimensional, perfused bioreactor has been designed and built that allows for the long-term culture of primary hepatocytes. Within the microfabricated flow channels of this reactor, cells self assemble over time into tissue structures that more closely mimic hepatic morphology and phenotype than conventional two-dimensional culture systems. By studying non-viral gene delivery in this system, quantitative experiments and experimentally-driven computational models can be developed that may better describe how a vector will perform in vivo. Methodologies in density gradient electrophoresis (DGE) have been adapted to obtain greater resolution in subcellular fractionation. An experimental scheme has been developed which utilizes a newly constructed DGE device that has demonstrated proof of principle for the separation and collection of the vesicular organelles that play an important role in gene delivery. Combined with quantitative downstream assays for both the DNA plasmid and the polymer carrier, vector dynamics can now potentially be tracked at cell entry, progressive stages of vesicular trafficking and escape, and nuclear import, providing data sets which may in turn lead to more accurate and predictive mathematical models. Through a systematic iteration of quantitative experiments and computational simulations, these models will be fine-tuned for different polymer carriers administered to the hepatic tissue constructs, potentially allowing for optimization of specific vector properties and increased success of non-viral approaches.