Quantitative analysis of non-viral gene therapy in primary liver culture systems

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2007.Includes bibliographical references (p. 161-172).Gene therapy has the potential to cure thousands of diseases caused by genetic abnormalities, provide novel combination therapies for cancers and viral infections, and offer a new and effective platform for next generation vaccines. However, after more than three decades of research and development efforts, clinical success has yet to be realized. Successful delivery of DNA is a crucial first step in attaining safe and effective gene therapeutics. While vectors based upon recombinant viruses have shown high delivery and transfection efficiencies, they may also pose certain health risks to patients, can be difficult to target to cell or tissue types of interest, and present difficulties for large-scale manufacturing. 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 and cannot provide adequate and sustained levels of gene expression. Continued efforts focus on producing next generation non-viral vectors that safely deliver therapeutic transgenes with the efficiency of their viral counterparts. Many barriers exist in the successful trafficking of these non-viral complexes to the nucleus.(cont.) 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. Our combined approach of quantitative vector trafficking and expression experiments coupled with computational simulation of vector specific mathematical models that describe every step of the gene delivery process has shown that a systems level approach can glean insight into the most rate-limiting steps for a given vector and generate hypotheses for future vector development and improvement. These studies have been extended to primary liver cultures, coupled with device development to attain a more clinically relevant model system and more spatial resolution to study intracellular vector trafficking and localization. A larger perfused 3-D liver bioreactor has been built that allows for long-term culture of primary hepatocytes that more closely mimic hepatic phenotype than in conventional 2-D cultures and for multiplexed quantitative measurement that is not possible in animal models.(cont.) A newly constructed density gradient electrophoresis device can separate vesicular organelles and track vector dynamics throughout the cell. These systems have provided more comprehensive data sets which show that vectors behave differently in different culture systems and that different vectors show unique cell trafficking dynamics. These results lend insight for future vector screening methodologies and provide vector specific mathematical models for primary cell transfection that can lead to further optimization of the polymer vectors studied in this work, which can contribute to the development of more efficient next generation in vivo delivery agents.by Nathan C. Tedford.Ph.D

Interrogating Signaling Nodes Involved in Cellular Transformations Using Kinase Activity Probes

Protein kinases catalyze protein phosphorylation and thereby control the flow of information through signaling cascades. Currently available methods for concomitant assessment of the enzymatic activities of multiple kinases in complex biological samples rely on indirect proxies for enzymatic activity, such as posttranslational modifications to protein kinases. Our laboratories have recently described a method for directly quantifying the enzymatic activity of kinases in unfractionated cell lysates using substrates containing a phosphorylation-sensitive unnatural amino acid termed CSox, which can be monitored using fluorescence. Here, we demonstrate the utility of this method using a probe set encompassing p38α, MK2, ERK1/2, Akt, and PKA. This panel of chemosensors provides activity measurements of individual kinases in a model of skeletal muscle differentiation and can be readily used to generate individualized kinase activity profiles for tissue samples from clinical cancer patients.Cell Migration ConsortiumNational Institutes of Health (U.S.) (GM064346)National Institutes of Health (U.S.). Tumor Cell Networks Center (U54-CA112967)National Science Foundation (U.S.) (NSF-0070319)National Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (Fellowship F32GM085909

Gene Delivery Properties of End-Modified Poly(ß-amino ester)s

Here, we present the synthesis of a library of end-modified poly(beta-amino ester)s and assess their utility as gene delivery vehicles. Polymers were synthesized using a rapid, two-step approach that involves initial preparation of an acrylate-terminated polymer followed by a postpolymerization amine-capping step to generate end-functionalized polymers. Using a highly efficient poly(beta-amino ester), C32, we show that the terminal amine can greatly affect and improve polymer properties relevant to gene delivery. Specifically, the in vitro transfection levels can be increased by 30% and the optimal polymer:DNA ratio lowered 5-fold by conjugation of the appropriate end group. The most effective modifications were made by grafting primary diamine molecules to the chain termini. The added charge and hydrophobicity of some derivatives enhanced DNA binding and resulted in the formation of polymer-DNA complexes less than 100 nm in diameter. In addition, cellular uptake was improved 5-fold over unmodified C32. The end-modified poly(beta-amino ester)s presented here are some of the most effective gene-delivery polycations, superior to polyethylenimine and previously reported poly(beta-amino ester)s. These results show that the end-modification of poly(beta-amino ester)s is a general strategy to alter functionality and improve the delivery performance of these materials