Nathan C. Tedford

Quantitative comparison of polyethylenimine formulations and adenoviral vectors in terms of intracellular gene delivery processes

An objective of designing molecular vehicles exhibiting virus-like transgene delivery capabilities but with low toxicity and immunogenicity continues to drive synthetic vector development. As no single step within the gene delivery pathway represents the critical limiting barrier for all vector types under all circumstances, improvements in synthetic vehicle design may be aided by quantitative analysis of the contributions of each step to the overall delivery process. To our knowledge, however, synthetic and viral gene delivery methods have not yet been explicitly compared in terms of these delivery pathway steps in a quantitative manner. As a first address of this challenge, we compare here quantitative parameters characterizing intracellular gene delivery steps for an E1/E3-deleted adenoviral vector and three polyethylenimine (PEI)-based vector formulations, as well as the liposomal transfection reagent Lipofectamine and naked DNA; the cargo is a plasmid encoding the β-galactosidase gene under a CMV promoter, and the cell host is the C3A human hepatocellular carcinoma line. The parameters were determined by applying a previously validated mathematical model to transient time-course measurements of plasmid uptake and trafficking (from whole-cell and isolated nuclei lysates, by real-time quantitative PCR), and gene expression levels, enabling discovery of those for which the adenoviral vector manifested superiority. Parameter-sensitivity analysis permitted identification of processes most critically rate-limiting for each vector. We find that the adenoviral vector advantage in delivery appears to reside partially in its import to the nuclear compartment, but that its vast superiority in transgene expression arises predominantly in our situation from postdelivery events: on the basis of per-nuclear plasmid, expression efficiency from adenovirus is superior by orders of magnitude over the PEI vectors. We find that a chemical modification of a PEI-based vector, which substantially improves its performance, appears to do so by enhancing certain trafficking rate parameters, such as binding and uptake, endosomal escape, and binding to nuclear import machinery, but leaves endosomal escape as a barrier over which transgene delivery could be most sensitively increased further for this polymer.

Quantitative analysis of cell signaling and drug action via mass spectrometry-based systems level phosphoproteomics

Protein phosphorylation is a primary form of information transfer in cell signaling pathways and plays a crucial role in regulating biological responses. Aberrant phosphorylation has been implicated in a number of diseases, and kinases and phosphatases, the cellular enzymes that control dynamic phosphorylation events, present attractive therapeutic targets. However, the innate complexity of signaling networks has presented many challenges to therapeutic target selection and successful drug development. Approaches in phosphoproteomics can contribute functional, systems‐level datasets across signaling networks that can provide insight into suitable drug targets, more broadly profile compound activities, and identify key biomarkers to assess clinical outcomes. Advances in MS‐based phosphoproteomics efforts now provide the ability to quantitate phosphorylation with throughput and sensitivity to sample a significant portion of the phosphoproteome in clinically relevant systems. This review will discuss recent work and examples of application data that demonstrate the utility of MS, with a particular focus on the use of quantitative phosphoproteomics and phosphotyrosine‐directed signaling analyses to provide robust measurement for functional biological interpretation of drug action on signaling and phenotypic outcomes.

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.

Vascular Endothelial Growth Factor (VEGF) and Platelet (PF-4) Factor 4 Inputs Modulate Human Microvascular Endothelial Signaling in a Three-Dimensional Matrix Migration Context

The process of angiogenesis is under complex regulation in adult organisms, particularly as it often occurs in an inflammatory post-wound environment. As such, there are many impacting factors that will regulate the generation of new blood vessels which include not only pro-angiogenic growth factors such as vascular endothelial growth factor, but also angiostatic factors. During initial postwound hemostasis, a large initial bolus of platelet factor 4 is released into localized areas of damage before progression of wound healing toward tissue homeostasis. Because of its early presence and high concentration, the angiostatic chemokine platelet factor 4, which can induce endothelial anoikis, can strongly affect angiogenesis. In our work, we explored signaling crosstalk interactions between vascular endothelial growth factor and platelet factor 4 using phosphotyrosine-enriched mass spectrometry methods on human dermal microvascular endothelial cells cultured under conditions facilitating migratory sprouting into collagen gel matrices. We developed new methods to enable mass spectrometry-based phosphorylation analysis of primary cells cultured on collagen gels, and quantified signaling pathways over the first 48 h of treatment with vascular endothelial growth factor in the presence or absence of platelet factor 4. By observing early and late signaling dynamics in tandem with correlation network modeling, we found that platelet factor 4 has significant crosstalk with vascular endothelial growth factor by modulating cell migration and polarization pathways, centered around P38α MAPK, Src family kinases Fyn and Lyn, along with FAK. Interestingly, we found EphA2 correlational topology to strongly involve key migration-related signaling nodes after introduction of platelet factor 4, indicating an influence of the angiostatic factor on this ambiguous but generally angiogenic signal in this complex environment.

Illuminating signaling network functional biology through quantitative phosphoproteomic mass spectrometry

Advances in protein phosphorylation analysis by mass spectrometry (MS) are enabling the generation of high quality, quantitative datasets of protein phosphorylation with a breadth of coverage and reproducibility not previously attainable. Comparisons of signaling responses in cells at a network level are now feasible and studies looking at cellular response to ligand stimulation, drug treatment or genetic modification are transforming our understanding of how cellular decision processes are encoded through the signaling network. The large and dynamic datasets acquired through MS-based phosphoproteomics can be combined with other types of biological data for computational modeling of cellular decision processes with direct biological relevance to cellular state and predictive of cellular response. Signaling analysis at a network level is just beginning. Challenges remain in validating and translating initial models generated using defined in vitro models to in vivo systems. The advent of higher throughput methods for validating models generated with MS will deepen our understanding of the relationship between signaling and disease and therefore the development and implementation of therapeutics.

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

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.