Natural tissues can have complex architectures characterized by the organization of multiple cells into structures, such as branching networks of the vascular or nervous systems. This cellular organization arises, in part, from spatial patterns in the expression of soluble factors, which create concentration gradients that determine cell differentiation during morphogenesis or direct cellular processes such as migration or axonal extension. Regenerative strategies for damaged tissue must recreate these complex architectures to restore function. Here, we explore patterned gene delivery as a method to create gradients of diffusible proteins. Using DNA encoding for soluble growth factors, patterned transfection can lead to localized and sustained secretion thereby creating concentration gradients as the factors diffuse. We have patterned gene expression in vitro using substrate-mediated delivery, a process that immobilizes DNA complexes to a cell-adhesive substrate, combined with soft lithography techniques. Non-viral DNA complexes were successfully deposited in patterns with widths ranging from 100 - 1000 µm. The patterned DNA complexes successfully transfected cells in similar patterns, with relatively high efficiencies (approximately 30 - 35 % transfected cells). The ability of spatially patterned gene expression to direct cellular processes was investigated using a co-culture model of neurons and accessory cells. Neurotrophic factors, such as nerve growth factor (NGF), promote neuron survival and induce axonal elongation, the latter of which occurs either along high concentrations or up concentration gradients. Primary dorsal root ganglia (DRG) neurons were dissected from 8-day chicken embryos, dissociated, and cultured on patterns of pNGF transfected HEK293T cells. Neurite outgrowth was 30-fold greater at the location of transfected cells as compared to regions directly outside the pattern with both 250 and 100 µm patterns, and sprouting outside the pattern of transfection was reduced significantly when the pattern width decreased from 250 to 100 µm. Total neurite length within the pattern was significantly higher than for neuronal co-cultures in the absence of a pattern, or with NGF protein added to the media, suggesting that the patterning can increase the rate of axonal elongation. The diffusion of proteins from a pattern of gene expression was mathematically modeled to determine how certain design parameters, such as transfection efficiencies, pattern dimensions, and types of accessory cells, affected the concentration gradients. Additionally, factors that influence transport, such as receptor/ligand binding, internalization, and interactions with the ECM were considered. This system is a platform to investigate cellular responses to patterns of gene expression, and correlate observed responses to concentration gradients based on mathematical modeling, and may be applied for the engineering of functional tissue replacements.