A model for the analysis of nonviral gene therapy.

Further understanding of the mechanisms involved in cellular and intracellular delivery of transgene is needed to produce clinical applications of gene therapy. The compartmental and computational model designed in this work is integrated with data from previous experiments to quantitatively estimate rate constants of plasmid translocation across cellular barriers in transgene delivery in vitro. The experimental conditions between two cellular studies were held constant, varying only the cell type, to investigate how the rates differed between cell lines. Two rate constants were estimated per barrier for active transport and passive diffusion. Translocation rates of intact plasmid across the cytoplasmic and nuclear barriers varied between cell lines. CV1 cells were defined by slower rates (0.23 h(-1) cytoplasmic, 0.08 h(-1) nuclear) than those of the HeLa cells (1.87 h(-1) cytoplasmic, 0.45 h(-1) nuclear). The nuclear envelope was identified as a rate-limiting barrier by comparing the rate of intact plasmid translocation at each barrier. Slower intact plasmid translocation in CV1 cells was correlated with a reduced absolute capacity for transgene efficiency in comparison with HeLa cells. HeLa cells were three times more efficient than CV1 cells at producing green fluorescent protein per intact plasmid delivered to the nucleus. Mathematical modeling coordinated with experimental studies can provide detailed, quantitative understanding of nonviral gene therapy.

Transition-state analysis of a Vmax mutant of AMP nucleosidase by the application of heavy-atom kinetic isotope effects.

The transition state of the Vmax mutant of AMP nucleosidase from Azotobacter vinelandii [Leung, H. B., & Schramm, V. L. (1981) J. Biol. Chem. 256, 12823-12829] has been characterized by heavy-atom kinetic isotope effects in the presence and absence of MgATP, the allosteric activator. The enzyme catalyzes hydrolysis of the N-glycosidic bond of AMP at approximately 2% of the rate of the normal enzyme with only minor changes in the Km for substrate, the activation constant for MgATP, and the Ki for formycin 5'-phosphate, a tight-binding competitive inhibitor. Isotope effects were measured as a function of the allosteric activator concentration that increases the turnover number of the enzyme from 0.006 s-1 to 1.2 s-1. The kinetic isotope effects were measured with the substrates [1'-3H]AMP, [2'-2H]AMP, [2'-2H]AMP, [9-15N]AMP, and [1',9-14C, 15N]AMP. All substrates gave significant kinetic isotope effects in a pattern that establishes that the reaction expresses intrinsic kinetic isotope effects in the presence or absence of MgATP. The kinetic isotope effect with [9-15N]AMP decreased from 1.034 +/- 0.002 to 1.021 +/- 0.002 in response to MgATP. The [1'-3H]AMP isotope effect increased from 1.086 +/- 0.003 to 1.094 +/- 0.002, while the kinetic isotope effect for [1',9-14C, 15N]AMP decreased from 1.085 +/- 0.003 to 1.070 +/- 0.004 in response to allosteric activation with MgATP. Kinetic isotope effects with [1'-14C]AMP and [2'-2H]AMP were 1.041 +/- 0.006 and 1.089 +/- 0.002 and were not changed by addition of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)