Optimizing siRNA efficacy through alteration in the target cell-adhesion substrate interaction
Published: 2014
Language: eng
Description: Short interfering RNA (siRNA) is a class of nucleotide drugs with a profound potential to improve patient health through its ability to silence the expression of specific genes at the post-transcriptional level. However, the clinical application of siRNA therapeutics remains hindered by a lack of efficient delivery systems that deposit siRNA into the cytoplasm of cells, a step necessary for siRNA’s silencing effect. Much research has focused on the development of siRNA delivery agents to overcome this challenge. There are no standard pre-clinical models for testing of siRNA delivery agents, and investigators have chosen to evaluate efficacy in a variety of systems including in vitro tissue culture and animal models. These systems have vastly different cellular microenvironments which may modulate cellular behavior and affect the response of cells to siRNA, thus altering the apparent efficacy of siRNA delivery agents.

The substrate on which cells adhere is one aspect of the microenvironment that has been previously shown to alter cellular behavior. In this work, we tested the hypothesis that changing the properties of cellular adhesion substrates can change the apparent efficacy of a siRNA delivery agent. Specifically, we used a commonly employed in vitro cationic lipid siRNA delivery vector and evaluated siRNA silencing efficacy in U251 cells seeded on alginate hydrogel surfaces. These surfaces were synthesized to have systematic variation in integrin ligand arginine-glycine-aspartate (RGD) density and elastic modulus. We found that an eightfold increase in RGD content of the alginate grown substrate increased siRNA knockdown efficacy from 25 ± 12% to 52 ± 10%, with constant concentrations of siRNA and delivery agent. We found no difference in siRNA mediated knockdown efficacy over the elastic modulus range tested (53-133 kPa).

These results indicate that the cell-adhesion substrate interaction can modulate siRNA protein silencing efficacy, a finding important for evaluation of siRNA therapeutics in the in vitro setting.
Notes: Keywords: Bioengineering.

Thesis M.D. with Honors Harvard Medical School 2014