Therapeutic proteins and small interfering RNA (siRNA) therapeutics are emerging technologies to increase beneficial protein levels or reduce disease causing protein expression. To develop new therapeutics, critical challenges relating to the cost of production and delivery to the cells of interest will need to be overcome. The cell membrane, as part of the endomembrane system, is a barrier to both secretion of therapeutic proteins from cells and entry of siRNA therapeutics into cells. Disruption of normal cell function due to environmental stresses is known to alter the function of the endomembrane system. The effects of this disruption on the production and function of biotherapeutics remains poorly understood. To inform protein production process and siRNA therapeutic designs, we examined how environmental stresses altered protein secretion and siRNA processing. Industrial scale production of many therapeutic proteins relies on mammalian cells grown in batch bioreactors. To maximize productivity, the bioreactor contents must be mixed. However, excessive mixing can damage cells through shear. As a result, pockets of low nutrients and oxygen, high or low temperature, and cellular waste products can form. The cellular response to these types of environmental stresses is often to increase the production of protective proteins, which when combined with the burden of therapeutic protein production, overwhelms the cellular protein quality control system and activates the Unfolded Protein Response (UPR). Using small molecule inhibitors and activators, this research investigated how the UPR-activated protein degradation pathways of autophagy and proteasomal degradation impacted protein production. Interestingly, we showed that increasing proteasomal degradation improved protein secretion. Obesity, smoking, hypertension, and diabetes activate the UPR in patients’ cells. For patients receiving siRNA-based therapeutics, activation of UPR is likely to alter the intracellular processing of the siRNAs. We compared the changes in function of cationic lipid delivered siRNAs between control cells and cells with the UPR activated. Using chemical inhibitors and activators, we found that endosome/autophagy crosstalk is linked to differences in siRNA accumulation, distribution, and activity. Our results will inform future siRNA therapeutic designs by reinforcing that accumulation of siRNAs in a cell does not necessarily correlate to silencing levels and that the disease state of individual patients may affect the activity of siRNA therapeutics.
Read
