Functional features defining the efficacy of cholesterol-conjugated, self-deliverable, chemically modified siRNAs

Progress in oligonucleotide chemistry has produced a shift in the nature of siRNA used, from formulated, minimally modified siRNAs, to unformulated, heavily modified siRNA conjugates. The introduction of extensive chemical modifications is essential for conjugate-mediated delivery. Modifications have a significant impact on siRNA efficacy through interference with recognition and processing by RNAi enzymatic machinery, severely restricting the sequence space available for siRNA design. Many algorithms available publicly can successfully predict the activity of non-modified siRNAs, but the efficiency of the algorithms for designing heavily modified siRNAs has never been systematically evaluated experimentally. Here we screened 356 cholesterol-conjugated siRNAs with extensive modifications and developed a linear regression-based algorithm that effectively predicts siRNA activity using two independent datasets. We further demonstrate that predictive determinants for modified and non-modified siRNAs differ substantially. The algorithm developed from the non-modified siRNAs dataset has no predictive power for modified siRNAs and vice versa. In the context of heavily modified siRNAs, the introduction of chemical asymmetry fully eliminates the requirement for thermodynamic bias, the major determinant for non-modified siRNA efficacy. Finally, we demonstrate that in addition to the sequence of the target site, the accessibility of the neighboring 3\u27 region significantly contributes to siRNA efficacy

Manipulation of Panx1 Activity Increases the Engraftment of Transplanted Lacrimal Gland Epithelial Progenitor Cells

Sjögren's syndrome is a systemic chronic autoimmune inflammatory disease that primarily targets the salivary and lacrimal glands (LGs). Currently there is no cure; therefore, cell-based regenerative therapy may be a viable option. LG inflammation is facilitated by extracellular ATP and mediated by the Pannexin-1 (Panx1) membrane channel glycoprotein. We propose that suppression of inflammation through manipulation of Panx1 activity can stimulate epithelial cell progenitor (EPCP) engraftment. The expression of pannexins in the mouse and human LG was assayed by qRT-PCR and immunostaining. Acute LG inflammation was induced by interleukin-1α (IL1α) injection. Prior to EPCP transplantation, IL1α-injured or chronically inflamed LGs of thrombospondin-1-null mice (TSP-1-/-) were treated with the Panx1-specific blocking peptide (10panx) or the self-deliverable RNAi (sdRNAi). The efficacy of cell engraftment and the area of inflammation were analyzed by microscopy. Panx1 and Panx2 were detected in the mouse and human LGs. Panx1 and proinflammatory factors were upregulated during acute inflammation at days 1 to 3 after the IL1α injection. The analysis of EPCP engraftment demonstrated a significant and reproducible positive correlation between the 10panx peptide or Panx1 sdRNAi treatment and the number of engrafted cells. Similarly, treatment of the LG of the TSP-1-/- mouse (mouse model of chronic LG inflammation) by either Panx1 or Caspase-4 (also known as Casp11) sdRNAi showed a significant decrease in expression of proinflammatory markers and the lymphocyte infiltration. Our results suggest that blocking Panx1 and/or Casp4 activities is a beneficial strategy to enhance donor cell engraftment and LG regeneration through the reduction of inflammation