RNA Nanotherapeutics for the Amelioration of Astroglial Reactivity.

Abstract

In response to injuries to the CNS, astrocytes enter a reactive state known as astrogliosis, which is believed to be deleterious in some contexts. Activated astrocytes overexpress intermediate filaments including glial fibrillary acidic protein (GFAP) and vimentin (Vim), resulting in entangled cells that inhibit neurite growth and functional recovery. Reactive astrocytes also secrete inflammatory molecules such as Lipocalin 2 (Lcn2), which perpetuate reactivity and adversely affect other cells of the CNS. Herein, we report proof-of-concept use of the packaging RNA (pRNA)-derived three-way junction (3WJ) motif as a platform for the delivery of siRNAs to downregulate such reactivity-associated genes. In vitro, siRNA-3WJs induced a significant knockdown of Gfap, Vim, and Lcn2 in a model of astroglial activation, with a concomitant reduction in protein expression. Knockdown of Lcn2 also led to reduced protein secretion from reactive astroglial cells, significantly impeding the perpetuation of inflammation in otherwise quiescent astrocytes. Intralesional injection of anti-Lcn2-3WJs in mice with contusion spinal cord injury led to knockdown of Lcn2 at mRNA and protein levels in vivo. Our results provide evidence for siRNA-3WJs as a promising platform for ameliorating astroglial reactivity, with significant potential for further functionalization and adaptation for therapeutic applications in the CNS.The authors wish to acknowledge J. Bernstock and G. Pluchino for their critical insights throughout the execution of the study. This work was funded by the European Research Council (ERC) under the ERC-2010-StG grant agreement n° 260511-SEM_SEM, the Bascule Charitable Trust (RG 75149 to SP), the International Foundation for Research in Paraplegia (RG 69318 to S.P.), Wings for Life (RG 82921 to S.P.) and a core support grant from the Wellcome Trust and Medical Research Council to the Wellcome Trust – MRC Cambridge Stem Cell Institute. LPJ was supported by a research training fellowship from the Wellcome Trust (RRZA/057 RG79423)