RhoA knockdown by cationic amphiphilic copolymer/siRhoA polyplexes enhances axonal regeneration in rat spinal cord injury model

Abstract

Spinal cord injury (SCI) results in permanent loss of motor and sensory function due to developmentally-related and injured-induced changes in the extrinsic microenvironment and intrinsic neuronal biochemistry that limit plasticity and axonal regeneration. Our long term goal is to develop cationic, amphiphilic copolymers (poly (lactide-co-glycolide)-g-polyethylenimine, PgP) for combinatorial delivery of therapeutic nucleic acids (TNAs) and drugs targeting these different barriers. In this study, we evaluated the ability of PgP to deliver siRNA targeting RhoA, a critical signaling pathway activated by multiple extracellular inhibitors of axonal regeneration. After generation of rat compression SCI model, PgP/siRhoA polyplexes were locally injected into the lesion site. Relative to untreated injury only, PgP/siRhoA polyplexes significantly reduced RhoA mRNA and protein expression for up to 4 weeks post-injury. Histological analysis at 4 weeks post-injury showed that RhoA knockdown was accompanied by reduced apoptosis, cavity size, and astrogliosis and increased axonal regeneration within the lesion site. These studies demonstrate that PgP is an efficient non-viral delivery carrier for therapeutic siRhoA to the injured spinal cord and may be a promising platform for the development of combinatorial TNA/drug therapy.

Physicochemical stability and transfection efficiency of cationic amphiphilic copolymer/pDNA polyplexes for spinal cord injury repair

“…In addition, the success of gene delivery carriers depends upon the choice of delivery route and residence time at the delivery site. In our previous study, we observed that intraspinally injected PgP/siRNA-Cy5 polyplexes were retained at the injury site up to 24 hours post-injection, while naked siRNA-Cy5 was undetectable after 6 hours, likely either as a result of degradation or diffusion away from injection site 18 . In this study, we used a hydrophobic dye (DiR) loaded into the micelle core to visualize PgP/pDNA polyplexes and evaluated longer time periods after injection of DiR-PgP/pDNA in SCI lesion sites.…”

“…Preservation of bioactivity during long-term storage is another important challenge for the clinical translation of non-viral vectors. Previously, we showed that PgP can maintain stable complexes with siRNA up to 4 weeks at 4 °C 18 . In the present study, PgP/pDNA polyplexes showed physico-chemical stability and retained transfection efficiency after storage for up to 5 months at 4 °C.…”

“…The compression SCI was performed as previously described by Gwak et al . 4 , 18 . The exposed T8~T9 spinal cord was compressed by a vascular clip (75 g force) for 10 min.…”

“…Toward this end, we recently reported the synthesis and characterization of poly (lactide-co-glycolide)-graft-polyethylenimine (PgP) and its ability to efficiently transfect pGFP in various neural cell lines and primary chick forebrain neurons in 10% serum condition in vitro , as well as in the normal rat spinal cord 17 . We have also shown that PgP can deliver siRNA targeting RhoA, a critical signaling pathway activated by multiple extracellular inhibitors of axonal regeneration, to the injured spinal cord and maintain RhoA knockdown for up to 4 weeks post-injection, reduce astrogliosis and cavitation, and increase axonal regeneration 18 . In this paper, we show that PgP/pDNA polyplexes remain stable and retain transfection activity after lyophilization as well as after storage for 6 months at 4 °C, important features for commercial and clinical application.…”

Deletion of the Nucleotide Exchange Factor Vav3 Enhances Axonal Complexity and Synapse Formation but Tampers Activity of Hippocampal Neuronal Networks In Vitro

“…In the light of the current knowledge, members of the RhoA subfamily of GTPases represent attractive candidates. For example, current studies focus on the therapeutic inhibition of RhoA, which is correlated with an enhanced neuronal survival and axon regeneration after lesion [65][66][67][68][69]. Progress in this research field may help to develop new therapies and strategies to treat CNS lesions.…”