Nanoparticle Delivery of Fidgetin siRNA as a Microtubule-based Therapy to Augment Nerve Regeneration

Timothy O Austin,Peter W Baas,Wenqian Yu,Andrew J Matamoros,Parimala Nacharaju,David J Sharp,Joel M Friedman,Adam J Friedman

doi:10.1038/s41598-017-10250-z

Timothy O Austin, Peter W Baas + Show 6 more

Open Access

https://doi.org/10.1038/s41598-017-10250-z

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Abstract

Microtubule-stabilizing drugs have gained popularity for treating injured adult axons, the rationale being that increased stabilization of microtubules will prevent the axon from retracting and fortify it to grow through inhibitory molecules associated with nerve injury. We have posited that a better approach would be not to stabilize the microtubules, but to increase labile microtubule mass to levels more conducive to axonal growth. Recent work on fetal neurons suggests this can be accomplished using RNA interference to reduce the levels of fidgetin, a microtubule-severing protein. Methods to introduce RNA interference into adult neurons, in vitro or in vivo, have been problematic and not translatable to human patients. Here we show that a novel nanoparticle approach, previously shown to deliver siRNA into tissues and organs, enables siRNA to gain entry into adult rat dorsal root ganglion neurons in culture. Knockdown of fidgetin is partial with this approach, but sufficient to increase the labile microtubule mass of the axon, thereby increasing axonal growth. The increase in axonal growth occurs on both a favorable substrate and a growth-inhibitory molecule associated with scar formation in injured spinal cord. The nanoparticles are readily translatable to in vivo studies on animals and ultimately to clinical applications.

Highlights

To use a relatively new nanoparticle delivery system, which we have tested on cultures of adult rat dorsal root ganglion (DRG) neurons, a well-accepted in vitro model for spinal cord injury
In the adult DRG neurons treated with fidgetin siRNA (56.28 ± 42.50), there was a 19% increase in mean microtubule mass relative to control siRNA (47.53 ± 36.56) per unit area of axon (Fig. 2C), which is about a third as much of an increase as we previously reported of the fetal cortical neurons with more complete fidgetin knockdown
Using adult primary DRG cultures, a broadly accepted in vitro model for evaluating cell biological hypotheses relevant to nerve regeneration, we set out to test whether partial knockdown of fidgetin has potential for providing therapeutic benefit

Summary

Introduction

To use a relatively new nanoparticle delivery system, which we have tested on cultures of adult rat dorsal root ganglion (DRG) neurons, a well-accepted in vitro model for spinal cord injury. Based on a hydrogel/sugar glass composite, our siRNA delivery platform is a hybrid nanoparticle capable of encapsulating and controllably releasing a broad range of therapeutically relevant materials ranging from gaseous nitric oxide to larger macromolecules such as chemotherapeutic agents and phosphodiesterase inhibitors[10]. The nanoparticles have been shown to be capable of delivering siRNA to tissues and organs[10]

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Results

Conclusion