Delayed administration of a bio-engineered zinc-finger VEGF-A gene therapy is neuroprotective and attenuates allodynia following traumatic spinal cord injury.

Sarah A Figley,Gary Lee,Dale Ando,Michael G Fehlings,Spyridon K Karadimas,Yang Liu,S Kaye Spratt,Martin Giedlin,Peter Fettes,Richard Surosky,Kajana Satkunendrarajah,Hatem E Sabaawy

doi:10.1371/journal.pone.0096137

Abstract

Following spinal cord injury (SCI) there are drastic changes that occur in the spinal microvasculature, including ischemia, hemorrhage, endothelial cell death and blood-spinal cord barrier disruption. Vascular endothelial growth factor-A (VEGF-A) is a pleiotropic factor recognized for its pro-angiogenic properties; however, VEGF has recently been shown to provide neuroprotection. We hypothesized that delivery of AdV-ZFP-VEGF – an adenovirally delivered bio-engineered zinc-finger transcription factor that promotes endogenous VEGF-A expression – would result in angiogenesis, neuroprotection and functional recovery following SCI. This novel VEGF gene therapy induces the endogenous production of multiple VEGF-A isoforms; a critical factor for proper vascular development and repair. Briefly, female Wistar rats – under cyclosporin immunosuppression – received a 35 g clip-compression injury and were administered AdV-ZFP-VEGF or AdV-eGFP at 24 hours post-SCI. qRT-PCR and Western Blot analysis of VEGF-A mRNA and protein, showed significant increases in VEGF-A expression in AdV-ZFP-VEGF treated animals (p<0.001 and p<0.05, respectively). Analysis of NF200, TUNEL, and RECA-1 indicated that AdV-ZFP-VEGF increased axonal preservation (p<0.05), reduced cell death (p<0.01), and increased blood vessels (p<0.01), respectively. Moreover, AdV-ZFP-VEGF resulted in a 10% increase in blood vessel proliferation (p<0.001). Catwalk™ analysis showed AdV-ZFP-VEGF treatment dramatically improves hindlimb weight support (p<0.05) and increases hindlimb swing speed (p<0.02) when compared to control animals. Finally, AdV-ZFP-VEGF administration provided a significant reduction in allodynia (p<0.01). Overall, the results of this study indicate that AdV-ZFP-VEGF administration can be delivered in a clinically relevant time-window following SCI (24 hours) and provide significant molecular and functional benefits.

Highlights

In North America, it is estimated that approximately 1.5 million individuals are currently living with spinal cord injury (SCI), with over 12,000 traumatic SCI cases occurring each year [1]
The primary injury is responsible for triggering all of the downstream events, it is widely accepted that the processes that take place in the ‘‘secondary injury’’ phase are predominantly responsible for a significant portion of the damage and degeneration that is associated with SCI, including inflammation, ischemia, lipid peroxidation, production of free radicals, disruption of ion channels, necrosis and programmed cell death [3,4,5]
The Vascular endothelial growth factor-A (VEGF-A)-activating zinc-finger transcription factor protein (ZFP) (32E-p65) – referred to as AdV-ZFP-vascular endothelial growth factor (VEGF) – is a 378 amino acid multi-domain protein that is composed of three functional regions: (1) the nuclear localization signal (NLS) of the large Tantigen of SV40, (2) a designed 3-finger zinc-fingered protein (32E) that binds to a 9 base-pair target DNA sequence (GGGGGTGAC) present in the human VEGF-A promoter region and (3) the transactivation domain from the p65 subunit of human NFkB, which is identical to VZ+434, subcloned into pVAX1 (Invitrogen, San Diego, CA) with expression driven by the human cytomegalovirus (CMV) promoter

Summary

Introduction

In North America, it is estimated that approximately 1.5 million individuals are currently living with SCI, with over 12,000 traumatic SCI cases occurring each year [1]. Spinal cord injury is divided into two events, to separate the physical and the cellular pathologies. The primary injury is responsible for triggering all of the downstream events, it is widely accepted that the processes that take place in the ‘‘secondary injury’’ phase are predominantly responsible for a significant portion of the damage and degeneration that is associated with SCI, including inflammation, ischemia, lipid peroxidation, production of free radicals, disruption of ion channels, necrosis and programmed cell death [3,4,5]. Radical alterations to the spinal microvascular architecture and function occur following SCI and contribute to the secondary injury. Hemorrhage, systemic hypotension, loss of microcirculation, disruption of the bloodspinal cord barrier (BSCB) and loss of structural organization, enhance the cellular damage post-injury [2,6]. Despite the fact that these secondary events are responsible for the majority of the damage associated with SCI, many of these pathways alternatively provide an opportunity to target with therapeutic interventions

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