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

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

Treatment of traumatic brain injury using zinc-finger protein gene therapy targeting VEGF-A

Vascular endothelial growth factor (VEGF) plays a role in angiogenesis and has been shown to be neuroprotective following central nervous system trauma. In the present study we evaluated the pro-angiogenic and neuroprotective effects of an engineered zinc-finger protein transcription factor transactivator targeting the vascular endothelial growth factor A (VEGF-ZFP). We used two virus delivery systems, adeno-virus and adeno-associated virus, to examine the effects of early and delayed VEGF-A upregulation after brain trauma, respectively. Male Sprague-Dawley rats were subject to a unilateral fluid percussion injury (FPI) of moderate severity (2.2-2.5atm) followed by intracerebral microinjection of either adenovirus vector (Adv) or an adeno-associated vector (AAV) carrying the VEGF-ZFP construct. Adv-VEGF-ZFP-treated animals had significantly fewer TUNEL positive cells in the injured penumbra of the cortex (p<0.001) and hippocampus (p=0.001) relative to untreated rats at 72h post-injury. Adv-VEGF-ZFP treatment significantly improved fEPSP values (p=0.007) in the CA1 region relative to injury alone. Treatment with AAV2-VEGF-ZFP resulted in improved post-injury microvascular diameter and improved functional recovery on the balance beam and rotarod task at 30 days post-injury. Collectively, the results provide supportive evidence for the concept of acute and delayed treatment following TBI using VEGF-ZFP to induce angiogenesis, reduce cell death, and enhance functional recovery. © Mary Ann Liebert, Inc..link_to_subscribed_fulltex

Electrophysiological assessment following AdV-ZFP-VEGF administration.

<p>(A) Representative tracings of MEP's recorded from the hindlimb at 8 weeks post-injury. (B) MEP quantification. Recordings were obtained from hindlimb biceps femoris. Stimulation was applied to the midline of the cervical spinal cord (0.13 Hz; 0.1 ms; 2 mA; 200 sweeps). Latency was calculated as the time from the start of the stimulus artifact to the first prominent peak. AdV-ZFP-VEGF did not result in improved MEP's. (C) H-Reflex quantification. Recording electrodes were placed two centimeters apart in the mid-calf region and the posterior tibial nerve was stimulated in the popliteal fossa using a 0.1 ms duration square wave pulse at a frequency of 1 Hz. The rats were tested for maximal plantar H-reflex/maximal plantar M-response (H/M) ratios to determine the excitability of the reflex. AdV-ZFP-VEGF administration did not significantly alter the H/M ratio. n = 6/group.</p

Transduction of AdV-eGFP/AdV-ZFP-VEGF into the spinal cord.

<p>(A) Photomicrographs showing a transverse section of rat spinal cord obtained adjacent to the injury site 10 days after spinal cord injury and AdV-eGFP injection. eGFP signal was detected in both the gray matter and white matter. (B) High-power (63X) confocal images show that the AdV-eGFP vector (green) transfected neurons (NeuN), astrocytes (GFAP), oligodendrocytes (CC1) and endothelial cells (RECA-1). Cells have been counter-stained with DAPI (blue) as nuclear marker. (C) Bar graph displays quantification of transduced cell types ± SEM, as identified by the cell-specific markers NeuN, GFAP, RECA-1 and CC1. (D) Evaluation of AdV-ZFP-VEGF gene transfer. Western blot showed that the NFκB p65 rabbit polyclonal antibody recognizes the p65 activation domain in the AdV-ZFP-VEGF treated animals. The higher molecular weight bands are endogenous NFκBp65 fragments, which are also recognized by the antibody; however, these bands are present in both the control and treatment groups. The lower band (arrow) corresponds to the AdV-ZFP-VEGF and was only present in the treated animals. Lower panel shows actin expression as a protein control. Scale bar: 1000 µm for A; 100 µm for B.</p

AdV-ZFP-VEGF does not improve open-field walking (BBB) scores following SCI.

<p>Open-field locomotion was assessed using the 21-point BBB scale. Animals were assessed weekly for 8 weeks following injury by blinded observers (n = 8/sham and AdV-ZFP-VEGF groups; n = 10/injured control and AdV-eGFP groups). The left and right limbs were scored individually, but the data presented is the average between left and right hindlimb recovery.</p