Efficacy of Herpes Simplex Virus Vector Encoding the Human Preproenkephalin Gene for Treatment of Facial Pain in Mice

Abstract

Aims: To determine whether herpes simplex virus-based vectors can efficiently transduce mouse trigeminal ganglion (TG) neurons and attenuate preexisting nerve injury-induced whisker pad mechanical hypersensitivity in a trigeminal inflammatory compression (TIC) neuropathic pain model.

Methods: Tissue transduction efficiencies of replication-conditional and replication-defective vectors to mouse whisker pads after topical administration and subcutaneous injection were assessed using quantitative real-time PCR (qPCR). Tissue tropism and transgene expression were assessed using qPCR and reverse-transcriptase qPCR following topical application of the vectors. Whisker pad mechanical sensitivities of TIC-injured mice were determined using graduated von Frey fibers before and after application of human preproenkephalin expressing replication-conditional vector (KHPE). Data were analyzed using one-way analysis of variance (ANOVA) and post hoc tests.

Results: Transduction of target TGs was 8- to 50-fold greater after topical application than subcutaneous injection and ≥ 100-fold greater for replication-conditional than replication-defective vectors. Mean KHPE loads remained constant in TGs (4.5-9.8 × 10(4) copies/TG) over 3 weeks but were below quantifiable levels (10 copies/tissue) within 2 weeks of application in other nontarget cephalic tissues examined. Transgene expression in TGs was maximal during 2 weeks after topical application (100-200 cDNA copies/mL) and was below quantifiable levels (1 cDNA copy/mL) in all nontarget tissues. Topical KHPE administration reduced TIC-related mechanical hypersensitivity on whisker pads 4-fold (P < .05) for at least 1 week.

Conclusion: Topically administered KHPE produced a significant antinociceptive effect in the TIC mouse model of chronic facial neuropathic pain. This is the first report in which a gene therapeutic approach reduced trigeminal pain-related behaviors in an established pain state in mice.

Fig 1

Efficiency of replication “conditional” KHZ and “defective” D3GFP vector delivery to the mouse trigeminal ganglion (TG). Mice were treated with replication-conditional vector KHZ (8.0 × 10⁶ PFU in 10 µL) and replication-defective vector D3GFP (3.0 × 10⁷ PFU in 10 µL) by subcutaneous injection (sc) into the whisker pad or topically following light abrasion of the whisker pad with a needle (T/N), Dremel (T/D), or Dermapen (T/DP). DNA was purified from ipsilateral TGs 3 to 4 days following administration. *P < .05 with respect to the first (left most) bar, ie, KHZ applied topically following light abrasion with the needle.

Fig 2

Tissue distribution of vector DNA. Vectors KHPE and KHZ were topically applied to the whisker pads of mice. Nucleic acid was purified from trigeminal ganglion (TG), pons, and medulla 3 days to 3 weeks after vector treatment. (a) Vector DNA was quantified by qPCR and average copies per ipsilateral tissue are shown as indicated. *P < .05 with respect to the TG day 3–4 data; **P < .01 with respect to pons or medulla day 3–4 day data. (b) cDNA was synthesized from RNA purified from the same tissues, and amounts of latency-associated transcript (LAT) and hENK cDNAs were quantified by qPCR. Average copies of LAT and hENK cDNA ± SEM derived from TGs are shown as indicated. *P < .05 with respect to day 3–4 data.

Fig 3

Viral expression in the trigeminal ganglion (TG). Ipsilateral TGs were examined days 3 and 10 after vector KHZ application. Sections were immunostained for β-galactosidase (a to c) and respective consecutive sections for HSV-1 infected cell proteins (d and e). Few transduced neurons (dark-brown stain) were evident on days 3 (a to c) and 10 (d and e) in formalin-fixed, paraffin-embedded sections. Hematoxylin & eosin counterstain. Black arrows: neurons. White arrows: satellite glial cells. Calibration: (a) 500 µm; (b to e) 100 µm.

Fig 4

KHPE reduced mechanical hypersensitivity. Mechanical allodynia was evident on the ipsilateral whisker pad prior to vector delivery. Ipsilateral whisker pads of trigeminal inflammatory compression (TIC) mice were then treated topically with experimental vector KHPE (n = 12), control vector KHZ (n = 7), or vehicle control (n = 5) at 8 days post-TIC. Behavioral changes were determined on ipsilateral (a) and contralateral (b) whisker pads twice a week for 17 days after vector treatment (pt) (ie, 25 days post-TIC) and are presented as means ± SEM. *P < .05. Note scale differences.

Fig 5

A viral vector pump delivered to the mouse trigeminal ganglion by vector at peripheral application to facial tissues produced KHPE for 2 to 3 weeks and inhibited mechanical allodynia in whisker pad caused by trigeminal inflammatory compression injury. ENK = enkephalin; HSV = herpes simplex virus.