Experimental Gene Therapy with Serine-Histogranin and Endomorphin 1 for the Treatment of Chronic Neuropathic Pain

Abstract

The insufficient pain relief provided by current pharmacotherapy for chronic neuropathic pain is a serious medical problem. The enhanced glutamate signaling via NMDA receptors appears to be one of the key events in the development of chronic pain. Although effective, clinical use of systemic NMDA antagonists is limited by adverse effects such as hallucinations and motor dysfunction. Opioids are also potent analgesics but their chronic use is accompanied by tolerance and risk of addiction. However, combination of NMDA antagonists and opioids seems to provide a stable pain relieve at subthreshold doses of both substances, eliminating development of side effects. Our previous research showed that combined delivery of NMDA antagonist Serine histrogranin (SHG) and endomorphin1 (EM1) leads to attenuation of acute and chronic pain. The aim of this study was to design and evaluate an analgesic potency of the gene construct encoding SHG and EM1. Constructs with 1SHG copy in combination with EM1, 1SHG/EM1, and 6SHG/EM1 were intraspinally injected to animals with peripheral nerve injury-induced pain (chronic constriction injury, CCI) or spinal cord injury induced pain (clip compression model, SCI) and tactile and cold allodynia were evaluated. AAV2/8 particles were used for gene delivery. The results demonstrated 6SHG/EM1 as the most efficient for alleviation of pain-related behavior. The effect was observed up to 8 weeks in SCI animals, suggesting the lack of tolerance of possible synergistic effect between SHG and EM1. Intrathecal injection of SHG antibody or naloxone attenuated the analgesic effect in treated animals. Biochemical and histochemical evaluation confirmed the presence of both peptides in the spinal tissue. The results of this study showed that the injection of AAV vectors encoding combined SHG/EM constructs can provide long term attenuation of pain without overt adverse side effects. This approach may provide better treatment options for patients suffering from chronic pain.

Keywords: endomorphin 1; gene therapy; nerve injury; neuropathic pain; serine histogranin; spinal cord injury.

Figure 1

Analgesic effect of individual gene constructs in chronic constriction injury (CCI) (A,B) and spinal cord injury (SCI) (C,D) animals. (A) Injection of 1SHG and 6SHG constructs attenuated tactile allodynia compared to GFP controls at 2 and 3 weeks post injection. (B) Cold allodynia was attenuated in all treated animals compared to GFP control at one through 4 weeks post injection. (C) Stronger analgesic effect of the gene construct was observed in SCI model with attenuation of tactile allodynia in all treated animals up to 8 weeks post injection. (D) Similarly, in cold allodynia test marked drop in cold sensitivity was observed in treated animals (RM ANOVA; ⁺p < 0.05, ⁺⁺p < 0.01 for 1SHG vs. GFP; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 for 6SHG vs. GFP; *p < 0.05, **p < 0.01, ***p < 0.001 for EM vs. GFP; ^•p < 0.05 for 6SHG vs. 1SHG).

Figure 2

Fluorescence-linked immunosorbent assay (FLISA) analysis of HEK 293 supernatant for the presence of serine histrogranin (SHG) and EM recombinant peptides after transduction with 1SHG/endomorphin1 (EM1), 6SHG/EM1 and GFP gene constructs. (A) SHG and EM1 peptides detected in samples from compound gene constructs transduced cell cultures. No signal detected in the control samples from GFP transduced cells. (B) The level of SHG in 6SHG/EM samples was significantly higher than in 1SHG/EM samples as expected. The level of EM was comparable between samples (ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001 vs. GFP control; ^###p < 0.001 for 6SHG/EM vs. 1SHG/EM).

Figure 3

Analgesic effect of compound gene constructs in CCI (A,B) and SCI (C,D) animals. (A) Tactile allodynia was attenuated at 3 weeks post injection in both 1SHG/EM1 and 6SHG/EM1 treatment groups and the effect sustained up to 5 weeks post injection. (B) Cold allodynia was attenuated in both treatment groups as well with effect observed 1 week post injection up to 5 weeks. (C,D) In SCI groups, both gene constructs demonstrated analgesic efficacy in tactile (C) and cold (D) allodynia tests with effect observed 2 week post injection and sustained till the end of experiment (RM ANOVA; ⁺p < 0.05, ⁺⁺p < 0.01, ⁺⁺⁺p < 0.001 1SHG/EM1 vs. GFP; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 6SHG/EM1 vs. GFP).

Figure 4

Comparison of the analgesic effect of singe and compound gene constructs at 5 weeks post injection in CCI animals (A,C) and 6 and 8 weeks post injection in SCI animals (B,D). In both groups and both pain modalities, no differences were observed at the baseline or pre-injection time points. In CCI group at 5 weeks post injection the effect of 6SHG/EM1 was significantly different from GFP controls (ANOVA; **p < 0.01). In SCI group all treated animals demonstrated reduction in hypersensitivity with significant differences compared to control GFP groups as well as inter-treatment differences. The effect was observed up to 8 weeks post injection of the constructs with 6SHG/EM1 as the most efficient construct reducing pain-like behavior (ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001 vs. GFP, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 between treatments).

Figure 5

Pharmacological attenuation of the 6SHG/EM1 analgesic effect. Intrathecal injection of anti-SHG (5 mg/ml; A,B) and naloxone (0.1 mg/ml; C,D) in SCI animals 8 weeks post treatment (12 weeks post injury) reduced the observed effect in the treated animals. Anti SHG reversed both tactile and cold allodynia attenuation; naloxone was effective in reversing cold allodynia (*p < 0.05, **p < 0.01, ***p < 0.001 vs. preinjection; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. GFP).

Figure 6

FLISA analysis of the spinal cord for the presence of recombinant SHG and EM1 peptides. (A) Both peptides were detected in the samples from 1SHG/EM1 and 6SHG/EM1 treated animals, with no signal from GFP treated animals. (B) OD signal of either peptide was enhanced in the samples from treated animals in both injury groups, with SHG signal significantly higher in 6SHG/EM1 samples compared to 1SHG/EM1 in SCI group. No differences between treatment samples for EM1 signal were observed as expected (ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001 vs. GFP; ^#p < 0.05 between treatments).

Figure 7

Immunohistochemical detection of recombinant SHG and EM1 peptides in the spinal cord of SCI animals. Both peptides were detected in the vicinity of the injection site with rostral and caudal spread within 1 mm. Top panel: Low magnification of spinal dorsal horn from animals after injection of the recombinant constructs. (A) NeuN and DAPI staining at the level of the injection site. SHG immunostaining was used to visualize the location of the construct. SHG positive fibers were detected in the lateral and medial side of the dorsal horn (arrows in B) and as a punctate cytoplasmic staining in NeuN cells (arrows in C). Bottom panel: Higher magnification of some of the neuronal and glial cells from the dorsal horn. Colocalization of NeuN with SHG (D) and EM1 (E) confirm transduction of neuronal cells (arrows). No significant colocalization with GFAP marker have been found (F).