Background
Exogenous opioids (e.g. morphine), and endogenous opioid peptides, such as β-endorphin (END) and enkephalins, are powerful analgesics in animals and humans [
1‐
3]. Compared to conventional exogenous agonists, endogenous opioids have several advantages. These include reduced probabilities of receptor downregulation and of paradoxical excitatory effects due to high non-physiological exogenous agonist concentrations at the receptor [
4]. Gene therapy is an attractive strategy to enhance continuous production of endogenous opioids. The most often used vectors are recombinant viruses. Several laboratories have employed herpes simplex virus (HSV) encoding preproenkephalin (PPENK) to increase enkephalin production. Transfection of the spinal cord, trigeminal or dorsal root ganglia (DRG) with such vectors resulted in the attenuation of nociceptive behaviors in animal models of acute and pathological pain [
5‐
13]. Similar effects were found after hind paw inoculation with HSV encoding endomorphin-2 [
14] or spinal application of adenoviral and adeno-associated vectors encoding END in rodents [
15,
16].
The immunogenicity of viral vectors, the possibility of recombination with wild-type viruses, activation of oncogenes, and their relatively small capacity for therapeutic DNA have led to the development of non-viral vectors, mostly plasmids [
17]. Gene-gun application or electroporation of plasmids encoding PPENK or END precursor proopiomelanocortin (POMC) reduced experimental pain in animals [
18‐
23]. However, plasmids may also cause undesirable effects such as the production of antibodies (Ab) against bacterial proteins, changes in gene expression caused by the antibiotic resistance markers and immune responses to CpG dinucleotide motifs [
24‐
26].
Non-viral, non-plasmid minimalistic immunologically defined gene expression (MIDGE) vectors may overcome these problems. MIDGE vectors are linear molecules containing only a promoter, a gene of interest and an RNA-stabilizing sequence, flanked by two short hairpin oligonucleotide sequences. Important advantages of MIDGE vectors over plasmids are small size, absence of antibiotic resistance genes and the relatively low occurrence of CpG sequences [
27,
28]. Gene gun delivery into dermis or onto the eye lid of MIDGE encoding interleukin-12 or cytotoxic T-lymphocyte-associated antigen-4 and interleukin-4 protected cats against experimental feline immunodeficiency virus or improved corneal graft survival in mice, respectively [
29,
30]. To ensure the effective transport to the nucleus and transgene expression, a nuclear localization sequence (NLS) can be attached to the MIDGE. Indeed, MIDGE-NLS encoding hepatitis B antigen enhanced antiviral immunity after intramuscular injection in mice [
28]. Also, intradermal administration of MIDGE-NLS encoding LACK antigen (Leishmania homolog of receptors for activated C kinase) was protective against parasitic infection in mice [
31].
The rationale underlying the present studies was to combine the advantages of MIDGE vectors and the powerful analgesic properties of opioids creating MIDGE-NLS encoding POMC to enhance the production of END for the control of prolonged inflammatory pain. We have previously shown that END-producing leukocytes accumulate in inflamed tissue, where, in response to experimental stress (swimming in cold water) or to local injection of releasing agents, the cells secrete this peptide [
32‐
40]. Released opioids bind to opioid receptors on peripheral sensory nerve terminals, resulting in local analgesia in animals and in humans [
41‐
46]. Importantly, these events occur in peripheral tissues and, therefore, lack side effects such as nausea, respiratory depression, dependence and addiction mediated by opioid receptors in the central nervous system [
2,
3]. Our major objective was to enhance the production and release of END in inflamed tissue using POMC-MIDGE-NLS to provide continuous relief of inflammatory pain.
Discussion
In the present study we examined the control of prolonged inflammatory pain with novel END-coding MIDGE vectors, which lack the disadvantages of classical viral and plasmid vectors. Although, POMC-MIDGE-NLS injected into inflamed tissue appeared to be taken up by leukocytes in vivo (Fig.
2, Fig.
4), its anti-hyperalgesic actions were rather moderate and not consistently reproducible (Fig.
3, Fig.
4, Fig.
5, Fig.
6). Also, these effects could not be enhanced by increasing END release through stress or by modifying POMC-MIDGE-NLS to code for multiple copies of END (Fig.
5, Fig.
6).
Previously we have consistently observed significant analgesic effects of both exogenous and leukocyte-derived opioids against mechanical stimulation in experimental and clinical inflammatory pain [
2,
3]. In the current study, i.pl. injection of POMC-MIDGE-NLS reversed mechanical hyperalgesia, i.e. nociceptive thresholds returned to the levels measured in contralateral noninflamed paws in some experiments (Fig.
3, Fig.
4A, Fig.
5). Nevertheless, the effects were rather moderate and not always reproducible (Fig.
4B, C, Fig.
6). This lack of consistently significant anti-hyperalgesic effects of POMC-MIDGE-NLS might be related to higher basal PPT (after CFA but before vector injections) and bigger within-group variability of responses in some experiments (Fig.
4B, Fig.
6). In some previous reports hyperalgesia was fully reversed by opioid peptide vector delivery. For example, intrathecal electroporation of modified POMC plasmids suppressed thermal hyperalgesia in a neuropathic pain model [
23], and PPENK-HSV applied to the paw skin reversed thermal hyperalgesia induced by pertussis toxin or capsaicin [
5,
11,
12]. However, similar to our results, the majority of studies using either HSV, adenovirus, adeno-associated or plasmid vectors encoding opioid peptides found only partial and modest reductions of thermal or mechanical hyperalgesia in long-lasting (weeks) or short-lasting (1-3 h) pain models [
8‐
10,
13,
15,
16,
18‐
23]. A reproducibility of anti-hyperalgesic effects has not been reported in those earlier studies.
POMC-MIDGE-NLS decreased hyperalgesia for about 2 days. This is much shorter than the effects measured for 2-20 weeks after injections of viral vectors encoding opioid peptides [
5,
7‐
14,
16], or up to 2 weeks after endomorphin-2-HSV or POMC plasmid applications [
14,
20,
23]. However, in other studies using END-coding adenoviral vector or POMC plasmids, attenuation of hyperalgesia was only measured in short-lasting (30 min-3 h) pain models [
15,
18,
19,
21,
22]. Thus, longer-lasting anti-hyperalgesic effects were mostly produced by viral vectors preferentially targeting neurons [
5,
7‐
14,
16]. In contrast, in our model MIDGE-NLS appeared to be taken up mostly by immune cells. At this stage of inflammation (5 days following CFA) monocytes/macrophages dominate, and their turnover rate of several hours correlates with the time-course of POMC-MIDGE-NLS-mediated anti-hyperalgesia. On the other hand, the 48 h-lasting elevation of nociceptive thresholds produced by POMC-MIDGE-NLS is superior to the 5-10 min-lasting effects after i.pl. END injection [
48]. Hence, some of our experiments demonstrated a clear prolongation of anti-hyperalgesic actions by POMC-MIDGE-NLS-based END delivery. Modification of POMC-MIDGE-NLS vectors to also transfect neurons might be an interesting future approach for improvement of their anti-hyperalgesic effects.
In some of our experiments POMC-MIDGE-NLS slightly elevated END content in immune cells accumulating in inflamed tissue. However, these effects were not always statistically significant or directly correlated with the attenuation of hyperalgesia, indicating variability in transfection and/or END production efficacy. Although there was some variability in the control END levels among experimental sets (Fig.
4) this does not seem to predict the efficacy of POMC-MIDGE-NLS in END production. For example, even though the control END levels in Fig.
4A and Fig.
4B were comparable, the END levels after different doses of POMC-MIDGE-NLS varied between the two graphs. Also, it seems that there was no direct correlation between the control END levels and the extent of hyperalgesia. Thus, despite differences in the control END levels in Fig.
4A, B and
4C, the basal PPT (after CFA but before control vector injection) were comparable among these three graphs. The lack of significant increases in the numbers of opioid-positive leukocytes suggests that when POMC-MIDGE-NLS transfected these cells it was mostly active in those already expressing the peptide. Other vectors have been shown to increase opioid peptide production in different tissues. For example, HSV-PPENK applied to skin enhanced the expression of PPENK, Met- or Leu-enkephalin in the spinal cord and primary afferent neurons in healthy animals [
5,
7,
11‐
13]. Spinal application of adenoviral vectors encoding END resulted in increased cerebrospinal fluid levels of the peptide [
15]. Importantly, while antinociceptive effects were assessed after induction of inflammation or neuropathy, the transfection efficacy of these viral vectors in peripheral neurons was primarily verified in healthy animals [
5,
7,
11‐
13,
16]. There was no clear explanation for these approaches and they make interpretation of the data difficult. Transfection efficacy of plasmid vectors was evaluated either directly in inflamed tissues treated with the vectors, or in the spinal cord after intrathecal electroporation. For instance, gene-gun application of plasmids coding for POMC or PPENK to inflamed rat paws or into the bladder wall, or intrathecal electroporation in animals with hind paw inflammation or with sciatic nerve injury increased levels of END or Met-enkephalin in the respective tissues, although cell types were not specified in those experiments [
18‐
23]. None of those previous studies reported transfection of immune cells with vectors encoding opioid peptide precursors. MIDGE vectors do not seem to possess particular characteristics in cell type targeting. In previous studies their transfection efficacy was examined predominantly in vitro in various cell lines. Accordingly, MIDGE-NLS encoding hepatitis B antigen transfected chicken hepatoma cells [
28], MIDGE-NLS encoding LACK antigen was successful in transfecting kidney COS-7 cells [
31], and MIDGE encoding IL-12 transfected a feline T lymphocyte line [
29]. Hence, it appears that if a vector is introduced into a cell line or tissue, cells are "forced" to express a transgene regardless of the physiological/pathological state of the tissue. This might explain some (scarce) staining in non-inflamed paws after β-galactosidase-MIDGE-NLS application in our current study. On the other hand, it is reasonable to assume that the type of transfected cells might be determined by the pathological condition of the tissue. Thus, in our studies it appears that when tissue is inflamed and infiltrated by leukocytes, these cells might be a major target for POMC-MIDGE-NLS.
Our studies do not provide direct evidence for the opioid-receptor selectivity and the site of anti-hyperalgesic actions of END coded by POMC-MIDGE-NLS. Testing specific and peripherally-restricted opioid receptor antagonists would be necessary to determine selectivity and site of the vector actions. However, because of the variable reproducibility of POMC-MIDGE-NLS-mediated anti-hyperalgesic effects, such experiments are difficult to perform. Similar concerns apply to the effects of POMC-MIDGE-NLS encoding 1 or 2 copies of END, as they produced only minor PPT elevations. Nevertheless, an action via peripheral opioid receptors is most likely. The fact that Abs recognizing endogenous END were reactive in our immunohistochemical and RIA experiments suggests that POMC-MIDGE-NLS-induced END is not different from authentic END and therefore should act at opioid receptors. We have previously shown that opioid peptides injected directly into inflamed tissue can produce antinociception via opioid receptors on peripheral sensory neurons [
2,
3,
48]. Furthermore, direct application to injured tissues of either HSV-PPENK or POMC plasmid resulted in peripheral opioid receptor-selective anti-hyperalgesic effects [
6,
7,
13,
21]. In contrast, applications of POMC-MIDGE-NLS (Fig.
6), POMC plasmid or of exogenous opioids into peripheral non-injured tissues [
2,
3,
21,
48] did not significantly change nociceptive thresholds. Most probably this is related to an intact perineural barrier and/or insufficient number or G-protein coupling of opioid receptors on sensory neurons in noninflamed tissues [
2,
3,
43,
44,
46].
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
HM designed and conducted most behavioral experiments, analyzed the data and wrote the manuscript. M. Schroff, DO and BW designed and constructed the MIDGE vectors. WB and DL performed some behavioral experiments. NS and MB executed radioimmunoassay. SAM carried out immunohistochemistry. HLR and AB commenced flow cytometry experiments. M. Schäfer participated in the study design. CS and BW conceived the study. CS participated in the data interpretation and writing of the manuscript. All authors have read and approved the final manuscript.