Intramuscular electroporation with the pro-opiomelanocortin gene in rat adjuvant arthritis
Arthritis Research & Therapy
1478-6354
Intramuscular electroporation with the pro-opiomelanocortin gene in rat adjuvant arthritis
I-Chuan Chuang 2
Chien-Ming Jhao 1
Chih-Hsun Yang 0
Hsien-Chang Chang 2
Chien-Wen Wang 2
Cheng-Yuan Lu 0
Yao-Jen Chang 0
Sheng-Han Lin 0
Pao-Lin Huang 3
Lin-Cheng Yang 3
Corresponding author: Lin-Cheng Yang (e-mail: lcyang 2
.hinet.net)
0 Gene Therapy Laboratory, Chang Gung Memorial Hospital , Kaohsiung, 833 , Taiwan
1 Institute of Biomedical Engineering, National Taiwan University Hospital , Taipei , Taiwan
2 Institute of Biomedical Engineering, National Cheng Kung University , Tainan , Taiwan
3 Gene Therapy Laboratory, Tajen Institute of Technology , Pingtung, 907 , Taiwan
Endogenous opioid peptides have an essential role in the intrinsic modulation and control of inflammatory pain, which could be therapeutically useful. In this study, we established a muscular electroporation method for the gene transfer of proopiomelanocortin (POMC) in vivo and investigated its effect on inflammatory pain in a rat model of rheumatoid arthritis. The gene encoding human POMC was inserted into a modified pCMV plasmid, and 0-200 µg of the plasmid-POMC DNA construct was transferred into the tibialis anterior muscle of rats treated with complete Freund's adjuvant (CFA) with or without POMC gene transfer by the electroporation method. The safety and efficiency of the gene transfer was assessed with the following parameters: thermal hyperalgesia, serum adrenocorticotropic hormone (ACTH) and endorphin levels, paw swelling and muscle endorphin levels at 1, 2 and 3 weeks after electroporation. Serum ACTH and endorphin levels of the group into which the gene encoding POMC had been transferred were increased to about 13-14-fold those of the normal control. These levels peaked 1 week after electroporation and significantly decreased 2 weeks after electroporation. Rats that had received the gene encoding POMC had less thermal hypersensitivity and paw swelling than the non-gene-transferred group at days 3, 5 and 7 after injection with CFA. Our promising results showed that transfer of the gene encoding POMC by electroporation is a new and effective method for its expression in vivo, and the analgesic effects of POMC cDNA with electroporation in a rat model of rheumatoid arthritis are reversed by naloxone.
electroporation; gene delivery; pro-opiomelanocortin; RT-PCR; skeletal muscle
Introduction
We are now in an era of molecular medicine in which
increasing focus is being placed on gene therapy as a
potential approach for the treatment of several disorders in
various types of patients. Several previous studies have
reported that multinucleate and post-mitotic myofibres in
skeletal muscle are capable of both long-term transgene
expression and systemic delivery of proteins to the blood
circulation. Gene delivery to skeletal muscle has therefore
been investigated as a method of creating a tissue
reservoir for the secretion of non-muscle proteins such as
growth hormone. Different functional genes, including
those that encode factor IX [
1–3
], erythropoietin [
4
],
kallikrein [
5
] and interleukin-12 [
6
], have been delivered to
skeletal muscle for potentially therapeutic purposes.
It has been shown that endogenous ligands, and
especially the opioid peptides, are expressed by resident
immune cells in inflamed peripheral tissue. Environmental
stimuli and endogenous substances such as
corticotropinreleasing hormone and cytokines can stimulate the release
of these opioid peptides, resulting in local analgesia and
suppression of the immune system [
7
]. A therapeutic
‘pain-killer gene’, encoding pro-opiomelanocortin (POMC),
ACTH = adrenocorticotropic hormone; bp = base pairs; CFA = complete Freund’s adjuvant; PBS = phosphate-buffered saline; POMC =
proopiomelanocortin; RIA = radioimmunoassay; RT–PCR = reverse transcriptase polymerase chain reaction.
R8
produces the opioid peptides β-endorphins, other shorter
endorphins, adrenocorticotropic hormone (ACTH) and
α-melanocyte stimulating hormone. Injection of POMC
cDNA with a gene gun has produced analgesic effects in
phase 2 of the formalin test [
8
]. The possibility exists that
this exogenous POMC-mediated analgesia could be used
for the control of chronic inflammatory pain such as
rheumatoid arthritis or osteoarthritis.
Various studies have focused on the application of gene
delivery using viral vectors such as adenovirus, retrovirus
and herpes simplex virus for muscle-based gene therapy.
However, the use of these viruses as vectors has been
hindered by viral cytotoxicity, host immune rejection after
repeat dosing, and limited transient transgene expression
[
9–12
]. Adenoviral vectors are able to infect both mitotic
myoblasts and post-mitotic immature myofibres and can be
prepared at high titres (109 to 1011 plaque-forming units/ml)
[
13,14
]. However, stability and long-term transgene
expression using first-generation adenoviral vectors have (...truncated)