Intramuscular Electroporation With the Pro-Opiomelancortin Gene
Intramuscular Electroporation With the Pro-Opiomelancortin Gene
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 pro-opiomelanocortin (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.
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, erythropoietin, kallikrein and interleukin-12, 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 corticotropin-releasing hormone and cytokines can stimulate the release of these opioid peptides, resulting in local analgesia and suppression of the immune system. A therapeutic 'pain-killer gene', encoding pro-opiomelanocortin (POMC), 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. 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. Adenoviral vectors are able to infect both mitotic myoblasts and post-mitotic immature myofibres and can be prepared at high titres (10 to 10plaque-forming units/ml). However, stability and long-term transgene expression using first-generation adenoviral vectors have been hampered by the immune rejection in muscle. In addition, novel mutant vectors were developed subsequently and such mutants have reduced the problems associated with viral cytotoxicity and immune rejection.
Electroporation is a physical method of introducing macromolecules into cells by applying a brief electrical pulse that causes transient changes in membrane permeability. Moreover, electroporation as applied to muscle has been found to be relatively safe, and has the additional advantage of not being restricted by post-mitotic muscle cells. On the basis of the principle of electroporation, we developed a new strategy to achieve the transfer of the gene encoding the intramuscular 'pain-killer' POMC in vivo and determined the optimal transfection efficiency. The safety of the procedure, in terms of potential damage to muscle tissue, and the effectiveness of the gene transfer (that is, analgesic potency) were assessed by using parameters including thermal hyperalgesia, serum ACTH, endorphin, paw swelling and muscle endorphin levels after intramuscular electroporation into rats in which arthritis was induced by the injection of complete Freund's adjuvant (CFA).
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 pro-opiomelanocortin (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.
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, erythropoietin, kallikrein and interleukin-12, 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 corticotropin-releasing hormone and cytokines can stimulate the release of these opioid peptides, resulting in local analgesia and suppression of the immune system. A therapeutic 'pain-killer gene', encoding pro-opiomelanocortin (POMC), 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. 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. Adenoviral vectors are able to infect both mitotic myoblasts and post-mitotic immature myofibres and can be prepared at high titres (10 to 10plaque-forming units/ml). However, stability and long-term transgene expression using first-generation adenoviral vectors have been hampered by the immune rejection in muscle. In addition, novel mutant vectors were developed subsequently and such mutants have reduced the problems associated with viral cytotoxicity and immune rejection.
Electroporation is a physical method of introducing macromolecules into cells by applying a brief electrical pulse that causes transient changes in membrane permeability. Moreover, electroporation as applied to muscle has been found to be relatively safe, and has the additional advantage of not being restricted by post-mitotic muscle cells. On the basis of the principle of electroporation, we developed a new strategy to achieve the transfer of the gene encoding the intramuscular 'pain-killer' POMC in vivo and determined the optimal transfection efficiency. The safety of the procedure, in terms of potential damage to muscle tissue, and the effectiveness of the gene transfer (that is, analgesic potency) were assessed by using parameters including thermal hyperalgesia, serum ACTH, endorphin, paw swelling and muscle endorphin levels after intramuscular electroporation into rats in which arthritis was induced by the injection of complete Freund's adjuvant (CFA).