Gene transfer into muscle tissue is currently being developed as a method for the production, secretion and delivery of therapeutic proteins. This methodology has been used to produce a variety of physiologically active proteins and may ultimately be applied to the treatment of several diseases. In this review, we consider several applications of this methodology and discuss approaches for modulating therapeutic protein production and secretion from muscle, using growth hormone as an example. In addition, factors limiting the effectiveness of muscle gene transfer are also discussed, as these shall determine the efficacy of muscle gene transfer when applied to humans.
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GS MacColl, G Goldspink, and PM Bouloux
F Tanriverdi, LF Silveira, GS MacColl, and PM Bouloux
GnRH and sex steroids play an important role in immune system modulation and development. GnRH and the GnRH receptor are produced locally by immune cells, suggesting an autocrine role for GnRH. Experimental studies show a stimulatory action of exogenous GnRH on the immune response. The immune actions of GnRH in vivo are, however, less well established. Oestrogen and androgen receptors are expressed in primary lymphoid organs and peripheral immune cells. Experimental data have established that oestrogens enhance the humoral immune response and may have an activating role in autoimmune disorders. Testosterone enhances suppressor T cell activity. Although there are some clinical studies consistent with these findings, the impact of sex steroids in autoimmune disease pathogenesis and the risk or benefits of their usage in normal and autoimmune-disordered patients remain to be elucidated. There are neither experimental nor clinical data evaluating functional GnRH-sex steroid interactions within the human immune system, and there is a paucity of data relating to GnRH analogues, hormone replacement therapy and oral contraceptive and androgen action in autoimmune diseases. However, a growing body of experimental evidence suggests that an extra-pituitary GnRH immune mechanism plays a role in the programming of the immune system. The implications of these findings in understanding immune function are discussed.
GS MacColl, FJ Novo, NJ Marshall, M Waters, G Goldspink, and PM Bouloux
The production of peptide hormones by skeletal muscle tissue is a promising area of gene therapy. Skeletal muscle myogenesis can be induced in vitro, resulting in the fusion of mononucleate myoblasts to form multinucleate myotubes, and delivery vectors are first tested in vitro. C2C12 myoblasts transfected with pcDNA3-GH, which used the human cytomegalovirus (CMV) promoter, secreted immunoreactive GH with comparable biological activity to pituitary GH. Mouse myeloid leukaemia cells, which express the mouse GH receptor were used for the bioassay, and activation of these cells by GH was measured by a colorimetric microculture tetrazolium assay. Cells were incubated with a tetrazolium salt (MTS) and an intermediate electron acceptor (phenazine methosulphate, PMS), and formazan production was measured as optical density (O.D.) at 490 nm. The efficiencies of several plasmid expression vectors were compared in differentiated and non-differentiated muscle cells, as a function of bioactive GH secreted by the transfected cells. Ten-day differentiated C2C12 myotubes transfected with pcDNA3E-GH, which used the CMV promoter and a rat myosin light chain enhancer element, secreted significantly more biologically active GH than myotubes transfected with pcDNA3-GH (0.82 O.D. units+/-0.06 vs 0.57+/-0.05 respectively, P<0.001). This was consistent with reduced CMV promoter activity in myotubes. Myoblasts transfected with pcDNA3-GH secreted more bioactive GH than 10-day transfected myotubes (1.1+/-0. 1 vs 0.77+/-0.07 respectively). However, the responses were indistinguishable (both 1.0+/-0.09) if both the myotubes and myoblasts had been transfected with pcDNA3E-GH. Substitution of the vector pMHLC-GH, which used a muscle-specific truncated rabbit myosin heavy chain promoter, and the myosin enhancer resulted in a marked decrease in the responses to the conditioned medium from fused myotubes compared with the vectors pcDNA3-GH and pcDNA3E-GH (0. 24+/-0.02 vs 0.57+/-0.05 vs 0.82+/-0.06 respectively). We concluded that the combination of CMV promoter and myosin light chain enhancer in pcDNA3E-GH had the greatest expression efficiency of the several plasmid vectors which we investigated.
P.-M. G. Bouloux, P. Munroe, J. Kirk, and G. M. Besser
Olfaction plays an important role in sexual maturation and behaviour in the animal kingdom. Evidence that it has the same importance in man is far less convincing and frequently dogged by such anecdotes as the role of pheromones in synchronizing menstrual cyclicity in convents and all-girl schools. Sexual immaturity and defective olfaction do however coexist as an inherited disorder in man (Kallmann's syndrome: Kallmann, Schoenfeld & Barrera, 1944; DeMorsier, 1954; White, Rogol, Brown et al. 1983; Hermanussen & Sippell, 1985; Chaussain, Toublanc, Feingold et al. 1988) and for many years the biological basis of these defects remained enigmatic. Recently, the interrelationship between sexual development and absence of olfaction has been clarified in three mammals: mouse, macaque and man.
Mouse and macaque
A neurobiological basis of hypogonadism and anosmia was first suggested by elegant immunohistochemical studies conducted by Schwanzel-Fukuda & Pfaff (1989a). In the mature mouse, nerve cells that produce
P.-M. G. Bouloux, D. Perrett, M. Sopwith, and G. M. Besser
An in-situ isolated rat adrenal perfusion technique has been devised to study the opioid control of neurally mediated adrenomedullary catecholamine release. Adrenomedullary catecholamine secretion was induced by electrical stimulation of the cut end of the left descending thoracic sympathetic chain on platinum electrodes. The half-maximal stimulatory potential (ED50) of the system was 8 V, 20 Hz, with 300μs pulse width. Basal release of catecholamine from the adrenal was constant using a perfusion flow rate of 100–300μl/min, but increased significantly with increasing perfusion temperature over the range 36–38 °C. Following repetitive 30-s stimulation of the left thoracic sympathetic chain, and 3-min fraction collections, the total amount of catecholamine released per fraction remained within 80–100% of the maximum release for up to eight consecutive stimuli. The release of catecholamines was completely blocked by hexamethonium (0·1 mmol/l), but recovered to preblockade values within two further stimuli. Using the ED50 and the first three stimuli as control, the effects of morphine (10 nmol/l–1 mmol/l), d-Ala2-MePhe4-Met-enkephalin-(O5)-ol (DAMME; 10 nmol/l–0·1 mmol/l) and naloxone (10 nmol/l– 10 μmol/l) on the response to the next three stimuli were compared. Morphine, DAMME or naloxone did not significantly alter the amount of catecholamine released by this form of stimulation. Therefore in the rat, under the conditions used, there is no evidence for mu (μ) or delta (†) opiate modulation of neurally mediated catecholamine release from the rat adrenal medulla.
J. Endocr. (1986) 111, 7–15
A. M. J. Lengyel, A. Grossman, P.-M. G. Bouloux, L. H. Rees, and G. M. Besser
Dopamine and morphine modulate GH and LH release, probably at a hypothalamic locus. To investigate this in more detail, we studied the influence of these substances on somatostatin and LH-releasing hormone (LHRH) release from rat hypothalamic fragments in vitro. Hypothalamic fragments were incubated in Earle's medium. After 60 min of preincubation, medium from two 20-min incubations was collected and somatostatin and LHRH levels measured by radioimmunoassay. Dopamine (10 nmol/l–0·1 mmol/l) induced a progressive increase (r = 0·41; P <0·01) in basal somatostatin levels. K + (30 mmol/l)-induced somatostatin release was also increased (r = 0·54; P <0·01) by increasing doses of dopamine. Metoclopramide (10 μmol/l) blocked the dopamine (1 μmol/l)-induced increase in somatostatin release. No significant relationship between dopamine and LHRH was found either basally or after K + (30 mmol/l) stimulation. Basal somatostatin was negatively correlated (r = −0·63; P <0·01) with morphine concentrations. No significant correlation was found after K+ (30 mmol/l) depolarization. Basal LHRH release was not influenced by morphine, while K +(30 mmol/l)-induced release was significantly lower than controls only at a concentration of 10 nmol/l. These results suggest that dopamine and morphine act at a hypothalamic level to modulate GH release through alterations in somatostatin secretion. Dopamine and morphine have no consistent effect on hypothalamic LHRH release.
J. Endocr. (1985) 106, 317–322