| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 14, 2002 as doi:10.1096/fj.01-0702fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
2
* Department of Molecular and Cellular Biology,
Center for Cell and Gene Therapy,
Children’s Nutrition Research Center, ASR-USDA Baylor College of Medicine, Houston, Texas 77030, USA; and
Valentis, Inc., The Woodlands, Texas 77381, USA
2Correspondence: Center for Cell and Gene Therapy/Room 1120N, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. E-mail: ada{at}bcm.tmc.edu
SPECIFIC AIMS
In this study of adult SCID mice, we show that growth pattern and body composition can be efficiently regulated long-term after the delivery of a plasmid-based, mifepristone-inducible GHRH expression system. Regulated animal growth occurred after a single electroporated injection of a mixture of two plasmids (10 µg of DNA)—one expressing the GeneSwitchTM regulator protein, the other an inducible growth hormone-releasing hormone (GHRH) gene—into the tibialis anterior muscles of adult SCID mice. Administration of the ligand mifepristone (MFP) up-regulated GHRH expression, as shown by elevations of IGF-I levels; when MFP dosing was withdrawn, IGF-I returned to baseline levels. Five cycles of IGF-I induction were observed during a 5 month period. Chronic MFP dosing for 25 days significantly increased lean body mass, weight gain, and bone mineral density compared with non-MFP-treated controls.
PRINCIPAL FINDINGS
1. Design of the muscle specific mifepristone inducible GHRH system
Growth hormone (GH) is released in a distinctive pulsatile pattern that has profound importance for its biological activity. This episodic pattern of secretion seems to be related to the optimal induction of physiological effects at the peripheral level. Secretion of GH is stimulated by the natural GH secretagogue GHRH and inhibited by somatostatin, both hypothalamic hormones. GH replacement therapy is widely used clinically with beneficial effects, but doses have often been supraphysiological and associated with side effects. These unwanted pathologies probably occur because treatment with recombinant GH protein raises basal levels of GH and abolishes its natural episodic pulses. In contradistinction, no side effects have been reported for therapies using recombinant GHRH. Due to its short half-life in vivo, frequent (1–3 times/day) intravenous, subcutaneous, or intranasal (requiring 300-fold higher dose) administrations of GHRH are necessary. Thus, recombinant GHRH protein administration is not practical as a chronic therapy. Gene therapy may overcome the limitations of recombinant GHRH protein use. Regulated expression of GHRH is desirable for use in animals under some circumstances and may be required to provide an acceptable safety margin for use in humans.
The GeneSwitchTM system for ligand-dependent induction of transgene expression is based on a carboxyl-terminally truncated progesterone receptor that fails to bind to its natural agonist, progesterone, but instead is activated by antiprogestins, such as MFP. Thus, the injected DNA requires MFP to be transcriptionally activated. The chimeric regulator protein of the GeneSwitchTM system consists of the ligand binding domain of the truncated human progesterone receptor fused to the DNA binding domain of the yeast GAL4 protein (which binds a specific 17 bp recognition sequence) and a transcriptional activation domain from the p65 subunit of human NF-
B. The gene for the GeneSwitch regulator protein was inserted into a myogenic expression vector, designated pGS1633, which is expressed constitutively under the control of a muscle-specific skeletal alpha-actin promoter. The GHRH plasmid (designated pGR1774) contains an inducible promoter that consists of six copies of the 17 mer Gal4 binding site fused to a minimal TATA box promoter. The GHRH coding sequence is a 228 bp fragment of mutated porcine GHRH cDNA, termed HV-GHRH. The HV-GHRH molecule displays a high degree of stability in serum, with a half-life of 6 h (vs. the natural GHRH, which has a 6–12 min half-life) and increased GH stimulatory activity in pigs. The muscle-specific GeneSwitch and inducible GHRH plasmids each have a 5' untranslated region that contains a synthetic intron and a 3' untranslated region/poly(A) site from the human GH gene.
2. GHRH GeneSwitch plasmids is regulated in vitro in primary myoblasts
To test the inducible GHRH system in vitro, primary chicken myoblasts were transfected with a mixture of the GHRH/GeneSwitch plasmids (GR1774/pGS1633 in a 10:1 w/w ratio). Negative controls were cells transfected by the GeneSwitch and GHRH plasmids (but not treated with MFP) or transfected by the inducible GHRH plasmid alone. Positive control were cells transfected by a constitutively expressed GHRH plasmid driven by a synthetic muscle-specific promoter (designated SP-GHRH). GHRH transcripts of the expected size of 0.35 kb were observed only in cells transfected with the GeneSwitch/inducible GHRH plasmids and treated with MFP and in cells transfected with the positive control. No GHRH transcripts were detected in cells not treated with MFP or in those transfected by the inducible GHRH plasmid alone.
3. GHRH/GeneSwitch is expressed and regulated in vivo, inducing IGF-I levels in treated animals
For long-term in vivo testing, plasmids for the GHRH/GeneSwitch system were delivered to the muscles of SCID mice. The left tibialis anterior muscle of mice was injected with 10 µg of the 10:1 mixture of pGR1774/pGS1633, followed by caliper electroporation. At 21 days postinjection, we started injecting intraperitoneal MFP (250 µg/kg) for 3 days. On day 4, the animals were bled and serum was used to measure IGF-I levels. After administration of MFP for 4 consecutive days, IGF-I levels increased from 1101 ± 34 ng/ml to 1798 ± 165 ng/ml (P<5x10-4). Significant changes in IGF-I levels were seen when the MFP group was compared with animals that received a ß-galactosidase plasmid (1087±65 ng/ml, P<6x10-4) and with animals that received the GHRH/GeneSwitch plasmids but not dosed with MFP (1171.79±42 ng/ml, P<0.001). Upon repeated administration of MFP using the same protocol (3 days induction, bleed the fourth day, recovery to background 7 days) over 149 days, serum IGF-I levels rose repeatedly from 1.1- to 1.7-fold over those exhibited by animals not dosed with MFP (Fig. 1
).
|
4. Body weight is increased and body composition is changed in chronically induced animals
Body weight was similar for all groups during the first 125 days of the study. From day 125 to day 149, the mice were dosed with MFP every day. On day 149, body weight was increased by 7.5% in chronically MFP-induced GHRH/GeneSwitch animals, which averaged 31.84 ± 0.12 g (P<0.03), compared with ß-gal controls (29.62±0.98 g) and with animals not induced with MFP (30.53±0.59 g). All values are average ± SE. Organs (lungs, heart, liver, kidney, stomach, intestine, adrenals, gonads, brain) were collected and weighed. Pituitary glands were dissected within minutes postmortem and weighed. The ratio of pituitary weight to total body weight increased after chronic stimulation of the GHRH/GeneSwitch by 20% (7.35±0.31x10-5) vs. ß-gal controls (6.13±0.46x10-5) and to animals not dosed with MFP (6.3±0.22x10-5), P < 0.035. There was no significant difference between the ß-gal-injected animals and animals injected with the GHRH/GeneSwitch system but not given MFP. The increase in pituitary weight is most likely due to somatotrophs hypertrophy, as it is known that GHRH is capable of stimulating the synthesis and secretion of GH from the anterior pituitary and has a specific hypertrophic effect on somatotrophs. At the end of the experiment, body composition was analyzed in vivo by dual-energy X-ray absorptiometry, using a high-resolution PIXImus scanner, and subsequently postmortem. Body composition studies by PIXImus (total body fat, non-bone lean tissue mass and bone mineral area, content, and density) showed significant changes in chronically MFP-induced animals injected with the GHRH/GeneSwitch system. Lean, non-bone body mass (Fig. 2
A) increased by 2.5% in GHRH/GeneSwitch animals + MFP (87.44±0.65% vs. ß-gal controls, 84.94±0.6%, and no MFP-treated animals, 84.88±0.3%, P<0.03). Fat mass (Fig. 2B
) decreased by 2% in GHRH/GeneSwitch animals (12.59±0.62% vs. ß-gal controls, 14.57±0.75%, and no MFP-treated animals, 15.09±0.3%, P<0.05).
|
5. Bone mineral density is significantly increased upon chronic induction of the GHRH/GeneSwitch system
Upon chronic stimulation of the GHRH/GeneSwitch system, significant changes occurred in bone area (Fig. 2C
), which increased by 7%, (12.81±0.14 cm (2) vs. ß-gal controls, 11.98±0.3 cm2, or no MFP-treated animals, 12.07±0.26 cm2, P <6x10-4), bone mineral content (Fig. 2D
) increased by 14.6% (0.755±0.012 g vs. ß-gal controls, 0.659±0.019 g, or no MFP-treated animals, 0.694±0.023 cm2, P<0.002), and bone mineral density increased by 6% (0.059±0.0007 g/cm2 vs. ß-gal controls 0.056±0.0009 g/cm2, or no MFP-treated animals, 0.057±0.0007 g/cm2, P<0.012). There was no overall difference among the animals injected with the ß-gal plasmid and animals injected with the GHRH/GeneSwitch plasmids but not given MFP. This supports the absence of GHRH expression by the GHRH/GeneSwitch plasmids in the absence of MFP dosing.
CONCLUSIONS
To obtain regulation of growth and body composition by gene therapy, we believed it was necessary to use an improved version of the plasmid-based GeneSwitch system. A significant improvement of our plasmid vector was the use of a cDNA that codes for a more stable GHRH analog, HV-GHRH. Enhanced biological potency and delivery in conjunction with proper gene expression-regulation over > 149 days reduces the theoretical quantity of GHRH needed to achieve physiological levels of GH production and secretion compared with recombinant GHRH therapies. Animals with the GHRH/GeneSwitch system did not experience side effects from the GHRH therapy or mifepristone administration and had normal biochemical profiles with no associated pathology or organomegaly. Upon chronic induction of the GHRH/GeneSwitch system, the increases in IGF-I levels, enhancement in growth by 7.5%, and changes in body composition (with increased lean body mass by 2.5% and decreased fat by 2%) were dramatic as to function. The effects of the stimulation of GHRH on bone metabolism were even more remarkable, with increased bone mineral density by 6%. That we show pituitary hypertrophy indicates that ectopic expression of myogenic GHRH vectors operates through the natural GH axis (stimulation of GH synthesis and secretion at the pituitary level). This long-lasting regulated therapy has the potential to replace classical GH therapy regimens and may stimulate the GH axis in a more physiologically appropriate manner. GHRH stimulates bone formation, and our therapy may be used to promote postfracture bone growth.Data show that GH plus IGF-I (delivered as recombinant proteins) synergistically increases lean muscle weight and total body weight, and were more effective in re-epithelialization of a burn wound than either GH or IGF-I alone. Studies also showed that long-term stimulation of the GH axis, which includes doses in the range given to humans during clinical trials on GH deficiency and to revert age-related physiological declines, has no overt deleterious effects on longevity and pathology in aged rodents. Prospects for the MFP-inducible GHRH/GeneSwitch system could be the efficient delivery of GHRH as a performance enhancer for supporting pediatric growth and a positive anabolic state, as well for treatment of burn, sepsis, large surgery, AIDS-induced cachexia, and improvement of general well being in the elderly.
|
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0702fje; to cite this article, use FASEB J. (January 14, 2002) 10.1096/fj.01-0702fje 
This article has been cited by other articles:
|
|
 |
|
  J. D. Veldhuis, J. N. Roemmich, E. J. Richmond, and C. Y. Bowers Somatotropic and Gonadotropic Axes Linkages in Infancy, Childhood, and the Puberty-Adult Transition Endocr. Rev., April 1, 2006; 27(2): 101 - 140. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Draghia-Akli and M. L. Fiorotto A new plasmid-mediated approach to supplement somatotropin production in pigs J Anim Sci, January 1, 2004; 82(13_suppl): E264 - 269. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Draghia-Akli, K. K. Cummings, A. S. Khan, P. A. Brown, and R. H. Carpenter Effects of plasmid-mediated growth hormone releasing hormone supplementation in young, healthy Beagle dogs J Anim Sci, September 1, 2003; 81(9): 2301 - 2310. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. S. Khan, M. L. Fiorotto, L.-A. Hill, P. B. Malone, K. K. Cummings, D. Parghi, R. J. Schwartz, R. G. Smith, and R. Draghia-Akli Nonhereditary Enhancement of Progeny Growth Endocrinology, September 1, 2002; 143(9): 3561 - 3567. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
