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NATURE BIOTECHNOLOGY VOL 17 DECEMBER 1999 http://biotech.nature.com 1179

Regulated expression of the growth hormone (GH) pathway isessential for optimal linear growth, as well as for homeostasis of car-bohydrate, protein, and fat metabolism. Growth hormone synthesisand its pulsatile secretion from the anterior pituitary is stimulated bygrowth hormone–releasing hormone (GHRH) and inhibited bysomatostatin, both hypothalamic hormones1. Growth hormoneincreases production of insulin-like growth factor-I (IGF-I) primar-ily in the liver, as well as other target organs. Both IGF-I and GH pro-vide feedback to the hypothalamus and pituitary to inhibit GHRHrelease and GH secretion. The endogenous rhythm of GH secretionbecomes entrained to the imposed rhythm of exogenous GHRH2.

Linear growth velocity and body composition respond to GH orGHRH replacement therapies in a broad spectrum of conditions,both in humans and in farm animals. The etiology of these condi-tions can vary significantly. In 50% of human GH deficiencies theGHRH–GH–IGF-I axis is functionally intact but does not elicit theappropriate biological responses in its target tissues. Similar pheno-types are produced by genetic defects at different points in the GHaxis3, as well as in non–GH-deficient short stature. In several condi-tions characterized by growth retardation in which theGHRH–GH–IGF–I axis is functional, such as Turner’s syndrome4,hypochondroplasia5, Crohn’s disease6, intrauterine growth retarda-tion7, or chronic renal insufficiency8, therapeutic administration ofGHRH or GH has been shown to be effective in promoting growth9.

In the elderly, there is considerable decrement in the activity of theGHRH–GH–IGF-I axis that results in reduced GH secretion andIGF-I production. These changes are associated with a loss of skeletalmuscle mass (sarcopenia), osteoporosis, increased fat deposition, anddecreased lean body mass2,10. It has been demonstrated that the devel-opment of these changes can be offset by recombinant GH therapy.

Current GH therapy has several shortcomings, however, includ-ing frequent subcutaneous or intravenous injections, insulin resis-tance, and impaired glucose tolerance11. Children treated with GHare vulnerable also to premature epiphyseal closure and slippage ofthe capital femoral epiphysis12. In domestic livestock, GHRH andGH stimulate milk production, increase feed-to-milk conversion,and sustain growth, primarily by increasing lean body mass13,14, andincrease overall feed efficiency. Hot and chilled carcass weights areincreased, and carcass lipid (percentage of soft-tissue mass) isdecreased by GHRH15.

Although GHRH protein therapy entrains and stimulates normalcyclical GH secretion with virtually no side effects16, the short half-life of the molecule in vivo requires frequent (one to three times perday) intravenous, subcutaneous, or intranasal (at a 300-fold higherdose) administrations. Thus, recombinant GHRH administration isnot practical as a chronic therapy. However, extracranially secretedGHRH, as a mature or a truncated polypeptide, is often biologicallyactive17, and a low level of serum GHRH (100 pg/ml) stimulates GHsecretion16. These characteristics make GHRH an excellent candi-date for gene therapeutic expression.

Direct plasmid DNA gene transfer is currently the basis of manyemerging therapeutic strategies because it avoids the potential prob-lems associated with viral genes or lipid particles18. Skeletal muscle isa preferred target tissue because the muscle fiber has a long life spanand can be transduced by circular DNA plasmids. Skeletal muscle-borne plasmids have been expressed efficiently over months or yearsin immunocompetent hosts19,20. Previously, we reported that humanGHRH cDNA could be delivered to skeletal muscle by an injectablemyogenic expression vector in mice, where it transiently stimulatedGH secretion over a period of two weeks31. We have now optimized

RESEARCH

Myogenic expression of an injectableprotease-resistant growth

hormone–releasing hormone augmentslong-term growth in pigs

Ruxandra Draghia-Akli1,4*, Marta L. Fiorotto2, Leigh Anne Hill1,4, P. Brandon Malone1,4, Daniel R. Deaver3,and Robert J. Schwartz1,4,5*

1Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030. 2USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030. 3The Pennsylvania State University, University Park, PA 16802. 4Applied Veterinary Systems, Houston, TX 77030.

5National Space Biological Research Institute, Houston, TX 77030. *Corresponding authors R.D.A. ([email protected]) and R.J.S. ([email protected]).

Received 3 June 1999; accepted 30 August 1999

Ectopic expression of a new serum protease-resistant porcine growth hormone–releasing hormone,directed by an injectable muscle-specific synthetic promoter plasmid vector (pSP-HV-GHRH), elicitsgrowth in pigs. A single 10 mg intramuscular injection of pSP-HV-GHRH DNA followed by electroporationin three-week-old piglets elevated serum GHRH levels by twofold to fourfold, enhanced growth hormonesecretion, and increased serum insulin-like growth factor-I by threefold to sixfold over control pigs. After65 days the average body weight of the pigs injected with pSP-HV-GHRH was ∼ 37% greater than theplacebo-injected controls and resulted in a significant reduction in serum urea concentration, indicatinga decrease in amino acid catabolism. Evaluation of body composition indicated a uniform increase inmass, with no organomegaly or associated pathology.

Keywords: growth hormone–releasing hormone, gene therapy, muscle-specific promoter, electroporation

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this injectable vector system (pSP-HV-GHRH) by incorporating apowerful synthetic muscle promoter21 coupled with a novel pro-tease-resistant GHRH molecule with a substantially longer half-lifeand greater GH secretory activity. We improved the vector deliveryto skeletal muscle via a highly efficient electroporation technology22.As shown here, this potent vector augments long-term growth inlarge mammals.

ResultsGHRH super-active analogs with increased secretagogue activityand stability. Growth hormone–releasing hormone has a short half-life in the circulation, in both humans and pigs23. We reasoned that byemploying GHRH analogs with a prolonged biological half-lifeand/or improved secretagogue activity, we might be able to achieveenhanced GH secretion. Therefore, GHRH mutants were generatedby site-directed mutagenesis of the porcine (1–40)OH form of thecDNA (Fig. 1A). A G15A substitution was used to increase α-helicalconformation and amphiphilic structure that decreases cleavage bytrypsin-like enzymes24. Also, GHRH analogs with the Ala15 substitu-tion display a four to five times higher affinity for the GHRH recep-tor25. We replaced Met27-Ser28 with Leu27-Asn28 (ref. 26) in orderto reduce loss of biological activity due to oxidation of the Met27,thus forming a triple amino acid substitution denoted as 15/27/28-GHRH. Dipeptidylpeptidase IV is the prime serum GHRH degrada-tive enzyme27. Lower-affinity dipeptidase substrates were created byfurther mutagenesis of 15/27/28-GHRH, and by replacing Ala2 withIle2 (TI-GHRH) or Val2 (TV-GHRH) or by replacing Tyr1 and Ala2with His1 and Val2 (HV-GHRH, His1-Val2-Ala15-Leu27-Asn28).

To test the biological potency of the mutated porcine GHRH(pGHRH) cDNA, we engineered plasmid vectors that can direct veryhigh levels of gene expression specific to skeletal muscle with the useof a synthetic muscle promoter, SPc5-12 (ref. 21). A 228 bp fragmentof pGHRH cDNA that encodes the 31–amino acid signal peptideand a mature peptide pGHRH (Tyr1-Gly40) or the GHRH mutants,followed by the 3′ untranslated region of the human GH (hGH)cDNA, was incorporated into myogenic GHRH expression vectors.Skeletal myoblasts were transfected with each construct. PurifiedGHRH moieties from the conditioned culture media were assayedfor biological potency by their ability to induce GH secretion by piganterior pituitary cell cultures. Media were collected from the pitu-itary cell cultures after 24 h and analyzed for porcine-specific GH byradioimmunoassay (RIA). Conditioned media from all cell culturestransfected with a GHRH construct were more effective in promot-ing GH secretion than the β-galactosidase (β-gal)–transfected cells(P < 0.01). The modified GHRH species (15/27/28-GHRH, TI-GHRH, TV-GHRH) showed 20–50% improvements in GH secre-tion over wild-type pGHRH, but these did not attain statistical sig-nificance, nor were they different from the positive control. Onlyone of the mutants, HV-GHRH, resulted in a substantial increase inGH secretagogue activity relative to wild-type pGHRH, as indicatedby pGH values as high as 1,600 ng/ml (P < 0.04; Fig. 1B).

The stability of the wild-type pGHRH and the analog HV-GHRH was then tested by incubation of GHRH peptides in porcineplasma, followed by solid-phase extraction and HPLC analysis24. Atleast 80% of the wild-type pGHRH was degraded within 60 min ofincubation in plasma (Fig. 1C). In contrast, incubation of HV-GHRH in pig plasma for up to 6 h showed that at least 75% of thepolypeptide was protected against enzymatic cleavage, indicating aconsiderable increase in the resistance of HV-GHRH to serum pro-tease activity.

Muscle injection of pSP-HV-GHRH increases porcine GHRH,GH, and IGF-I serum levels over two months. We asked if the opti-mized protease-resistant pSP-HV-GHRH vector could effect in vivo,long-term expression of HV-GHRH and stimulate secretion of GHand IGF-I. Schematic maps of pSP-HV-GHRH, the wild-type con-

struct (pSP-wt-GHRH, positive control), and an Escherichia coli β-gal expression vector, pSPβgal (placebo control), are shown inFigure 2A. Three-week-old castrated male pigs were anesthetized,and a jugular vein catheter was inserted using sterile techniques forthe collection of serial blood samples with minimal discomfort tothe animal. Plasmid expression vector DNA (10 mg of DNA of pSP-HV-GHRH, pSP-wt-GHRH or pSPβgal) was injected directly intothe left semitendinosus muscle. The injected muscle was clampedwith a caliper and electroporated using the previously optimizedconditions of 200 V/cm with four pulses of 60 ms, as described28.

In vivo activity of the myogenic expression vectors was first evaluat-ed by measuring the GHRH serum concentration. In pigs injected withpSP-HV-GHRH, GHRH concentration had increased by seven dayspostinjection (Fig. 2B) and was 150% of the β-gal control level by 14days (652 ± 77 pg/ml versus 420 ± 13 pg/ml). pSP-HV-GHRH serumGHRH reached a plateau value by 60 days that was twofold to threefoldgreater than the injected control value (P < 0.002). An indirect measureof the total amount of GHRH secreted by the pSP-HV-GHRH–injectedpigs was derived by correcting for the increase in body weight betweenday 0 and day 60, assuming blood volume is 8% of body weight. This isnecessary to adjust for the increase in the distribution space of the pep-tide that results from the rapid growth of the animals. Total vascularGHRH was threefold higher than that of the β-gal–injected controlpigs (Fig. 2C) (1,426 ± 10 ng versus 267 ± 25 ng, P < 0.034). By com-parison, the pSP-wt-GHRH–injected pigs required 45 days to demon-strate an increase in GHRH secretion and had attained a twofold, albeitnonsignificant (P < 0.16), increase after 60 days.

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Figure 1. GHRH superactive analogs increase GH secretagogueactivity and stability. (A) Comparison of the porcine wild-type (1-40)OH amino acid sequence (p wt-GHRH) with the HV-GHRH analog.(B) Pig GH release in porcine primary pituitary culture was stimulatedby GHRH species isolated from conditioned media of skeletal musclecells transfected with myogenic expression vectors driving porcineGHRH analogs. Amino acid substitutions of G15A, M27L, and S28Nare represented by GHRH-15/27/28. Substitutions as in 15/27/28,plus A2I, are represented by TI-GHRH. Substitutions as in 15/27/28,plus A2V, are represented by TV-GHRH. Substitutions as in 15/27/28,plus Y1H and A2V, are represented by HV-GHRH. The constructcoding for E. coli β-gal was used as a negative control. As a positivecontrol, cells were stimulated with 10 ng of recombinant hGHRH(1-44)NH2 (*P < 0.038 for HV-GHRH versus wild-type pGHRH-conditioned medium; P < 0.005 for all GHRH species versus the β-galnegative control). (C) During a 6 h incubation in porcine plasma, HV-GHRH is more stable than wild-type pGHRH.

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Very young animals have high levels of GH, which decrease afterbirth until puberty. Blood samples taken every 15 min over a 6 hperiod at 7 and 14 days following the initial plasmid injections wereassayed, and a mean 6 h GH value was calculated. The changes in themean 6 h GH concentration from days 0 to 7 and days 7 to 14 postin-jection were calculated for each animal (Fig. 2D). The GHRH-injected pigs showed a less dramatic decline or even an increase intheir average GH value after 7 days. The average changes in GH con-centrations were: HV, +1.52 ng/ml; wild type, -0.73 ng/ml; β-gal, -3.2 ng/ml. From 7 to 14 days postinjection, GH concentrations

changed by: HV, +1.09 ng/ml; wild type, -4.42 ng/ml; β-gal, -6.88ng/ml. The change in mean GH concentrations over the 14 day peri-od is statistically significant for HV-GHRH–injected pigs comparedwith β-gal controls (P < 0.033).

Another indication of increased systemic levels of GH is anincrease in serum IGF-I concentration. We observed that the level ofserum IGF-I started to rise three days postinjection in pigs injectedwith pSP-HV-GHRH (Fig. 2E). By 21 days, serum IGF-I concentra-tions were approximately threefold higher than in β-gal–injectedpigs. This threefold increase was maintained over 60 days (P < 0.03).

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Figure 2. Single injections of GHRH myogenic expression vectors in vivo increases porcine GHRH, GH, and IGF-I serum levels over two months.(A) All constructs contain the SPc5-12 synthetic promoter and the 3′ untranslated region (UTR) of hGH. The HV-GHRH construct was used as amodel of mutated protein and compared with the wild-type pGHRH as a positive control, and with the β-gal construct as a negative control. (B)Serum pGHRH concentrations in pSP-GHRH–injected and pSPβgal-injected control pigs (for pigs injected with pSP-HV-GHRH versus pSPβgalat individual time points: *P < 0.02, #P < 0.002, and ¶for total experimental period, P < 0.002). (C) Total circulatory GHRH in pSP-GHRH–injectedpigs versus control pSPβgal–injected control pigs; the total serum values reflect the absolute, growth-associated increase in distributionvolume over the experimental period (for pigs injected with pSP-HV-GHRH versus pSPβgal: *P < 0.05 at individual time points; #P < 0.004 for totalexperimental period). (D) pSP-HV-GHRH injected pigs showed an increase in plasma pGH levels compared with pSPβgal-injected control pigs(*P < 0.033). (E) Plasma IGF-I concentrations after direct intramuscular injection of pSP-GHRH constructs. Values were significantly higher forthe entire experimental period in pSP-HV-GHRH–injected pigs than in pSPβgal-injected control pigs (*P < 0.03).

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Figure 3. Myogenic GHRH expression vectorsenhance pig growth. (A) Average body weight gainover two months was enhanced in pSP-GHRH–injected pigs (P < 0.014). (B) Feed efficiencywas improved in the pSP-GHRH–injected pigscompared with pSPβgal-injected controls (*P < 0.05for pigs injected with pSP-wt-GHRH versus pSPβgal;#P < 0.02 for pigs injected with pSP-HV-GHRH versuspSPβgal). (C) Comparison between a pSP-HV-GHRH–injected animal (right) and a pSPβgal-injectedcontrol pig (left), 45 days postinjection.

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By comparison, pigs injected with pSP-wt-GHRH showed anaverage 1.6-fold increase in their serum IGF-I level at 21 days rel-ative to control values, progressively increasing further by 60 dayspostinjection, although this value was not statistically differentfrom the β-gal control value (P < 0.39).

Myogenic GHRH expression vectors enhance pig growth. ThepGH secreted systemically after intramuscular injection of myo-genic pSP-GHRH expression vectors augmented growth over 65days: animals injected with pSP-wt-GHRH on average were 22%heavier than the β-gal controls (37.12 kg versus 29.37 kg, P <0.26), whereas the pigs injected with pSP-HV-GHRH were 38% heav-ier (41.77 kg; P < 0.014) (Fig. 3A). Feed (kilograms of food consumedper kilogram weight gain) also improved by 20% in pigs injected withGHRH constructs compared with controls: 2.11 ± 0.01 in pSP-HV-GHRH (P < 0.02), 2.15 ± 0.11 in pSP-wt-GHRH (P < 0.05), 2.37 ±0.07 in pSPβgal pigs (Fig. 3B). Body composition studies by dual-energy X-ray absorptiometry (total body fat, nonbone lean tissuemass, and bone mineral content measurements), total body 40K (leanbody mass estimation), neutron activation analysis (for total carcassnitrogen determination)29, and quantitative dissection of organs(heart, lung, liver, spleen, brain, kidney, pancreas, adrenal glands,stomach, and intestines) showed a proportional increase of all bodycomponents in GHRH-injected animals. There were no signs oforganomegaly or associated pathology. A photograph of a pSPβgal-injected control pig and a pSP-HV-GHRH–injected pig after 45 daysis shown in Figure 3C.

The serum biochemical profile of pSP-HV-GHRH-injected pigs(Table 1) demonstrated a significant decrease in serum urea com-pared with pSP-wt-GHRH or pSPβgal-injected control pigs (P <0.006) and is indicative of decreased amino acid catabolism. Serumglucose, creatinine, and total protein levels were similar among pigsinjected with pSP-GHRH or pSPβgal, suggesting that renal, liver,and pancreatic functions were not altered adversely.

DiscussionAmong the nonviral techniques developed for gene transfer in vivo, thedirect injection of plasmid DNA into muscle is simple, inexpensive,and safe. The relatively low expression levels of the transferred DNAexpression vectors have limited the application of this methodology.Previously, these levels were insufficient to ensure systemic physiologi-cal concentrations of secreted proteins such as hormones, neurotroph-ic factors, or coagulation factors in large mammals. In order to obtaingrowth of a large mammal by gene therapy, we believed that it was nec-essary to increase the potency of the myogenic vector system. Werecently described a strategy for the construction and the characteriza-tion of synthetic muscle promoters by the random assembly of E-boxes, myocyte-specific enhancer-binding nuclear factor–2 (MEF-2),transcription enhancer factor–1 (TEF-1), and serum response element(SRE) sites21. Several synthetic promoters were identified whose tran-scriptional activity in terminally differentiated skeletal muscle greatlyexceeded that of the natural myogenic skeletal α-actin gene promoterand viral promoters. Analysis of direct intramuscular injection ofSPc5-12–driven DNA plasmid in normal mouse skeletal musclerevealed a sixfold to eightfold increase in activity over the ubiquitouslyexpressed cytomegalovirus promoter even after a month. As shown inFigure 2, SPc5-12 was capable of eliciting moderate increases in piggrowth and IGF-I levels by driving wild-type pGHRH production.

Some individual amino acid substitutions leading to protease-resistant GHRH molecules have been tested previously in farm ani-mals and humans27,30. We found that the combination of five aminoacid substitutions in the novel GHRH analog HV-GHRH constructresulted in increased GH secretagogue activity (as shown in theassays with pig anterior pituitary somatotrophic cells) in compari-son with four other GHRH analogs (Fig. 1) and was more resistantto serum proteases.

Electrogene therapy allows genes to be efficiently transferred andexpressed in desired organs or tissues, and it may represent a newapproach for gene therapy that does not require the use of viral genesor particles. The electroporation system has been used previously inrodents and small animals, as well as humans, and does not appearto cause significant distress. Compared with classical gene therapytechniques, electrogene therapy increased transfection efficiencyover 100-fold and allowed for prolonged HV-GHRH expression over60 days in pigs.

Enhanced biological potency and delivery reduce the theoreticalquantity of GHRH plasmid needed to achieve physiological levels ofGH production and secretion. Treated pigs did not experience anyadverse effects of therapy, had normal biochemical profiles, anddeveloped no associated pathology or organomegaly. The profoundincreases in IGF-I levels and enhancement in growth over twomonths indicate that the myogenic expression of the HV-GHRHvector has the potential to replace classical GH therapy regimens andmay stimulate the GH axis in a more physiologically appropriatemanner. The HV-GHRH molecule, which displays a high degree ofstability and GH secretory activity in pigs, also may be useful inhuman clinical medicine because the serum proteases that degradeGHRH are similar in most mammals.

Experimental protocolDNA constructs. The plasmid pSPc5-12 contains a 360 bp SacI/BamHI frag-ment of the SPc5-12 synthetic promoter21 in the SacI/BamHI sites of pSK-GHRH backbone31. The wild-type and mutated pGHRH cDNAs wereobtained by site-directed mutagenesis of human GHRH cDNA (in vitroAltered Sites II Mutagenesis System, Promega Corporation, Madison, WI),and cloned into the BamHI/HindIII sites of pSK-GHRH. The GHRH cDNAis followed by the 3′ untranslated region of hGH.

Cell culture. Minimal Essential Medium (MEM), heat-inactivated horseserum (HIHS), gentamicin, Hanks’ Balanced Salt Solution (HBSS), and lipo-fectamine were obtained from Gibco BRL (Grand Island, NY). Primarychicken myoblast cultures were obtained and transfected as described31,32.After transfection, the medium was changed to MEM that contained 2%HIHS, and the cells were allowed to differentiate. Media and cells were har-vested 72 h postdifferentiation. One day before harvesting, cells were washedtwice in HBSS and the media changed to MEM, 0.1% bovine serum albumin.Conditioned media were treated by adding 0.25 volumes of 1% trifluo-roacetic acid (TFA) and 1 mM phenylmethylsulfonylfluoride, frozen at -80°C, lyophilized, purified on C-18 Sep-Columns (Peninsula Laboratories,Belmont, CA), relyophilized, and used in RIA or resuspended in media con-ditioned for primary pig anterior pituitary cell culture. The pig anterior pitu-itary culture was obtained as described33.

Plasma proteolytic activity on GHRH molecules. Briefly, chemically syn-thesized HV-GHRH was prepared by peptide synthesis. Pooled porcine plas-ma was collected from normal pigs and stored at -80°C. At the time of thetest, the porcine plasma was thawed, centrifuged, and allowed to equilibrateat 37°C. Mutant and wild-type GHRH samples were dissolved in the plasmato a final concentration of 100 µg/ml. Immediately after the addition of theGHRH, as well as 15, 30, 60, 120, 240, and 360 min later, 1 ml of plasma waswithdrawn and acidified with 1 ml of 1 M TFA. Acidified plasma was purifiedon C-18 affinity SEP-Pak columns, lyophilized, and analyzed by HPLC usinga Waters 600 multi-system delivery system, a Waters intelligent sampleprocessor, type 717 and a Waters spectromonitor 490 (Waters SolventCorporation, Millipore Corporation, Milford, MA). The mobile phase was(A) 0.1% TFA in H2O (B) 0.1% TFA in 95% acetonitrile, and 5% water; the

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Table 1. Plasma biochemical profile of GHRH-injected pigs and placebo1

Glucose Urea Creatinine Total protein (mg/dl) (mg/ml) (mg/ml) (g/dl)

pSPβgal 99.2 ± 4.8 9.0 ± 0.9 0.82 ± 0.06 4.60 ± 0.22pSP-wt-GHRH 97.5 ± 8.0 8.3 ± 1.0 0.83 ± 0.06 4.76 ± 0.35pSP-HV-GHRH 104.8 ± 6.9 6.9 ± 0.5 0.78 ± 0.04 4.88 ± 0.23

1The pSP-HV-GHRH-injected pigs had a significant decrease in serum urea concen-tration relative to controls (P < 0.01). No other differences were statistically significant.


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gradient was 80% (B) over 30 min. The flow rate was 0.75 ml/min. Peptidepeaks were detected at 214 nm. The percentage of peptide degraded at thevarious time points was assessed by integrated peak measurements.

Animal studies. Three groups of two three- to four-week-old mixed-breedbarrows (Yorkshire χ Landrace χ Hampshire χ Duroc) were studied in twoindependent studies. The animals were individually housed with ad libitumaccess to water, and were fed a 24% protein and 3.0 kcal digestible energy/gdiet (Producers Cooperative Association, Bryan, TX) provided at 6% of theirbody weight daily (approximately 90% of ad libitum intake). The animalswere weighed at 8:30 AM every other day before receiving their daily ration.The protocol was approved by the Baylor College of Medicine Animal Careand Use Committee and conducted in accordance with the National ResearchCouncil’s Guide for the Care and Use of Laboratory Animals.

Intramuscular injection of plasmid DNA. The pigs were randomlyassigned to one of the three treatments. The pigs fasted overnight and wereimplanted with one polyethylene jugular catheter under general isofluraneanesthesia using sterile technique. Endotoxin-free plasmid (Qiagen, Valencia,CA) preparations of pSPc5-12–HV–GHRH, pSPc5-12–wt–GHRH, andpSPc5-12βgal were diluted to 1 mg/ml in phosphate-buffered saline, pH 7.4.While anesthetized, each pig received a 10 mg injection of plasmid directlyinto the semitendinosus muscle. Two minutes after injection, the muscle wasclamped with calipers and electroporated as described28. Blood for biochem-ical analyses was drawn from the jugular catheter of each pig 3, 7, 14, 21, 28,45, and 60 days postinjection. At 65 days postinjection animals were killedand internal organs and the injected muscle were quantitatively dissected,weighed, frozen in liquid nitrogen, and stored at -80°C. Back fat thicknesswas measured following standard procedures. The empty carcass (wholebody with all internal organs removed) was weighed, frozen, and stored forneutron activation analysis.

Porcine GHRH, GH, and IGF-I assays. Porcine GHRH was measuredusing a heterologous human assay system (Peninsula Laboratories); sensitiv-ity of the assay is 1 pg/sample. Plasma porcine GH was measured with aporcine-specific double antibody procedure RIA (Pennsylvania StateUniversity, University Park, PA). The sensitivity of the assay is 4 ng/sample.Plasma IGF-I was measured using a heterologous human immunoradiomet-ric assay (DSL [Diagnostic System Labs], Webster, TX).

Body composition assessment. Body composition measurements29 wereperformed either under anesthesia 30 and 60 days postinjection (dual-energyX-ray absorptiometry and total body 40K), or postmortem (dissection, back-fat thickness assessment, and neutron activation analysis).

Statistics. Data were analyzed using the Microsoft Excel statistics package.Values shown in the figures are the mean ± standard error of the mean.Specific p values were obtained using Student’s t-test or analysis of variance.A P < 0.05 value was set as the level of statistical significance. Mean values foreach of the SP-GHRH groups were compared independently of thepSPβgal–injected controls, using two-tailed, two-sample with unequal vari-ance tests.

AcknowledgmentsWe thank Jim Cunningham, Frankie Biggs, Craig Stubblefield, and MichaelStubblefield for excellent care of the animals; Dr. Harry Mersmann, RomanShipaylo, and Dr. Ken Ellis for the body composition measurements; CharlesMcDonald and Dr. Richard Cook for the synthesis of the HV-GHRH polypeptide;Jana Peters for the GH assays; and Dr. Craig Delaughter for carefully reviewing thismanuscript. We acknowledge support for this study from Applied VeterinarySystems Inc. (Houston, TX), The Texas Advanced Technology Program, and theNational Space Biological Research Institute (NASA), the US Department ofAgriculture, Agricultural Research Service under Cooperative Agreement number58-6250-6001. The contents of this publication do not necessarily reflect the viewsor policies of the US Department of Agriculture nor does mention of trade names orcommercial products or organizations imply endorsements by the US government.

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