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Vaccine 28 (2010) 1905–1910

Contents lists available at ScienceDirect

Vaccine

journa l homepage: www.e lsev ier .com/ locate /vacc ine

ffects of maternal plasmid GHRH treatment on offspring growth

mir S. Khana,∗, Angela M. Bodles-Brakhopa, Marta L. Fiorottob, Ruxandra Draghia-Akli a

VGX Pharmaceuticals, Inc., 2700 Research Forest Drive, The Woodlands, TX 77381, USAUSDA ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

r t i c l e i n f o

eywords:lectroporationene therapyrowth hormone releasing hormonelasmidwine

a b s t r a c t

To differentiate prenatal effects of plasmid growth hormone-releasing hormone (GHRH) treatment frommaternal effects mediated by lactation on long-term growth of offspring, a cross-fostering study wasdesigned. Pregnant sows (n = 12) were untreated (n = 6) or received either a Wt-GHRH (n = 2) or HV-GHRH(n = 4) plasmid. At birth, half of each litter was cross-fostered (treated to controls and controls to treatedonly). Piglets from plasmid-injected sows were heavier at birth (HV-GHRH, 1.65 ± 0.07 kg; Wt-GHRH,1.46 ± 0.05 kg vs. Controls, 1.27 ± 0.03 kg; P ≥ 0.001) and at weaning (Wt-GHRH, 6.01 ± 0.21 kg and HV-GHRH, 6.34 ± 0.15 kg vs. Controls, 5.37 ± 0.14 kg; P ≥ 0.02, respectively). Control piglets cross-fostered toplasmid-injected sows grew faster to weaning (Wt-GHRH, 5.61 ± 0.15 kg and HV-GHRH, 5.70 ± 0.29 kg

vs. Controls, 5.08 ± 0.22 kg; P > 0.05, respectively). Piglets from plasmid-injected sows that suckled oncontrol sows were larger than control piglets on control sows (Wt-GHRH, 5.93 ± 0.20 kg and HV-GHRH,6.2 ± 0.19 kg vs. Controls, 5.08 ± 0.22 kg; P > 0.05, respectively), but smaller than their littermates left ontheir treated mothers. The observed improvements were maintained until the end of the study when theoffspring were 170-day-old. The results suggest that the improved growth of offspring of GHRH plasmid-

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treated sows pre-weanineffects on the pituitary co

. Introduction

A gene therapy approach in combination with electropora-ion (EP) offers an alternative or complimentary approach toonventional treatment strategies for modulation of growth, devel-pment and health. Indeed, we have previously demonstratedhat the single administration of a commercially available plas-

id that encodes a growth hormone-releasing hormone (GHRH)LifeTideTMSW5), when administered to pregnant sows increasedurvival and production parameters in their offspring for threeequential parities [1]. Furthermore, we have shown that plasmidHRH administration with half of the dose previously used aloner in combination with porcine somatotrophin (pST) was effectiven improving survivability and production parameters (Person, ineview, BMC Vet. Res.).

It is likely that multiple mechanisms are responsible for these

ffects of increased maternal GHRH production on offspring sur-ival and growth. The GHRH/growth hormone (GH)/insulin-likerowth factor-I (IGF-I) axis plays an important role in the regulationf growth and development [2], as well as in the physiology of preg-

∗ Corresponding author at: VGX Pharmaceuticals, Inc., 2700 Research Forest Drive,uite 180, The Woodlands, TX 77381, USA. Tel.: +1 281 296 7300x107;ax: +1 281 296 7333.

E-mail address: akhan@inovio.com (A.S. Khan).

264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2009.10.093

ttributable to improved maternal performance, while after weaning thenent are relevant.

© 2009 Elsevier Ltd. All rights reserved.

nancy and lactation [3]. In previous studies, we have shown thatthere is transplacental transfer of GHRH from mother to fetus thatcan directly impact fetal development [4]. We have also demon-strated that in the offspring of plasmid GHRH-treated pregnantanimals there is a change in pituitary lineage as demonstrated byan increased number of somatotrophs and lactotrophs [5], with-out pituitary hyperplasia. It is also known that GHRH treatmentimproves lactation [6] and we have shown that the injection ofcows with plasmid GHRH results in improved lactation perfor-mance [7]. However, the extent to which the improved growthand survivability of the offspring is attributable to improvementsin maternal physiology during pregnancy and lactation vs. thoseresulting from the changes to the offspring themselves is uncer-tain.

In order to differentiate between the effects of plasmid GHRHadministration on the mother vs. the indirect effects on offspringwe carried out a cross-fostering study in which offspring bornto treated sows were cross-fostered to untreated control sowsat birth and vice versa. The offspring were then followed to 170days of age when they were euthanized and body compositionscompared. We demonstrate that administration of plasmid GHRH

to gestating gilts improved offspring survival and body composi-tion and that this could be attributed to both a prenatal benefiton offspring intrauterine growth and development, and improvedpost-natal performance linked to better maternal lactation perfor-mance.

1906 A.S. Khan et al. / Vaccine 28

Table 1Grouping of cross-fostered offspring. There were 6 control sows with 63 offspring,2 Wt-GHRH-treated sows with 18 offspring and 4 HV-GHRH-treated sows with42 offspring. Of the control offspring, 15 were cross-fostered to Wt-GHRH and 17were cross-fostered to HV-GHRH sows; the remaining 31 stayed within their treat-ment group. Of the Wt-GHRH offspring 10 were cross-fostered to control sows;the remaining 8 stayed within their treatment group. Of the HV-GHRH offspring 22were cross-fostered to control sows; the remaining 20 stayed within their treatmentgroup.

Groups Sow treatment Cross-fostered to:

1 Control Control2 Control Wt-GHRH3 Control HV-GHRH4 Wt-GHRH Wt-GHRH

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. Materials and methods

.1. Animals and study design

This study followed 140 pigs born to 12 first parity sows ofhich six served as untreated controls, two were treated with alasmid encoding the wild-type GHRH peptide (Wt-GHRH), andour were administered a plasmid encoding a protease-resistantHRH (HV-GHRH) [8]. The dam line was from a commerciallyvailable Landrace X Yorkshire crossbred female mated to a com-ercial terminal line Duroc crossbred boar. Newborn piglets were

ubdivided into a total of seven groups according to the cross-ostering plan summarized in Table 1. At birth, approximatelyalf of each litter was cross-fostered, so that treated and con-rol sows nursed both their own offspring and offspring fromn alternative treatment (i.e. half the piglets from four controlows were replaced with piglets from the four HV-GHRH-treatedows and vice versa and half the piglets from the remainingwo control sows received half the litter of the two Wt-GHRH-reated sows and vice versa). Sows were housed in groups of 6–8nimals per pen during the gestation period and then individ-ally in farrowing crates during lactation. At birth piglets wereagged for identification. Males were castrated at 5–7 days ofge. Piglets were suckled until they were 21 days of age, whenhey were weaned and group housed in pens. The offspring wereeighed at birth and at routine intervals until the end of the

tudy when they were 170-days-old. All studies were carried outt a Research and Development farm (Burton, TX) and animalsere maintained in accordance with National Institutes of Healthuide, U.S. Department of Agriculture, and Animal Welfare Actuidelines.

.2. Diet and food intake

All diets were fed ad libitum (base mixes from Suidae, Greens-urg, Indiana, mixed at Ludemann Grocery and Mill, Brenham,exas). The sows were fed a gestation diet containing 14% proteinrom the time they were treated until parturition. After partu-ition, the sows were transferred to a lactation diet containing6% protein. For the offspring, a creep-feed diet of 23% proteinith high lysine was introduced 1 week prior to weaning andaintained until 4–5 days after weaning. A prestarter diet of 21%

rotein was then introduced for a period of 10 days, followed bystarter diet of 18% protein until the offspring reached approxi-ately 20 kg. The animals were then maintained on a grower diet

f 16% protein until the end of the study. Food intake was mea-ured per pen and is calculated over a 106-day period from studyays 64–170.

(2010) 1905–1910

2.3. DNA construct

The myogenic plasmid expressing porcine GHRH was pre-viously described [1,9]. Briefly, plasmid expression was drivenfrom a muscle-specific SPc5-12 synthetic promoter [10]. Wild-typeporcine GHRH cDNA was cloned into the BamHI/HindIII sites ofpSPc5-12, to generate pSP-GHRH [8]. The 3′ polyadenylation anduntranslated region of human (h)GH was cloned downstream ofGHRH cDNA. The plasmid was produced under good manufactur-ing practices (GMP) (VGX Pharmaceuticals, Inc., The Woodlands,TX) and formulated in sterile water for injection with 1% HPLCpurified low molecular weight poly-l-glutamate sodium salt. Toobtain pSP-HV-GHRH, the porcine GHRH cDNA modified to ren-der the resulting peptide (HV-GHRH) more protease resistant andextend its half-life, was cloned into the BamHI/HindIII sites of pSP-GHRH, followed by the 3′-untranslated region and poly(A) signal ofthe hGH gene. HV-GHRH is a GHRH analog with amino acids His1and Val2 substituted with Tyr1 and Ala2, Gly15 substituted withAla15, and Met27 and Ser28 with Leu27 and Asn28.

2.4. Treatment

Twelve pregnant gilts between 76 and 90 days of gestation wereadministered in the semimembranosus leg muscle with 5 mg ofGHRH plasmid using a 21 gauge hypodermic needle. Post-injection,EP was applied through a five electrode array consisting of 21 gaugeelectrodes using a CELLECTRA® constant current device, 0.5 A, 5pulses, 60 ms/pulse with 1 s interval between pulses. Treated giltswere anaesthetized, before the plasmid treatment, with Zoletil®

(1 ml per 100 kg) and Ketamine (0.25 ml per 100 kg) given byIM injection by the attending veterinarian. None of the offspringreceived any direct treatment with plasmid GHRH.

2.5. Biochemistry analysis

Blood was withdrawn from the offspring by jugular punctureat 50 and 170 days of age. Plasma was analyzed for: creatinine,total protein, chloride, potassium, sodium, blood urea nitro-gen, glucose, calcium, alkaline phosphatase, bilirubin, inorganicphosphorus, globulin, cholesterol, creatinine phosphokinase andalbumin (Antech Diagnostics; Irvine, CA).

2.6. Weight and meat analysis

At 170 days of age, the pigs were killed by captive bolt at theMeat Science Department at Texas A&M University (College Station,TX). The organs were dissected and weighed, and meat analyzed [5].

2.7. Data analysis and statistics

The plasmid GHRH treatment groups were compared with thecontrols. In addition, the cross-fostered groups were comparedwith those that were not cross-fostered. A Microsoft Excel statisticsanalysis package was used. Values shown in the tables and figuresare the mean ± SEM. Specific P values were obtained by comparisonusing the Student’s t-test, or ANOVA with correction for multi-ple comparisons, as appropriate; P < 0.05 was set as the level ofstatistical significance.

3. Results

This pilot study aimed to differentiate between the pituitaryeffects of plasmid GHRH treatment from the maternal effects medi-ated by lactation on the offspring. To this end, offspring fromgestating sows treated with either Wt- or HV-plasmid GHRH werecompared to offspring from untreated controls.

A.S. Khan et al. / Vaccine 28

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period from 64 to 170 days of age was calculated on a per animal

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ig. 1. Birth and wean weight. (A) Birth weight and (B) wean weight of offspringrom control, Wt-GHRH- and HV-GHRH-treated sows, where *P < 0.005 comparedo controls.

The offspring of gestating sows administered either Wt- or HV-HRH plasmid were heavier at birth and at weaning compared

o untreated controls as shown in Fig. 1. The average numbersf piglets born per sow were not different between treatmentroups (control, 9 ± 1 piglets; Wt-plasmid GHRH, 9 ± 4 piglets,

V-plasmid GHRH, 11 ± 2 piglets). At weaning offspring from Wt-HRH plasmid-treated sows were 12% heavier than controls, whileffspring from HV-GHRH plasmid-treated sows were 18% heavier.

For the cross-fostered groups (Fig. 2), weight at weaningapproximately 20 days of age), and after weaning at approxi-

ig. 2. Weight at weaning. Weight of offspring from control, Wt-GHRH- and HV-GHRH-trer to an alternative treatment as indicated. Control offspring cross-fostered to Wt-GHRHostered to control sows, *P < 0.05. Wt-GHRH offspring cross-fostered to Wt-GHRH or conows, †P < 0.05. HV-GHRH offspring cross-fostered to HV-GHRH and control sows were sig

(2010) 1905–1910 1907

mately 32 days of age, followed similar trends: control offspringthat were cross-fostered to either Wt- or HV-GHRH-treated sowswere heavier than control offspring that were suckled with con-trol sows (P < 0.05). For offspring from Wt- and HV-GHRH sowscross-fostered to control sows there was a trend towards lowerweight gain when compared with offspring that were suckled bytheir birth mothers, although this difference did not attain sta-tistical significance. Nonetheless, their weights at weaning weregreater than those of the control offspring (P < 0.05). From weaningto 170 days of age the percentage increases in weight for con-trol offspring cross-fostered to Wt-GHRH plasmid-treated sowswas 10.50% (n = 15), while control offspring cross-fostered to HV-GHRH plasmid-treated sows was 12.20% (n = 16) compared tocontrols that were not cross-fostered (n = 30). Increases in weightfor Wt-GHRH plasmid-treated offspring that remained on theirbirth mother was 20.32% (n = 8), while Wt-GHRH plasmid-treatedoffspring cross-fostered to control sows was 16.73% (n = 10) com-pared to controls that were not cross-fostered. Increases in weightfor HV-GHRH plasmid-treated offspring that remained on theirbirth mother was 27.85% (n = 19), while HV-GHRH plasmid-treatedoffspring cross-fostered to control sows was 22.05% (n = 22) com-pared to controls that were not cross-fostered.

The offspring of treated sows, regardless of cross-fostering,were heavier than those of untreated controls until the end ofthe study at 170 days of age, with controls on average weigh-ing 125 ± 1.74 kg, Wt-plasmid GHRH treated on average weighing129 ± 2.15 kg, and HV-plasmid GHRH treated on average weigh-ing 136 ± 1.89 kg. There was no statistical difference in weight atthe end of the study for offspring of control sows that were cross-fostered to Wt-GHRH plasmid-treated sows compared to offspringof control sows left on their control mother (P = 0.37). However,for offspring of control sows that were cross-fostered to HV-GHRHplasmid-treated sows there was a significant increase in weightcompared to controls not cross-fostered at the end of the 170-day study period (132.5 ± 1.6 kg vs. 124.3 ± 2.6 kg, respectively,P = 0.01).

The amount of feed consumed by the animals over a 106-day

basis. The average feed intake per pig based on prenatal treat-ment, irrespective of cross-fostering was: for controls, 244.84 kg(n = 53); for offspring of the Wt-GHRH-treated sows, 281.38 kg(n = 17); for offspring of HV-treated sows, 286.77 kg (n = 38). The

ated sows at weaning (day 21) cross-fostered (CF) either within their own treatmentsows or to HV-GHRH sows were significantly heavier than control offspring cross-trol sows were significantly heavier than control offspring cross-fostered to controlnificantly heavier than control offspring cross-fostered to control sows, ‡P < 0.05.

1908 A.S. Khan et al. / Vaccine 28 (2010) 1905–1910

Table 2Total protein and albumin levels. Biochemistry analyses were performed on plasma samples collected from offspring of control, Wt-GHRH plasmid-, and HV-GHRH plasmid-treated sows. Total protein and albumin levels were significantly different among offspring of control and treated sows at 50 and 170 days of age as indicated. All otherparameters measured were not significantly different among groups. For offspring that were cross-fostered outside of their own group there were no significant differences.

Age Total protein Albumin

50 days Controln = 58

5.21 ± 0.38 – 3.21 ± 0.41 –

Wt-GHRHn = 18

5.62 ± 0.30 P < 0.001 3.64 ± 0.30 P < 0.001

HV-GHRHn = 39

5.53 ± 0.29 P < 0.001 3.42 ± 0.29 P < 0.01

170 days Controln = 50

7.07 ± 0.56 – 3.82 ± 0.39 –

Wt-GHRHn = 14

7.68 ± 0.31 P < 0.001 4.07 ± 0.38 P < 0.05

HV-GHRH 7.33 ± 0.29 P < 0.01 4.01 ± 0.20 P < 0.005

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eed to gain ratio calculated as the total weight of feed consumedivided by the total weight gain measured over the same periodas greater for offspring from GHRH-treated sows (2.94 for HV-HRH and 2.76 for Wt-GHRH) compared to offspring from controlows (2.44). When analyzed based on cross-fostering groupinghe average feed intake per pig was: for non-cross-fostered con-rols, 215.72 kg (n = 29); for non-cross-fostered offspring from

t-GHRH-treated sows, 274.56 kg (n = 8); for non-cross-fosteredffspring from HV-GHRH-treated sows, 224.13 kg (n = 17); for con-rol offspring cross-fostered to HV-GHRH-treated sows, 236.95 kgn = 21); for control offspring cross-fostered to Wt-GHRH-treatedows, 258.71 kg (n = 10); for HV-GHRH-treated offspring cross-ostered to control sows, 215.07 kg; for WT-GHRH-treated offspringross-fostered to control sows, 232.97 kg. The feed to gain ratio waslso analyzed based on cross-fostering grouping and was greater forffspring from GHRH-treated sows (2.63 for HV-GHRH and 3.07 fort-GHRH) compared to controls (2.39). For control offspring cross-

ostered to HV-GHRH-treated sows the feed to gain ratio was 2.38,hereas for control offspring cross-fostered to Wt-GHRH-treated

ows the feed to gain ratio was 2.53. For offspring from HV-GHRH-reated sows cross-fostered to control sows the feed to gain ratioas 2.34, whereas for Wt-GHRH offspring cross-fostered to control

ows the feed to gain ratio was 2.64.Plasma biochemistry analyses were carried out at 50 and 170

ays of age. All of the offspring from GHRH-treated sows had signif-cantly greater total protein and albumin concentrations (Table 2)

able 3issection analysis of body composition. Body composition was assessed from yield da70-day-old.

Assessment – yield data Controln = 29

Carcass weight (kg) 93.00 ± 2.11Carcass length (in.) 34.03 ± 0.22Average backfat thickness (in.) 1.03 ± 0.04

Assessment – cuts Controln = 14

Side weight (kg) 91.21 ± 3.90Belly (kg) 12.84 ± 0.91Spare ribs (kg) 2.69 ± 0.16

Assessment – quality traits Controln = 29

Cook loss (%) 36.52 ± 0.77Marbling score (1–5) 1.62 ± 0.15Average drip loss (%) 6.78 ± 0.37

tatistical significance from control was determined by the Student’s t-test.

less than the total number of animals per group due to insufficient quantity or a

compared to untreated control offspring at both ages. Plasma glu-cose concentrations were normal at both time points for all groups(normal range 65–95 g/dl). A comparison of total protein and albu-min for cross-fostering groups at day 50 and day 170 revealed thatthere were no significant differences between offspring left on theirmother and offspring that were cross-fostered. All other parame-ters measured fell within normal ranges and were not significantlydifferent.

Measures of body composition and parameters of meat qualityare summarized in Table 3. All measures of yield and cut data weresignificantly greater in offspring from HV-GHRH-treated sows com-pared to controls, regardless of cross-fostering. Although offspringfrom Wt-GHRH-treated sows showed a trend towards greater cutscompared to controls, this difference was not statistically signifi-cant. Meat quality traits also were significantly enhanced by GHRHtreatment: percentage cook loss was reduced in offspring fromHV-GHRH-treated sows, whilst marbling scores and drip loss werereduced in offspring from Wt-GHRH-treated sows compared tocontrols. There were no differences among the other meat qual-ity traits that were measured (shear force – a measure of thetenderness of meat, color scores, firmness) although there wasa trend toward lower shear force, suggesting more tender meat,

with offspring from HV-GHRH-treated sows < Wt-GHRH-treatedsows < controls.

We also compared mortality among groups. For controls, fatalpathology ranged from sudden death, crippled, prolapse, enteri-

ta, cuts, and meat quality traits at the end of the study when the offspring were

Wt-GHRH HV-GHRHn = 32 n = 9

91.21 ± 1.37 100.22 ± 1.78 P = 0.0434.08 ± 0.18 34.86 ± 0.17 P = 0.031.08 ± 0.03 1.25 ± 0.09 P = 0.04

Wt-GHRH HV-GHRHn = 15 n = 6

94.27 ± 1.88 103.38 ± 1.50 P = 0.0114.63 ± 0.55 17.48 ± 0.36P = 0.00023.00 ± 0.10 3.33 ± 0.13 P = 0.007

Wt-GHRH HV-GHRHn = 32 n = 9

35.89 ± 0.89 33.80 ± 1.44 P = 0.051.14 ± 0.06P = 0.001 1.5 ± 0.177.90 ± 0.27 P = 0.02 7.28 ± 0.63

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A.S. Khan et al. / Vac

is, swollen joints and bleeding ulcer in 7 out of 63 pigs (11%).reatment with either Wt-GHRH or HV-GHRH plasmid reducedortality with only 1 out of 18 (6%) and 2 out of 42 (5%) pigs,

espectively, having a fatal pathology. There were no significantifferences between the groups as calculated by a chi-squaredest.

. Discussion

We have previously shown that a plasmid-mediated GHRHreatment to the mother during pregnancy impacts pituitary lin-ages and post-natal growth and development of the offspring.evertheless, some effects of growth and immune function in theffspring could not be completely explained by these pituitaryhanges. In this pilot study the impact on the offspring of the admin-stration of plasmid GHRH to gestating sows was investigated in aross-fostering study. The use of a large animal model in this caseerves to demonstrate the feasibility and efficacy of a gene therapyased administration with EP.

The GHRH/GH/IGF-I axis is multifaceted with many hormonesnvolved in complex feedback and control mechanisms from direct-ng gene expression to the regulation of mature peptide action [11].he GHRH/GH/IGF-I axis plays a key role in the co-ordination ofrotein and energy metabolism during growth, with GH treatment

ncreasing protein synthesis and decreasing protein degradation,ither via the regulation of IGF-I or by direct action. It is alsonown that prenatal and post-natal nutrition play a large role inegulating growth, including through developmental control of theomatotropic hormone axis [12]. This study provides some novelnsights into the pre- and post-natal regulation of the developmentf the GHRH/GH/IGF-I axis and its consequences for growth andevelopment.

In addition, this study provides evidence for the application ofene therapy administered intramuscularly via EP as an alternativer complimentary method to conventional treatment options foroth veterinary and human use. Plasmid-based gene therapy haseveral advantages over conventional drug or protein-based treat-ent methods and viral-based strategies. Conventional treatment

ptions, such as GH can be expensive, requiring repeat administra-ions. Viral-based gene therapies present other problems, such asmmune and inflammatory responses, toxicity, limited large scaleroduction yields, and limitations in the size of the carried ther-peutic genes. The use of non-viral vectors, used in conjunctionith improved EP devices that enable a high level of transfection of

he non-viral vectors supersedes many of these issues. The intra-uscular administration of low doses of plasmid GHRH with EP

esults in production of the GHRH protein by skeletal muscle cellsollowed by its release into the circulation and stimulation of pitu-tary GH production and release. We have previously shown thathe administration of plasmid GHRH results in long-term expres-ion without it being expressed in organs other than the injecteduscle [5].Treatment with plasmid GHRH during pregnancy induces

etabolic changes in the mother that improve nutrient utilizationnd lactation and leads to stronger, more vigorous offspring [3].lasmid GHRH may also act directly on the developing fetus as weave shown that GHRH protein can cross the placenta and impactituitary weight, increase somatotroph and lactotroph numbers,nd increase the post-natal growth rate of the offspring [4,13].njection of sows with plasmid GHRH improves intrauterine weight

ain and as a result piglets are larger at birth and weaning, and reacharket weight earlier compared to controls (P < 0.001) [5]. Mater-

al lactation performance has also been shown to be enhanced byH treatment in late gestation. It enhances mammary tissue growth

14] and we have demonstrated greater milk production in plasmid

(2010) 1905–1910 1909

GHRH-treated cows compared to untreated controls [7]. We havealso demonstrated that treatment of pregnant animals with plas-mid GHRH results in a substantial decrease in offspring morbidityand mortality (P < 0.01), which has always represented a major eco-nomic loss. In a more recent study, a single administration of thecommercially available plasmid GHRH (LifeTideTMSW5) to gestat-ing gilts enhanced body composition parameters of the offspringwhile decreasing their morbidity and mortality over three consecu-tive pregnancies [1]. In all of these studies, no adverse effects relatedto treatment were noted in treated sows or offspring.

It is known that pigs reared in smaller litters survive at a higherrate and reach market weight sooner than those reared in largelitters. Therefore, cross-fostering of piglets is commonly used tostandardize and improve body weight at weaning and avoid thedetrimental consequences of piglets that would otherwise haveto be reared in large litters. However, it has been reported thatcross-fostered pigs gained less weight to 21 days of age (5.9 kg vs.6.1 ± 0.14 kg) and took longer to reach 105 kg (195 days vs. 191 ± 1.4days) compared to pigs that were not cross-fostered (P = 0.02) [15].Cross-fostered pigs also had significantly lower survival rates to21 and 42 days of age than non-cross-fostered pigs even thoughthey had a similar birth vigor score. Earlier research indicated thatsuccess in cross-fostering can be achieved if stronger rather thanweaker pigs from a litter are cross-fostered [16]. Here we showthat the negative effects of cross-fostering can be negated by pre-natal treatment of the sow with plasmid GHRH. Treatment of sowswith plasmid GHRH resulted in stronger piglets that were betterable to withstand the early stages of life, and had reduced mor-tality compared to controls. Moreover, the increased weight atbirth of offspring from GHRH-treated sows was associated with anincreased weight at weaning and at slaughter. Importantly, cross-fostering of offspring from controls to GHRH-treated sows alsoresulted in rates of weight gain that were greater than for piglets leftto suckle on their own control mother. The results from this studyindicate that this effect can be attributed purely to the improvedlactation and milk production of plasmid GHRH-treated sows.

Offspring from GHRH plasmid-treated sows, either non-cross-fostered or when cross-fostered to control sows, were heavier thannon-cross-fostered offspring from control sows. Cross-fosteringof offspring from GHRH plasmid-treated sows to control sows,demonstrates that the lactation capacity of untreated control sowsis not sufficient to sustain the growth advantage that was acquiredin utero. Although the higher birth weights, like the greater weightgain from birth to weaning, likely resulted from an increase in nutri-ent supply to the offspring, in both instances we cannot exclude thepossibility that offspring of plasmid GHRH-treated sows were ableto utilize nutrients for growth with greater efficiency. These off-spring have the further benefit that their pituitary would remain apositive influence on growth and development long after weaninghas occurred.

This study combined with previously published reports in het-erogeneous populations that are more representative of humanpopulations and diseases [7,17–19] demonstrates the potential ofplasmid GHRH gene therapy for human use. To date many attemptshave been made to extend the half-life of GH and GHRH ther-apeutics in order to provide sustained IGF-I levels with feweradministrations [20–22]. As yet, none of these approaches havebeen able to match the long-term effects that can be exerted bya single administration of plasmid with EP. Understanding theGHRH/GH/IGF-I axis and the effects of treatment with plasmidGHRH in a large animal model will facilitate the transition of

this technology and its beneficial effects to humans. The admin-istration of plasmid GHRH by EP in farm animal setting providesevidence of the efficacy of this technique not only to improvemilk and food production but also its potential use for humantherapies.

1 cine 28

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cknowledgements

This study was funded by VGX Pharmaceuticals, Inc., Theoodlands, TX 77382 (formerly ADViSYS) and in part from U.S.

epartment of Agriculture, Agricultural Research Service underooperative Agreement number 58-6250-6001. The contents ofhis publication do not necessarily reflect the views or policiesf the U.S. Department of Agriculture, nor does mention of tradeames, commercial products or organization imply endorsementy the U.S. Government. We would like to thank the Meat Scienceepartment at Texas A&M University, College Station, TX; Drs. Dougern and Patricia Brown for support and invaluable expertise; and

he Rosenbaums at the R&D research farm, TX.Disclosure: ASK is an employee of VGX Pharmaceuticals, Inc.

nd owns stock and/or stock options in this company; AMBB isn employee of VGX Pharmaceuticals, Inc. and owns stock and/ortock options in this company; no competing financial interestsxist for MIF; RDA was an employee of VGX Pharmaceuticals, Inc.nd owns stock and/or stock options in this company; RDA is annventor on patents and patent applications assigned or licensed toGX Pharmaceuticals, Inc.

eferences

[1] Person R, Bodles-Brakhop AM, Pope MA, Brown PA, Khan AS, Draghia-AkliR. Growth hormone-releasing hormone plasmid treatment by electropo-ration decreases offspring mortality over three pregnancies. Mol Ther2008;16:1891–7.

[2] Frutos MG, Cacicedo L, Fernandez C, Vicent D, Velasco B, Zapatero H, et al.Insights into a role of GH secretagogues in reversing the age-related decline inthe GH/IGF-I axis. Am J Physiol Endocrinol Metab 2007;293:E1140–52.

[3] Draghia-Akli R, Fiorotto ML. A new plasmid-mediated approach to supplementsomatotropin production in pigs. J Anim Sci 2004;82:E264–9.

[4] Fiorotto ML, Lopez R, Oliver WT, Khan AS, Draghia-Akli R. Transplacental trans-

fer of a growth hormone-releasing hormone peptide from mother to fetus inthe rat. DNA Cell Biol 2006;25:429–37.

[5] Khan AS, Fiorotto ML, Cummings KK, Pope MA, Brown PA, Draghia-AkliR. Maternal GHRH plasmid administration changes pituitary cell lineageand improves progeny growth of pigs. Am J Physiol Endocrinol Metab2003;285:E224–31.

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(2010) 1905–1910

[6] Shingu H, Hodate K, Kushibiki S, Ueda Y, Touno E, Shinoda M, et al. Hormonaland lactational responses to growth hormone-releasing hormone treatment inlactating Japanese Black cows. J Dairy Sci 2004;87:1684–93.

[7] Brown PA, Bodles-Brakhop AM, Draghia-Akli R. Effects of plasmid growthhormone releasing hormone treatment during heat stress. DNA Cell Biol2008;27:629–35.

[8] Draghia-Akli R, Fiorotto ML, Hill LA, Malone PB, Deaver DR, Schwartz RJ.Myogenic expression of an injectable protease-resistant growth hormone-releasing hormone augments long-term growth in pigs. Nat Biotechnol1999;17:1179–83.

[9] Draghia-Akli R, Li XG, Schwartz RJ. Enhanced growth by ectopic expression ofgrowth hormone releasing hormone using an injectable myogenic vector. NatBiotechnol 1997;15:1285–9.

10] Li X, Eastman EM, Schwartz RJ, Draghia-Akli R. Synthetic muscle promoters:activities exceeding naturally occurring regulatory sequences. Nat Biotechnol1999;17:241–5.

11] Breier BH. Regulation of protein and energy metabolism by the somatotropicaxis. Domest Anim Endocrinol 1999;17:209–18.

12] Kappeler L, De Magalhaes FC, Leneuve P, Xu J, Brunel N, Chatziantoniou C,et al. Early postnatal nutrition determines somatotropic function in mice.Endocrinology 2009;150:314–23.

13] Khan AS, Fiorotto ML, Hill LA, Malone PB, Cummings KK, Parghi D, et al. Non-hereditary enhancement of progeny growth. Endocrinology 2002;143:3561–7.

14] Sejrsen K, Purup S, Vestergaard M, Weber MS, Knight CH. Growth hormone andmammary development. Domest Anim Endocrinol 1999;17:117–29.

15] Stewart TS, Diekman MA. Effect of birth and fraternal litter size and cross-fostering on growth and reproduction in swine. J Anim Sci 1989;67:635–40.

16] Neal SM, Irvin KM. The effects of crossfostering pigs on survival and growth. JAnim Sci 1991;69:41–6.

17] Bodles-Brakhop AM, Brown PA, Pope MA, Draghia-Akli R. Double-blinded,placebo-controlled plasmid GHRH trial for cancer-associated anemia in dogs.Mol Ther 2008;16:862–70.

18] Brown PA, Bodles-Brakhop A, Draghia-Akli R. Plasmid growth hormone releas-ing hormone therapy in healthy and laminitis-afflicted horses-evaluation andpilot study. J Gene Med 2008;10:564–74.

19] Brown PA, Bodles-Brakhop AM, Pope MA, Draghia-Akli R. Gene therapy by elec-troporation for the treatment of chronic renal failure in companion animals.BMC Biotechnol 2009;9:4.

20] Clemmons DR. Long-acting forms of growth hormone-releasing hormone andgrowth hormone: effects in normal volunteers and adults with growth hor-mone deficiency. Horm Res 2007;68(Suppl. 5):178–81.

21] Ionescu M, Frohman LA. Pulsatile secretion of growth hormone (GH) persists

during continuous stimulation by CJC-1295, a long-acting GH-releasing hor-mone analog. J Clin Endocrinol Metab 2006;91:4792–7.

22] Teichman SL, Neale A, Lawrence B, Gagnon C, Castaigne JP, Frohman LA. Pro-longed stimulation of growth hormone (GH) and insulin-like growth factorI secretion by CJC-1295, a long-acting analog of GH-releasing hormone, inhealthy adults. J Clin Endocrinol Metab 2006;91:799–805.