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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 2, 2003 as doi:10.1096/fj.02-0671fje. |
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2
* Department of Molecular and Cellular Biology, Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, Texas, USA;
Advisys, Inc., The Woodlands, Texas, USA; and
USDA-ARS Children Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA
2Correspondence: Research Team Leader, Advisys, Inc., 2700 Research Forest Dr., Suite 180, The Woodlands, TX 77381, USA. E-mail: ruxandradraghia{at}advisys.net
SPECIFIC AIMS
We report here a very efficient in vivo transfer of reporter and therapeutic plasmid DNA into porcine muscle fibers using electric pulses of low field intensity. Dose and time courses were performed. Our studies show that by optimizing the electroporation method, favorable physiological changes such as enhanced weight gain and improved body composition can be obtained at extremely low plasmid doses in a large mammal.
PRINCIPAL FINDINGS
1. Improved delivery method for porcine
Direct intramuscular (i.m.) plasmid DNA injection followed by electroporation is a method for the local and controlled delivery of plasmid DNA into skeletal muscle. It has the advantage that it uses low plasmid quantities (as low as 0.1 mg) rather than the high quantities typically used with passive delivery modalities. It has been shown that the degree of permeabilization of the muscle cells depends on the electric field intensity, length of pulses, shape and type of electrodes, and cell size. The porcine muscle fibers are quite large and consequently more suitable for electropermeabilization than rodent muscle. In this report, we show that a single injection of an optimum dosage of plasmid, followed by electroporation with i.m. applicators, in a large mammal is sufficient to produce therapeutic plasma hormone levels with biologically significant effects on the growth and development of the animal. The age of the animal was also considered. These results could be extrapolated to other gene therapy applications.
We evaluated the type of electrode needed to achieve a physiologically significant level of a secreted reporter protein in 4–5 kg pigs using either external caliper electrodes or injectable electrodes. Reporter vectors that express secreted embryonic alkaline phosphatase (SEAP) were used in these studies at a dose of 2 mg pSP-SEAP/animal. Six-needle array electrodes were compared with standard caliper electrodes. Conditions of 6 pulses, 200 V/cm, 60 ms/pulse previously tested as being the most effective in pigs were applied in all tests. SEAP values were measured on days 0, 3, and 7 postinjection. Seven days postinjection, SEAP levels were 9.33 ± 2.26 ng/(mL·kg) in plasmid-injected and caliper-electroporated animals compared with 0.02 ± 0.005 ng/(mL·kg) in vehicle-injected animals (Fig. 1
). Using the 6-needle array, we obtained a 19-fold increase in SEAP values compared with calipers (177.41±18.44 ng/(mL·kg), P<0.0035). When using the same number of pulses but lower voltage (100 V/cm) and the 6-needle electrodes, the average SEAP to 144.64 ± 11.82 ng/(mL·kg) after 7 days, P < 0.006.
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When longissimus dorsi and semitendinosus muscles were injected using similar conditions, expression from the semitendinosus muscle was slightly higher. Skin and muscle from the injected pigs were collected at the end of the experiment (at 53 days postinjection) and histologically analyzed. Picro-sirius red staining was used to estimate scar tissue (collagen content). At 100–200 V/cm, no skin or muscle damage was seen when the needle-type electrodes were used.
2. Increased efficiency using needle-type electroporation delivery for therapeutic proteins
GHRH stimulates the production and release of GH from the anterior pituitary, which in turn stimulates production of IGF-I from the liver and other target organs. In previous studies from our laboratory, young pigs weighing 4–5 kg were injected with 10 mg myogenic vector expressing a mutated form of GHRH stable to proteases (pSP-HV-GHRH) and electroporated using a caliper electrode.
In our current study, we wanted to determine the best age at which to inject young pigs. Groups of piglets were injected with 2 mg pSP-HV-GHRH using the 6-needle array electrodes at different ages: birth, 7, 14, and 21 days of age. Each animal received one injection. The group injected at 14 days of age demonstrated the greatest weight gain (statistically significant different from PBS controls at every time point (final weights: 25.8±1.5 kg vs. 19.7±0.03 kg, P<0.013)). The next best group was injected at 7 days of age and weighed 21.9 ± 1.5 kg at 53 days of age, P < 0.02.
In a parallel study we explored the possibility of reducing the plasmid quantity needed to achieve improved growth and changes in the metabolic and hormonal profile of pigs. Groups of four piglets were injected at 10 days of age with pSP-HV-GHRH (3 mg, 1 mg, 0.1 mg) and electroporated using a 6-needle array electrode (Fig. 2
). The group injected with 0.1 mg of plasmid had the greatest weight gain, with statistically significant differences to controls at 53 days of age (22.4±0.8 kg vs. 19.7±0.03 kg, P<0.012). To confirm these results, groups of 10 pigs were injected with 0.1 or 1 mg of the pSP-HV-GHRH construct. A group of 20 pigs was used as control. The animals were followed for 100 days. The group injected with 0.1 mg was on average 9% heavier than controls, 81.88 ± 2.72 kg vs. 75.06 ± 1.64 kg, P < 0.023, whereas the group injected with 1 mg plasmid was 7% heavier than controls, P < 0.034.
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One animal in the group injected at 21 days and one animal injected with the highest plasmid dose (3 mg) developed neutralizing antibodies against the mutated HV-GHRH and showed reduced rates of weight gain (at 53 days postinjection 15.6 kg and 15.95 kg, respectively, vs. > 21 kg for the paired animal in the same treatment group). No other group developed neutralizing antibodies. Thus, the minimal plasmid dose (0.1 mg) with injection at optimum age using the 6-needle electrode resulted in the best growth performances. Note that in previous studies from our laboratory, we used 100-fold more, i.e., 10 mg pSP-HV-GHRH with the caliper electrodes to produce similar changes.
An indication of increased systemic levels of GHRH and GH is an increase in serum IGF-I concentration. We observed that the level of serum IGF-I started to rise 3 days postinjection in pigs that received the 0.1 and 1 mg doses of pSP-HV-GHRH. By 35 days after injection (age of animals: 45 days), serum IGF-I concentrations were 
10-fold higher in pigs injected with 0.1 mg and 7-fold higher in pigs injected with 1 mg plasmid compared with controls (P<0.007 and P<0.04, respectively). Inhibition of the GHRH axis by neutralizing antibodies was associated with lower IGF-I levels.
In pSP-HV-GHRH-injected pigs, under optimum conditions serum urea decreased (8.36±1.33 to 9.67±1.27 mg/mL in pSP-HV-GHRH-injected pigs vs. 11.14±1.9 mg/mL in controls, respectively (P<0.05)), indicating decreased amino acid catabolism. Serum glucose levels were similar between the plasmid pSP-HV-GHRH-injected pigs and controls; insulin levels were normal and within the control range. The fact that these animals have a normal carbohydrate metabolism is important, as most livestock and/or patients under recombinant GH therapy develop impaired glucose metabolism and insulin resistance.
3. Body composition is changed in pigs that received GHRH treatment
Necropsy data showed a proportional increase of all internal organs in GHRH-injected animals (heart, lung, liver, spleen, brain, adrenals, stomach, kidney, pancreas, intestine). Body composition studies were performed by dual-energy X-ray absorptiometry (total body fat, nonbone lean tissue mass, and bone mineral content) and 40K analysis (lean body mass). The final body composition was different: animals injected with pSP-HV-GHRH at different ages gained proportionally less fat than controls and were leaner at the end of the study (4.34±0.04 g of fat gained/kg of fat free mass gained per day for injection at birth, 4.4±0.04 g for injection at 7 days vs. controls 5.63±0.34 g, P<0.05). Bone mineral density was higher in animals injected 14 days after birth (0.363±0.005 g/cm2 vs. 0.329±0.003 g/cm2 in controls, P<0.004), consistent with the overall greater efficacy of the treatment at this age. All other treated groups had similar trends in their body composition, without statistical significance due to interanimal variability.
Treated pigs did not experience any side effects from the therapy, had normal biochemical profiles, and manifested no associated pathology or organomegaly. From a functional standpoint, the increases in IGF-I levels, enhancement in growth, and changes in body composition were remarkable. The effects of the stimulation of GHRH secretion on bone metabolism, as manifested by a 10% increase in bone mineral density, is also significant. These results indicate that ectopic expression of myogenic HV-GHRH vectors has the potential to replace classical GH therapy regimens and may stimulate the GH axis in a more physiologically appropriate manner. The HV-GHRH molecule, which displays a high degree of stability and GH secretory activity in pigs, may also be useful in human clinical medicine. However, it is important to establish a minimal plasmid dose on a pertinent model in order to avoid the unwanted pathology associated with antibody development.
CONCLUSION
In this report, we have shown that as little as 0.1 mg plasmid delivered under the proper electroporation conditions could have an important biological impact, enhancing animal growth and modifying body composition and metabolism in the injected animals. Larger plasmid quantities are not necessary and may be detrimental through development of neutralizing antibodies and strong negative feedback onto the GHRH-GH-IGF-I axis. We predict that these improvements to the electroporation methodology in a large animal model will constitute a step forward to human gene delivery, eliminating the need for viral vectors or liposomes that often act as antigens when they are used as gene therapy vectors.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0671fje; to cite this article, use FASEB J. (January 2, 2003) 10.1096/fj.02-0671fje 
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