Recombinant growth hormone (GH) was one of the first proteins to be synthesized via DNA recombinant techniques in the late seventies. It was also one of the first proteins to be used in studies of animal models for gene therapy, already in the eighties. This was due to the real therapeutic need for GH, combined with the fact that its detection by well-known immunoassay methods is facile and sensitive. Moreover, evident phenotypic effects can be observed and measured in several animal models (e.g., dwarf mice), some of which have GH deficiencies that closely resemble their human counterparts.
GH gene therapy has the potential advantage of circumventing laborious and expensive purification processes, quality control procedures and the repetitive injections that are required in the conventional treatment. The ideal situation would, of course, be to introduce the deficient protein into the circulation via a mechanism that resembles the natural process. These treatments have not yet reached the clinical stage for humans, the major challenge being to achieve a sustainable and regulated
Primary keratinocytes are one of the most attractive vehicles for gene transfer and gene therapy. They are among the most accessible cells in the body and can be serially propagated in culture; the procedure for their transplantation is already well established, e.g., for burn patients, and the therapy can be reversed by excision of the genetically modified tissue. Cutaneous gene therapy has already been demonstrated to be a powerful tool for the successful treatment of severe skin disorders such as epidermolysis bullosa 1. Keratinocytes can also act as cellular bioreactors, secreting GH or other proteins systemically. When primary human keratinocytes were retrovirally transduced with the human (hGH) or mouse (mGH) growth hormone genes in our laboratory, they exhibited high and stable
Among the various
The GH gene therapy approach based on
A much better option would appear to be the direct administration of naked plasmid DNA, a methodology that has been successfully adopted by several authors in the last decade. Analysis of growth parameters such as body weight, organ weight (quadriceps muscles, liver, kidneys, heart and spleen) and total (nose-to-tail) length of the animals have been used to study the endocrine and local (autocrine/paracrine) effects of hGH in greater depth after intramuscular DNA administration in immunodeficient dwarf mice (lit/scid) 6. Although the majority of the strategies described for GH gene therapy are still limited by the absence of an appropriate mechanism for regulating
In this chapter, we review the results reported in five studies from our laboratory, primarily involving
2. Ex vivo strategies
In 1995, the Canadian group mentioned above reported that the growth defect of dwarf mice (Snell dwarf) could be partially corrected by implanting microencapsulated allogeneic myoblasts engineered to secrete mouse growth hormone. The encapsulation of these mouse myoblasts into GH-deficient Snell dwarf mice provided a completely homologous system 4. The plasmid pKL-mGH, encoding mGH cDNA under the regulation of human β-actin promoter and also containing the neomycin resistance gene, was used to transfect the mouse myoblast cell line C2C12. G418 resistant clones were selected and screened for the level of mGH in the culture medium. Clone Myo-45, which secreted 147 ng of mGH/106 cells/day, was selected for encapsulation. Microcapsules were implanted via a 22G catheter into the peritoneal cavity and, by the end of the 3rd week, the body weight of the dwarf mice had increased about 1.6-fold and the increase in body length had doubled compared to the control group. There were also significant increases in the levels of non-esterified free fatty acids (a measure of the lipolytic effect of the capsule-delivered mGH), while peripheral organ weights and tibial growth plate thickness were also significantly greater. The authors hypothesized that most of the capsule-derived mGH was sequestered in the liver through the hepatic GH receptors, inducing the secretion of hepatic insulin-like growth factor I (IGF-I), which mediates most of the GH-dependent systemic metabolic effects. After 5 weeks, however, a lack of further growth in weight or length was evident in all of the mice. According to the authors, this was not due to the absence of mGH transgene expression by the encapsulated cells, but rather to the non-responsiveness of the mice to the hormone at this age (13-15 weeks). In fact, a second implantation of freshly-prepared capsules on day 42 did not result in any further growth enhancement. Moreover, encapsulated cells retrieved at the end of the experiment (178 days) continued to secrete
Fibroblasts are also a potentially interesting cell type for
The same Chinese research group subsequently used primary porcine fetal fibroblasts, transduced with the same pGH-carrying vector described above, to enhance the weight gain of growth-retarded Tao-Yuan Swine, a local breed in Taiwan that is slow growing and fat, but palatable8. Immortalized fibroblasts were avoided because of their tumorigenic potential, even though they have been used quite successfully in several different studies in mice. The transduced primary cells were encapsulated with the same type of alginate-poly-L-lysine-alginate membranes used previously for mice myoblasts 4 and then implanted into the peritoneal cavity of the swine, resulting in a significant increase in weight gain already on day 16 post-implantation, even though no increase in serum pGH could be detected. The use of immunoprotective microcapsules thus constitutes a simple method of delivering recombinant genes
Our research group has focused its
Conventional epidermal sheets of these mGH-secreting keratinocytes, prepared by us using the classical technique of Barrandon et al.2, showed a drop in secretion rates of >80% simply due to detachment of the epithelium from its substratum. Replacement of this conventional grafting methodology by organotypic raft cultures14 completely overcame this problem. Employing a similar
Several hypotheses can be raised as the possible cause(s) of this immediate suppression or blocking of exogenous GH in the circulation of lit/scid mice after the grafting. These include:
a limited mGH circulatory half-life in lit/scid;
a rapid clearance from the bloodstream due to a specific binding or selective transfer;
impediments due to poor vascularization, to a fast inflammatory process or to an unidentified specific barrier;
the occurrence of extremely efficient apoptotic events, transgene inactivation or promoter failure;
partial immune reactivity spontaneously developed by the immunodeficient mice, leading to some production of B and T cells (i.e., “leakiness”).
Although several of these hypothetical mechanisms could be rationally or even experimentally excluded, so far we have been unable to positively prove the existence of any one of them. This led us think that comparative tests carried out by injecting mGH-expressing naked DNA, enhanced by
In vivo approach
Adenovirus administration is an effective way of delivering therapeutic genes since they can be produced and purified in a concentrated form, which facilitates
Concerning GH deficiency and dwarf animals, an adenoviral vector containing rat GH (rGH) cDNA was used in 1996 by the Houston group mentioned above to induce constitutive GH expression in hepatocytes of GH-deficient lit/lit mice 5. When the recombinant adenoviral vector, controlled by the human elongation factor 1-α (EF1α) promoter, was administered via the tail vein of this animal at a dose of 108 pfu (plaque-forming units), high levels of GH were detected in the serum for at least 7 weeks. This viral dose led to an unbelievably high peak of the serum GH level of 1.9 µg/ml, which then decreased to ~125 ng/ml during the next 2 weeks, remaining at that level for the duration of the experiment (7 weeks). A viral dose of 109 pfu induced an even higher serum GH level of ~35 µg/ml, which then decreased to ~2.5 µg/ml over the next two weeks and stabilized at 1 µg/ml for the duration of the study. The authors attributed the decrease in GH expression in part to the ability of the host’s immune system to recognize and subsequently eliminate the virally transduced cells. Little dwarf mice treated with 108 pfu of rGH adenovirus showed an increase in circulating IGF-I levels (from 61 to 238 ng/ml in 3 weeks). Although the serum GH levels increased dramatically, the serum IGF-I levels did not follow the same pattern. This may be due to the presence of reduced IGFBP-3 in GH-deficient humans and animals. A rapid weight gain, resulting in a body weight comparable to that of normal age-matched lit/+ animals, was achieved by 5 weeks of treatment. Total body length was also indistinguishable from that of lit/+ mice within 7 weeks of treatment and body composition was normal, with a reduction in the percentage of fat and increase in the water and protein contents. The animals treated with rGH adenoviruses exhibited slight, but significant, increases in liver and kidney sizes and a tendency to increase fasting blood glucose and insulin levels, which are known effects in response to prolonged exposure to high levels of GH. It was suggested, however, that the induced hepatic and renal hyperplasia warranted further investigation. According to the authors, the little mice dwarf phenotype could be corrected, with minimal side-effects, by constitutive GH expression achieved through
In a subsequent study, carried out in 1999 by groups at the National Institute of Health (NIH, Bethesda, MD), an adenovirus encoding mGH cDNA was injected into the quadriceps muscle or submandibular ducts of mGH-deficient Snell dwarf mice to obtain a homologous system and thus avoid possible side effects resulting from species differences 16. When the adenoviral vector was used
More recently, in 2008, a new generation of double-stranded adeno-associated viral vectors (dsAAV) encoding mGH cDNA driven by a universal promoter (CMV) were used by a group coordinated by Johns Hopkins University School of Medicine to prepare viral particles that were injected into GHRHKO mice, a model of isolated GH deficiency due to generalized ablation (knock-out, KO) of the GHRH gene 17
18. These genetically modified dsAAV can infect dividing and non-dividing cells
In an initial study, GHRHKO mice were injected intraperitoneally with either a single dose (low dose) or two doses (high dose) of 1 x 1011 viral particles at the 10th and 11th days of age and were followed up to the 6th or 24th week of life17. Body weight and length of both viral-treated groups became normal at 6 months of age and normal femoral and tibial lengths, body composition and weight of organs (liver, spleen, heart and kidney) were also obtained. At week 6, serum GH levels were higher in mice receiving both virus doses compared with controls, while they were normal at week 24. This is consistent with the results of previous studies showing that long-term expression by this type of viral vector is limited to the liver and skeletal and cardiac muscle. This was confirmed by the detection of GH mRNA in these same tissues of the GHRHKO mice. Nevertheless, serum IGF levels were significantly higher in both virus-treated groups compared to the control group at week 24, showing that the expressed GH is still functional in GH-deficient and immunocompetent mice and that no resistance to its effect was developed over time, since a species-specific GH cDNA was used. The use of a universal promoter is obviously a limitation for any clinical application of this approach because of the well-known long-term risks associated with excessive unregulated GH secretion. The authors concluded that, while the applicability of these findings is still very far from any possible clinical trial in humans, these new AAV vectors offer a good starting point for the development of novel regulated viral gene delivery systems for GH administration. Such systems could be based on the use of inducible promoters that can be regulated at will or of tissue-specific promoters, providing good systemic delivery together with limited local expression.
In a subsequent study, the same group utilized a dsAAV expressing mGH cDNA under the control of a muscle creatine kinase regulatory cassette in order to ensure adequate systemic delivery in conjunction with muscle-specific expression 18. A low-dose (0.5 x 1011 pfu) and a high-dose (1 x 1011 pfu) of virus were injected into the right quadriceps muscle of GHRHKO mice at the age of day 10. Virus-injected GHD mice showed a significant (P<0.05) increase in body length and weight, however without becoming fully normal, and a significant (P<0.05) reduction in visceral fat at week 6 of age. Quantitative RT-PCR showed that GH mRNA expression in the quadriceps muscle of animals treated with the high-dose of virus was significantly higher than in the gastrocnemius and cardiac muscles or the kidney and liver of the same mice. At 6 weeks of age, serum GH and IGF-I levels in both treated groups were not significantly higher than those in the control mice. This study showed that, although the strategy of vector-mediated GH therapy is still not applicable at the clinical stage, systemic GH delivery to GHD animals is possible via a single injection of viral particles derived from dsAAV using an approach that limits GH expression to skeletal muscle. In fact, it is known that widespread gene expression can result in toxicity 19.
An alternative system for delivering genes
A research group from a company in Texas has been a pioneer in GH naked DNA administration. They designed a muscle-specific gene medicine, composed of a hGH expression plasmid containing the chicken skeletal α-actin promoter complexed with a protective, interactive, non-condensing (PINCTM) delivery system, to be administered intramuscularly in hypophysectomized rats 21. This polymeric PINC gene delivery system consists of polyvinilpyrrolidone (PVP), which protects plasmids from extracellular nuclease degradation and facilitates the uptake of the plasmid by muscle cells. To test the
Another strategy used for
As already mentioned, our group started working with the administration of naked DNA and has described a strategy for
The DNA-injected quadriceps muscles presented a 48.1 % weight increase versus the saline-injected control (P<0.001), whereas the weight increase of non-injected quadriceps of treated animals was 31.0 % (P<0.005). DNA-injected quadriceps showed a 45.5 % increase compared to the non-injected quadriceps of the same treated mice (P<0.001). These data point to local (autocrine and/or paracrine) and systemic (endocrine) effects as a result of hGH DNA injection.
Another long-term experiment carried out with immunocompetent dwarf little mice (lit/lit), fail to produce the same degree of statistical significance. The body weight increase was somewhat lower (~21%) and the increase in body weight ceased after approximately one month. However, up to the 32nd day, the slope of the growth curve was 0.048 ± 0.038 g/mouse/day and the difference relative to the control curve (slope = 0.038 ± 0.016) was statistically significant (P<0.01).
This treatment with a single injection of 50 µg of pUC-UBI-hGH in lit/scid mice was also compared to regular injections of recombinant hGH (5 µg/twice a day/animal) during 30 days. The two different strategies provided a similar response in terms of weight variation, when comparing the body weight gain of 0.094 g/mouse/day for the naked DNA system and of 0.095 g/mouse/day for recombinant protein injected daily, while the slope for the control (saline injection followed by electroporation) was 0.22 g/mouse/day (manuscript in preparation)8.
We have thus shown that intramuscular naked DNA hGH administration can be effective for promoting the growth of dwarf “little” mice, a model of human isolated growth hormone deficiency (IGHD). More must still be done, however, especially in terms of achieving long-lasting, sustained serum levels of the therapeutic protein.
As far as we know, the treatment of systemic protein deficiencies via gene therapy has not yet reached the stage of successful clinical applications, even for other diseases like hemophilia 24. As shown here, however, total or partial correction of growth defects has been achieved in several animal models for GH gene therapy by the use of a variety of different
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