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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 2, 2003 as doi:10.1096/fj.03-0018fje. |
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Biotechnology Department, IPEN-CNEN, University of São Paulo, São Paulo, SP, Brazil
2Correspondence: Biotechnology Department, IPEN-CNEN, University of São Paulo, Avenida Professor Lineu Prestes, 2242, Cidade Universitária, 05508-900, São Paulo, SP, Brazil. E-mail: pbartoli{at}ipen.br
SPECIFIC AIMS
In this study we aimed to demonstrate that grafts of primary human keratinocytes, genetically modified to secrete hGH, can lead to sustained levels of circulating hGH in immunodeficient dwarf mice (lit/scid) resulting in phenotypic alterations, e.g., increases in their weight. As such, the grafted mice could serve as a model for hGH gene therapy.
PRINCIPAL FINDINGS
1. In vitro secretion of hGH by genetically modified primary human keratinocytes
The retroviral LXSN vector containing hGH cDNA under the control of the LTR promoter was used to produce a replication-defective retrovirus generated by transfecting GP+E86 ecotropic packaging cells with hGH vector (pLhGHSN) DNA via the CaCl2 method and subsequently infecting amphotropic GP+env+AM12 packaging cells. Eighteen colonies of the infected cells were grown separately and hGH virus-producing clones #1 and 6, designated AM12pLhGHSN-1 and -6, were selected on the basis of the viral titer (1.0x105 and 2.3x106 CFU/mL) and hGH concentration in the culture medium (216 and 53 ng/mL, respectively). Primary human keratinocytes isolated from newborn foreskin were infected with the hGH virus by incubation with the cloned producer cells. While clone-1 apparently leads to keratinocytes producing higher hGH secretion than clone-6 (4.04±0.22 vs. 2.75±0.77 µg hGH/106 cells/day), the difference in hGH secretion was not found to be significant according to the Student’s t test (P=0.05). Conditioned media derived from two different transductions of keratinocytes using clone-1 were subjected to Western blot analysis. The main band of the conditioned media corresponded to that of the International Standard of recombinant hGH; a slower migrating form present in both conditioned media was probably due to a dimer. Geneticin (G418) was used to specifically select transduced keratinocytes. The results show that the highest hGH expression was obtained via selection with 0.6 mg G418/mL. A set of different infections of keratinocytes (n=6) in conjunction with G418 selection at this concentration confirmed the high reproducibility of our transduction and selection conditions and provided an average in vitro secretion of 6.62 ± 0.57 µg hGH/106 cells/day (RSD=8.7%).
2. Levels of hGH in the circulation of lit/scid mice grafted with hGH-secreting keratinocytes
Epithelial sheets prepared from hGH-secreting, primary human keratinocytes were grafted onto the midback of immunodeficient dwarf mice (lit/scid), in which a single transversal fissure was made for introduction of the graft, prepared by wrapping a 78 cm2 epithelial sheet around a 2 x 2 cm paraffinized gauze to support the transplant. A group of 12 animals was grafted with the transduced keratinocytes and hGH circulatory levels were determined over a 12 day period. Since we had found that such keratinocytes consistently secreted hGH at high levels in vitro (
38 µg hGH/78 cm2 undetached epithelium/day) and their secretion declined relatively quickly to low levels after grafting, we determined plasma levels of the hormone not only at intervals of 2–3 days, but also shortly after grafting (at 4 h). This point indeed provided the highest hGH concentration (1.5 ng/mL), after which the hGH secretion declined to a relatively constant level (on day 3) of 0.2–0.3 ng/mL (average 0.23±0.078 ng/mL, for n=5), which was maintained until day 12 without showing a tendency to decrease. Even though this level of hGH in the circulation was quite low, the difference with the control group (12 animals grafted with nontransduced primary human keratinocytes) was highly significant (P<0.001). It is notable that the sensitivity of the hGH assay (DELFIA) used is on the order of 0.01 ng/mL, i.e., the minimal detectable concentration is 20-fold lower than the lowest hGH concentration observed in sera from the animals grafted with hGH-secreting keratinocytes.
3. Weight increase of lit/scid mice grafted with hGH-secreting keratinocytes
A second set of lit/scid mice treated similarly was used to determine the effect of grafting transduced, hGH-secreting keratinocytes on body weight. As shown in Fig. 1
, the mice grafted with hGH-secreting keratinocytes showed an increase in body weight (0.060 g/animal/day) significantly higher (P<0.01) than that of weight-matched controls (0.023 g/animal/day). The increases in body weight were preceded in both groups by a considerable decrease in weight on day 2. In a previous study in which mice received three subcutaneous injections per day of hGH-conditioned culture medium (equivalent to 0.16 µg hGH/day), a similar decrease in weight was observed, also on the second day after treatment.
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4. Comparison between weight increase and hGH circulatory levels in a different mouse model
As established in earlier studies, two daily intraperitoneal injections of 5 µg hGH to "little" mice lead to an average weight increase of 0.195 ± 0.010 g/animal/day (n=5, 10-day assays); this corresponds to an increase from the initial weight of 15%. Measurements of the hGH levels in the circulation of mice so treated indicated an average integrated concentration of 1.9 ng hGH/mL.
CONCLUSIONS
The present study has led to the highest in vitro secretion of hGH so far reported for genetically modified, primary human keratinocytes (7 µg/106 cells/day or 38 µg/graft/day). As far as we know, in vitro hGH secretion levels of >1 µg/106 cells/day had not been reported for such cells. A secretion of up to 5.3 µg/106 cells/day, however, has been described for a carcinoma-derived immortalized keratinocyte cell line (SCC-13). Immunodeficient dwarf mice (lit/scid) used in this study were for the first time used for grafting of hGH-secreting human epithelium, showing a steady level of hGH in the circulation of 0.2–0.3 ng/mL for the entire 12 day experiment. This is the longest period so far observed for in vivo secretion of hGH by primary human keratinocytes. Circulatory levels of 0.6 ng hGH/mL and 1 ng hGH/mL have been reported by others using transfected primary human keratinocytes or the previously mentioned immortalized cell line, respectively. In no case was secretion of hGH by the grafts accompanied by an increase in body weight of the experimental animals. The rate of in vivo secretion by the grafts in our studies was estimated to be 1.91 ng hGH/h. This is only 0.4% of an in vitro secretion rate, corrected for a possible 70% loss due to detachment of the epithelium. Even though the efficiency with which epidermally secreted proteins are taken up by the circulation has been estimated by others to be as low as 1–5%, we have no explanation for such low production or for the decline in secretion that occurred in the first 3 days, starting from circulatory levels of 1.5 ng hGH/mL. Despite the relatively low levels of hGH in their circulation (0.2–0.3 ng/mL), the dwarf mice (lit/scid) exhibited a significant weight increase during the 12 day test period. This is consistent with the hGH-induced weight gain previously obtained in "little" mice (lit/lit), 0.195 g/mouse/day, which produced hGH levels in the circulation of 1.9 ng/mL. We found the maximum weight gain shown by heterozygous littermates (lit/+) to be 0.80 g/mouse/day, with natural circulatory GH levels on the order of 6 ng/mL. It is also of interest that plotting the daily weight increases obtained for lit/scid, lit/lit, and lit/+ mice against the respective circulatory GH levels leads to a significant correlation (r=0.9980; P<0.05). These results suggest it is possible to develop a superior animal model for gene therapy based on a more careful choice and control of various factors, including utilization of pseudotyped vectors for obtaining higher retroviral titers and of epidermal sheets particularly enriched with keratinocyte stem cells whose active fraction should be routinely examined via clonal analysis, as well as a more efficient grafting methodology based, for example, on organotypic raft cultures. The mutant mouse (lit/scid) should be highly useful in preclinical studies of such gene therapy.
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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.03-0018fje; doi: 10.1096/fj.03-0018fje 
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