Ultrasensitive in vivo bioassay detects bioactive human growth hormone in transduced primary human keratinocytes

Abstract

An improved in vivo body weight gain bioassay for the potency determination of human growth hormone (hGH) has been set up in "little" mice (lit/lit), a mutant derived from the C57BL/6J strain. This improved assay now has a detection limit of the order of 0.05 micrograms/mouse/day, which corresponds to a sensitivity about 20-fold higher than that of the most sensitive in vivo assay reported up to now: the tibia test in hypophysectomized rats or mice. This sensitivity was achieved mainly by introduction of a careful pre-assay selection and of a three injections per day schedule. The utilization of these conditions in a 2x2 factorial assay design allowed the potency determination of recombinant DNA-derived hGH (rec-hGH) in bacterial extracts with acceptable accuracy and precision, together with the greatest economy of material, only 0.24 mg of unknown and standard hormone preparation being sufficient for an entire 10-animal assay. This contrasts to a minimum of 2.7 mg that are necessary for an economical assay in hypophysectomized rats. The same assay procedure was also used to demonstrate the in vivo bioactivity of hGH secreted into a culture medium from transduced human primary keratinocytes. The growth curve constructed with n = 8 little mice presented a highly significant correlation (r = 0.939, p < 0.001) and a slope = 0.016 g/mouse/day. It was thus possible to prove, for the first time, the in vivo bioactivity of rec-hGH secreted by transplantable human epidermal cells, utilized as an experimental model for somatic gene therapy.

Increases in weight of growth hormone‐deficient and immunodeficient (lit/scid) dwarf mice after grafting of hGH‐ secreting, primary human keratinocytes

“…The increases in body weight were preceded in both groups by a considerable decrease in weight on day 2. It is of interest that 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 (18). In that study, however, the slope of the weight variation curve for the test group was about fourfold lower than that in the present experiment, whereas the slope of the control group was essentially zero.…”

“…As established in previous studies (18,26), two daily 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 in the initial weight of ~15%. Figure 4 shows the hGH levels over a 24 h period in the circulation of "little" mice receiving two injections of 5 µg hGH.…”

“…We have previously shown that GH-deficient dwarf "little" mice (lit/lit) are very sensitive to extremely low concentrations of hGH in their circulation (18). In the present study we have used, for the first time, immunodeficient dwarf mice (lit/scid) to determine whether grafting of transduced, hGH-secreting primary human keratinocytes can lead to prolonged in vivo secretion of the hormone and subsequent phenotypic alterations.…”

Different ex Vivo and Direct in Vivo DNA Administration Strategies for Growth Hormone Gene Therapy in Dwarf Animals

“…Grafted mice also showed an increase in body weight (0.060 g/animal/day), which was significantly higher (P<0.01) than that of the controls (0.023 g/animal/day). This was the first time that continuous in vivo secretion of the hormone and subsequent phenotypic alterations due to the grafting of transduced, hGH-secreting primary human keratinocytes had been demonstrated in lit/scid mice, an animal model known to be very sensitive to low concentrations of hGH 13 . 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.…”

Correção fenotípica do nanismo avaliada por diferentes parâmetros de crescimento após administração de DNA plasmidial em modelo animal de deficiência isolada do hormônio do crescimento

“…• Ao final do experimento de longa duração (6 • Os órgãos do grupo tratado com DNA plasmidial que apresentaram as maiores • Os níveis de mIGF-I do grupo tratado com DNA plasmidial foram significativamente superiores em relação ao grupo salina, após 1 (P<0,01), 2 (P<0,001), 3 (P<0,001), 5 (P<0,01) e 6 (P<0,001) meses de tratamento.…”