Phenotypic correction of dwarfism in immunodeficient dwarf mice following intramuscular hGH plasmid administration analyzed via

different growth parametersHiguti, E.1; Cecchi, C.R.1; Oliveira N. A. J.2; Lima, E.R.1; Bartolini P.1; Peroni, C.N.1 1 Biotechnology Department, IPEN-CNEN, São Paulo, SP, Brazil

OBJECTIVESOBJECTIVESThe aim of this study is to obtain the maximum phenotypic correction after hGH-DNA administration followed by electroporation in immunodeficient dwarf mice (lit/scid).

MATERIALS AND METHODSMATERIALS AND METHODS

RESULTS AND DISCUSSIONRESULTS AND DISCUSSION

The first hGH-DNA administration reached a 35% weight increase, against 11% for the control group up to 104 days, when the body weight of treated mice had reached a plateau. Then a second administration was carried out obtaining a further 10% increase, while the third one at day 160 provided only 5% increase. The final weight gain stabilized at ~46 % after 188 days: catch-up growth towards normalization was 22.6 %. Non-treated scid mice with the same age were utilized as a normal growth control (Figure 1).

Animals: The mutant strains of CB17-Ghrhr lit/+ Prkdc scid/Bm (lit/scid) and scid mice were obtained from Dr. W. Beamer (The Jackson Laboratory, Bar Harbor, ME, USA) and bred in our animal house.

Plasmid: The pUC-UBI-hGH plasmid, containing the genomic hGH sequence (hGH - gDNA) of 2152 bp, under the control of the Ubiquitin C promoter, was amplified using E. coli DH5α and purified by Nucleobond Xtra-Midi kit from Machnerey-Nagel (Düren-Germany).

Bioassay procedures: Lit/scid mice were submitted to 50 µg hGH-gDNA or saline administration, followed by electrotransfer, using previously optimized parameters (8 pulses of 90 V/cm and 20 ms, separated by 0.5 s intervals). This procedure was repeated on days 104 and 160 of a 6-month experiment. The body weight variation of the animals was determined during the entire assay. At the end, the blood was withdrawn for hGH, mouse insulin-like growth factor I (mIGF-I) and glucose determinations. Nose-to-tail, tail and femurs lenghts were also measured.

mIGF-I: Serum mIGF-I levels were measured using the Quantikine mouse-rat IGF-I kit (R&D Systems, MN, USA).

Radioimmunoassay: hGH levels were measured by specific radioimunoassay, carried out using NIDDK reagents (Dr A. F. Parlow, National Hormone and Pituitary Program, Torrance, CA, USA), with a sensitivity of 0.1 ng/ml.

Statistical analysis: Quantitative variables, shown as the mean ± SD, were analyzed by the unpaired Student’s t test or two-way ANOVA and Bonferroni’s post hoc tests (Prism version 5.0, GraphPad Software Inc., La Jolla, CA, USA). A P value < 0.05 was considered to be statistically significant.

BACKGROUNDBACKGROUNDThe growth hormone deficiency (GHD) is the most common pituitary hormone deficiency. It is a chronic condition that requires growth hormone (GH) reposition for an extended period of time. Our group investigates an alternative strategy of gene therapy for this treatment based on the in vivo administration of a plasmid containing the human growth hormone (hGH) gene.

Supported by:

CONCLUSIONCONCLUSION

2 Lillehei Heart Institute, Department of Medicine, University of Minnesota, Minneapolis, MN, USA

cnperoni@ipen.br

Analyzing growth parameters more directly related to linear growth, a 16.4 % increase (catch-up = 26.6 %) for nose-to-tail, a 16.4 % increase (catch-up = 24.5 %) for tail and a 24.3 % increase (catch-up = 55.5 %) for femur length were obtained. An example of the dissected right femurs can be seen in Figure 2.

Figure 2: Dissected right femurs of lit/sci mice after pUC-UBI-hGH (p) or saline (s) administrations, followed by electroporation, and of non-treated lit/scid (l/c) and scid (s) mice.

The hGH determination was performed during the entire assay and the levels of the group that received hGH-DNA were higher than of the saline group (Figure 3), on 6 month the difference between DNA-treated and saline was significantly (P<0.001).

Figure 3: hGH levels in the serum of lit/scid mice after pUC-UBI-hGH ( ) or saline ( ) administrations, followed by electroporation.

The release of mIGF-I, the main physiological mediator of GH on growth, is associated to circulating GH levels. As expected, during the whole assay, the mIGF-I levels of the hGH-DNA group were significantly higher than those of the saline group, but still lower than non-dwarf scid mice.

Table 1: Plasmatic levels of mIGF-I in lit/scid mice after pUC-UBI-hGH or saline administrations, followed by electroporation , and in non-treated lit/scid and scid mice.

These results are very promising and pave the way to more effective preclinical assays for the treatment of GHD, via plasmid DNA muscular administration.

Supra-physiological expression of GH can be diabetogenic, once the GH can inhibit insulin, therefore plasma glucose determination was performed to verify the safety of this treatment. Glucose levels were not significantly different between DNA-hGH and saline groups (data no shown).

Animal groupTime

(month)mIGF-I

(ng/mL) ± SDStatistical

significance*non-treated lit/scid initial 27.7 ± 9.3

saline

1 21.8 ± 7.5

2 24. 7 ± 1.5

3 27.0 ± 5.0

5 27.3 ± 9.8

6 36.1 ± 13.5

pUC-UBI-hGH

1 146.7 ± 73.2 P<0.01

2 119.2 ± 26.5 P<0.001

3 161.7 ± 18.9 P<0.001

5 158.3 ± 54.8 P<0.01

6 241.6 ± 67.4 P<0.001

scid

1 683.3 ± 52.0

2 716.7 ± 142.2

3 485.0 ± 78.6

5 1233.3 ± 28.8

6 443.9 ± 97.9

Glucose: Concentration of glucose in the plasma was determined with the Glucose PAP Liquiform System (Labtest, MG, Brazil).

Catch-up growth (CG): This parameter was calculated using the body weight (g) or nose-to-tail (cm), tail (cm) or femur (mm) lenght , according to the formula:

CG = (Wt - WC)/(Wn – Wc) x 100 where: Wt = final weight or lenght of the treated group; Wc = final weight or lenght of the control (saline-treated) group; Wn = final weight or lenght of a normal co-aged animal group (scid mice, in this case).

* Significance levels comparing with saline at the same treatment time.

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Figure 1. Weight variation of lit/scid mice after pUC-UBI-hGH ( ) or saline ( ) administrations, followed by electroporation, and of non-treated scid ( ) mice.