Prenatal Treatment of X-Linked Hypohidrotic Ectodermal Dysplasia using Recombinant Ectodysplasin in a Canine Model

Abstract

X-linked hypohidrotic ectodermal dysplasia (XLHED) is caused by defects in the EDA gene that inactivate the function of ectodysplasin A1 (EDA1). This leads to abnormal development of eccrine glands, hair follicles, and teeth, and to frequent respiratory infections. Previous studies in the naturally occurring dog model demonstrated partial prevention of the XLHED phenotype by postnatal administration of recombinant EDA1. The results suggested that a single or two temporally spaced injections of EDI200 prenatally might improve the clinical outcome in the dog model. Fetuses received ultrasound-guided EDI200 intra-amniotically at gestational days 32 and 45, or 45 or 55 alone (of a 65-day pregnancy). Growth rates, lacrimation, hair growth, meibomian glands, sweating, dentition, and mucociliary clearance were compared in treated and untreated XLHED-affected dogs, and in heterozygous and wild-type control dogs. Improved phenotypic outcomes were noted in the earlier and more frequently treated animals. All animals treated prenatally showed positive responses compared with untreated dogs with XLHED, most notably in the transfer of moisture through paw pads, suggesting improved onset of sweating ability and restored meibomian gland development. These results exemplify the feasibility of ultrasound-guided intra-amniotic injections for the treatment of developmental disorders, with improved formation of specific EDA1-dependent structures in dogs with XLHED.

Fig. 1.

Numbers of meibomian glands per centimeter of eyelid in WT, untreated heterozygous (Het), and XLHED dogs, and XLHED dogs treated in utero with Fc:EDA1 at the indicated embryonic developmental days (E32/45, E45, E55). Untreated WT (white circles) and heterozygous (black and white circles) dogs were pooled for the analysis. Values are the mean ± S.E.M. One-way ANOVA assuming Gaussian distribution, and comparing conditions to a control (WT or XLHED) with Dunnett’s multiple comparison test. ^nsP > 0.05; ***P < 0.001; ****P < 0.0001.

Fig. 2.

Mean frequency of ocular discharge requiring the administration of ophthalmic lubricants in WT, treated, and untreated XLHED dogs over a 20-week observation period. Values are the mean ± S.E.M. One-way ANOVA assuming Gaussian distribution, and comparing conditions to untreated XLHED with Dunnett’s multiple-comparison test, and nonparametric Kruskal-Wallis test with Dunn’s multiple-comparison test. Both sets of statistical results are displayed, with those believed to be less relevant shown in brackets. ^nsP > 0.05; **P < 0.1; *****P < 0.0001. Het, heterozygous.

Fig. 3.

Hair coverage over the dorsal cranium. Each dog depicted here is representative of all of the dogs in each treatment group. (A) In the WT dog (left), there is complete hair coverage over the forehead, whereas most is missing in the XLHED dog. (B) There is subjectively more hair coverage (arrows) on the forehead of this 8-week-old XLHED puppy from the IUTxE32/45 group when compared with the XLHED control in (A). (C) At 6 months of age, the increased hair coverage (arrows) shown in this XLHED dog from the IUTxE45 was present to the same degree as when the dog was 8 weeks old (data not shown). (D) There was little to no improvement of hair coverage in any of the XLHED dogs from the IUTxE55. The forehead of a 6-month-old dog from this group is shown here.

Fig. 4.

Average number of teeth in WT, heterozygous (Het), treated, and untreated XLHED dogs. (A) Deciduous teeth. (B) Adult teeth. Gray circles in the XLHED group indicate dogs coming from an independent experiment and included in the statistical analysis. In the untreated and treated XLHED groups, identical symbols (black upright triangles, inverted triangles, circles, diamonds, or squares) indicate the same animal in (A and B). Values are the mean ± S.E.M. A one-sample t test first assessed whether the average number of teeth in XLHED dogs differed from normal (28 in pups, 42 in adults). Thereafter, groups of treated dogs were compared with untreated XLHED dogs by one-way ANOVA with Dunnett’s multiple-comparison test. ^nsP > 0.05; ****P < 0.0001.

Fig. 5.

MCC in WT, heterozygous (Het), treated, and untreated XLHED dogs. Data indicated with gray circles originate from untreated animals analyzed in an independent experiment (Casal et al., 2007) but were included in the statistical analysis. Values are the mean ± S.E.M. One-way ANOVA assuming Gaussian distribution and comparing conditions to a control (XLHED) with Dunnett’s multiple comparison test: ^nsP > 0.05; ***P < 0.001.

Fig. 6.

Representative images preadministration and postadministration (Post) of pilocarpine with the Moisture Map (A) and the Paw Print Sensor (B) assays for normal dogs, XLHED dogs, and treated XLHED dogs from top to bottom, respectively. There is an appreciable difference between preadministration and postadministration of pilocarpine with the normal group and the treated group, which does not exist with the affected group. Primarily, there are more spots, spots may be larger, and spots may be darker. More spots are interpreted as an increased number of sweat glands or more productive sweat glands.

Fig. 7.

Difference in paw print intensity after administering pilocarpine, as an indirect measure to assess sweat function (mean ± S.D.). The gray circle indicates one heterozygote animal. One-way ANOVA assuming Gaussian distribution, and comparing conditions to a control (XLHED) with Dunnett’s multiple-comparison test. ^nsP > 0.05; *P < 0.05; ***P < 0.001.