Evaluation of prenatal intra-amniotic LAMB3 gene delivery by adenoviral and AAV vectors in a mouse model of Herlitz junctional epidermolysis bullosa

Background: Prenatal gene therapy has been considered for diseases such as Herlitz junctional epidermolysis bullosa (H-JEB), a lethal genodermatosis caused by absence of any of the three subunits of the basement membrane protein laminin-5. H-JEB presents from birth with widespread blistering of skin and mucosae and leads to death typically within the first year of life. In this study, we investigated in a mouse model whether H-JEB could be treated by gene delivery in utero.

Methods: Adenovirus type 5- and AAV2-derived vectors carrying a reporter gene or LAMB3 cDNA encoding the beta3 chain of laminin-5 were generated, tested for stability in amniotic fluid and evaluated in vitro on primary LAMB3-deficient murine keratinocytes and in vivo by injection into the amniotic cavity of LAMB3-deficient fetal mice at day 14 post coitum. The adenoviral and AAV vectors were administered alone or in combination at doses of 5×10exp9 and 7×10exp10 particles per fetus, respectively. Tissue samples of treated homozygous animals including skin and mucous membranes were investigated for transgene expression by quantitative RT-PCR and immunofluorescence staining. The extent of tissue separation in the skin was assessed histologically.

Results: In the presence of amniotic fluid the infectivity of adenoviral vectors was slightly reduced, while AAV-mediated gene delivery proved to be unaffected. In vivo, adenoviral vectors infected preferentially the fetal epidermis, whereas AAV delivered the transgene mainly to mucous membranes of the airways and upper gastrointestinal tract. The LAMB3 transgene was expressed in target epithelia of newborn LAMB3-deficient mice, and the transgenic beta3 chain was shown to assemble with the endogenous alpha3 and gamma2 chains of laminin-5, resulting in detectable amounts in the basement membranes of skin and mucosae. However, even combined delivery of the two vector types led only to a minor increase of the life span of homozygous LAMB3-deficient mice.

Conclusions: Although our data indicate that prenatal combined administration of adenoviral and adeno-associated LAMB3 vectors may offer therapeutic benefit in a mouse model of H-JEB, this model does not appear suitable for long-term investigations of the therapeutic concept.