Evaluation of prenatal intra-amniotic LAMB3 gene delivery in a mouse model of Herlitz disease

Abstract

Prenatal gene therapy has been considered for Herlitz junctional epidermolysis bullosa (H-JEB), a lethal genodermatosis caused by the absence of any of the three subunits of laminin-5, resulting from birth in widespread blistering and erosions of skin and mucosae. To investigate this strategy in an animal model, adenovirus type 5-and adeno-associated virus (AAV) type 2-derived vectors carrying a b-galactosidase reporter gene or LAMB3 cDNA encoding the b3 chain of laminin-5 were generated, tested for stability in amniotic fluid and evaluated in vitro on murine H-JEB keratinocytes, and in vivo by prenatal injection into the amniotic cavities of laminin-5 b3-deficient mice. The different vectors were administered individually or combined at maximum doses on day 14 post coitum. Adenoviral vectors infected preferentially the foetal epidermis, whereas AAV delivered the transgene mainly to mucous membranes of the airways and the upper digestive tract. The LAMB3 transgene was expressed in target epithelia of newborn laminin-5 b3-deficient mice, and the transgenic b3 chain was shown to assemble with its endogenous partner chains, resulting in detectable amounts of laminin-5 in the basement membranes of skin and mucosae and in a lower extent of tissue separation in the skin. However, only combined delivery of the two vector types led to a minor increase of the life span of H-JEB mice. Failure to rescue diseased animals was, at least in part, due to abandonment of any conspicuous pup by the heterozygous mother. This is the first study of a prenatal gene therapy approach to a heritable blistering disorder. Although our findings indicate that prenatal combined administration of adenoviral and adeno-associated LAMB3 vectors provides therapeutic benefit to H-JEB mice, this animal model appears unsuitable for long-term investigations of the therapeutic concept.

Attenuation of Severe Generalized Junctional Epidermolysis Bullosa by Systemic Treatment with Gentamicin

“…For further details, see the online supplementary material (www.karger.com/doi/10.1159/000499906) [23, 24] (Fig. 1).…”

Early intra-amniotic gene transfer using lentiviral vector improves skin blistering phenotype in a murine model of Herlitz junctional epidermolysis bullosa

“…The genotype of the heterozygous/homozygous LAM-B3 IAP animals was verified by PCR of the LAMB3 gene and the IAP insertion with a template of genomic DNA from tail samples as previously described. 16 …”

“…Although the majority of efforts to transduce skin by the intraamniotic route have achieved efficient gene expression, the duration of expression has been limited to 3-4 weeks, corresponding to the time required for a complete epidermal turnover cycle consistent with failure to transduce the skin stem cell population. [12][13][14][15][16][17] Effective gene therapy for skin disorders will require gene transfer to the skin stem cell populations to achieve long-term expression due to rapid cellular turnover inherent to the epidermis. There have been two types of stem cells identified in the epidermis: the basal cell and the bulge stem cell.…”

A New Mouse Model of Junctional Epidermolysis Bullosa: The LAMB3 628G>A Knockin Mouse

“…So far, treatment of JEB has only been supportive. Future therapeutic options may arise from bone marrow stem cells (Tolar et al, 2009) or from gene therapy, e.g., by means of autologous epidermal stem cells modified ex vivo (Mavilio et al, 2006;Di Nunzio et al, 2008) or by prenatal gene transfer (Mühle et al, 2006;Endo et al, 2012). Before clinical application, such therapeutic approaches have to be evaluated in animals.…”

“…Mouse models lacking the a3, b3, or g2 chain of laminin-332 exist (Kuster et al, 1997;Ryan et al, 1999;Meng et al, 2003), but affected animals die shortly after birth. Thus, long-term effects of therapeutic approaches cannot be evaluated in these models (Mühle et al, 2006). There is one mouse strain with a hypomorphic mutation of LAMC2 resulting in non-Herlitz JEB in homozygous animals (Bubier et al, 2010); however, LAMC2 mutations cause only a minority of human JEB cases (Schneider et al, 2007).…”