Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Continuously renewing tissues, such as the epidermis, are populated by a hierarchy of dividing transient amplifying cells, which are maintained by stem cells. Transient amplifying cells divide to maintain the tissue, but they are limited to a finite number of cell divisions before they differentiate and are sloughed. Only the stem cells remain for the life of the tissue. Thus, it is critical to target stem cells when designing gene therapy regimes for genetically inherited diseases, such as epidermolysis bullosa simplex (EBS). Unfortunately, isolating pure epithelial stem cells has been problematic. In this study, we used rapid adherence to collagen type IV to successfully enrich for epidermal stem cells from adult human skin. These preselected stem cells were slow to proliferate, but they ultimately formed large colonies. When recombined with the dermal substrate AlloDerm, the stem cells re-formed a stratified squamous epidermis within 1 week after raising the AlloDerm to the air-liquid interface. These organotypic cultures grew continuously and, even after 6 weeks in culture, they maintained a proliferative basal layer. When transduced with a retroviral LacZ vector, preselected stem cells formed beta-galactosidase-positive clones in submerged and organotypic cultures. Transduced cells showed persistent expression through 12 weeks in organotypic culture, demonstrating the feasibility of using preselected stem cells for gene therapy. Currently, we are developing two models of EBS to test a gene therapy approach, which is based on the premise that EBS stem cells with a mutant keratin (K)14 gene corrected to wild type will have a growth advantage over noncorrected EBS stem cells.
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PMID:Transduction of a preselected population of human epidermal stem cells: consequences for gene therapy. 1035 57

Gene therapy of large genes (e.g. plectin and collagen genes) is hampered by size limitations for insertions of the currently used viral vectors. To reduce the size of these insertions spliceosome-mediated RNA trans-splicing (SMaRT), which provides intron-specific gene-correction at the pre-RNA level, can be an alternative approach. To test its applicability in skin gene therapy, SMaRT was used in the context of the 4003delTC mutation in the collagen XVII gene (COL17A1) causing generalized atrophic benign junctional epidermolysis bullosa. A beta-galactosidase (beta-gal) trans-splicing assay system was established using intron 51 of COL17A1 as the target for trans-splicing. In this system, intron 51 is flanked by the 5'exon and the 3'exon of the beta-gal gene, the latter containing two in-frame stop codons. Cotransfection of a pre-trans-splicing molecule consisting of the binding domain of intron 51 and the 3'exon of beta-gal without the stop codons resulted in a 300-fold increase of beta-gal activity compared to controls. A 2-3-fold increase in efficiency was obtained through an elongation of the binding domains. Replacement of the complete 3'end of the COL17A1 gene was shown using a collagen XVII mini-gene construct. The beta-gal assay was used in human keratinocytes to evaluate the influence of a keratinocyte-specific spliceosome background. Reverse transcription polymerase chain reaction and beta-gal activity assay showed functional correction of the stop-codons in cultured human keratinocytes and in an immortalized GABEB cell line harbouring the 4003delTC mutation. These results demonstrate that SMaRT is feasible in a keratinocyte-specific context and therefore may be applied in skin gene therapy.
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PMID:Development of spliceosome-mediated RNA trans-splicing (SMaRT) for the correction of inherited skin diseases. 1263 Dec 45

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 beta-galactosidase 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 murine H-JEB keratinocytes, and in vivo by prenatal injection into the amniotic cavities of laminin-5 beta3-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 beta3-deficient mice, and the transgenic beta3 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.
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PMID:Evaluation of prenatal intra-amniotic LAMB3 gene delivery in a mouse model of Herlitz disease. 1687 Dec 30