Development and application of tissue-directed medicinal resources based on biotechnology

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For therapy of human genetic diseases, the research had been performed to establish the enzyme and gene replacement methods and to discover the medicinal resources for the selective tissue targeting affected with lysosomal enzyme deficiencies. During the term of project, new findings were obtained as follows :1. Protective protein/cathepsin A (PPCA) is a multifunctional glycoprotein that exhibits the protective effects on lysosomal neuraminidase (Neur) and β-galactosidase (β-Gal), and serine carboxypeptidase (cathepsin A ; Cath A) activity on a subset of neuropeptides including endothelin-1 (ET-1). Galactosialidosis (GS) is a human PPCA deficiency with autosomal recessive genetic trait, accompanied by the simultaneous decrease of these enzyme activities and multiple clinical manifestations including neurological abnormalities. Histochemical analysis of the autopsied GS brain against ET-1, one of the putative endogenous substrates of the Cath A, was performed. ET-1-like immunoreactivity … More in the neural cells with GS was demonstrated to increase abnormally.2. Simultaneous expression of ET-1 precursor protein and ET-B receptor cDNAs caused the accumulation of ET-1 in the GS fibroblastic cell line.3. PPCA gene disruption in a human astrocytoma cell line was performed using the targeting vector containing the drug-resistant gene cassette between the two loxP sequences. The cell line with one disrupted allele of PPCA gene was obtained.4. Homology modeling of human Neur was performed on the basis of X-ray structure of microbial neuraminidases. Structural effects of amino acid substitutions identified in human Neur deficiency (sialidosis) predicted that the domain in which some substitutions locate might contribute to the interaction with PPCA molecule.5. Sandhoff disease is a GM2-gangliosidoses caused by the genetic defect of the β-subunit of lysosomal β-hexosaminidase. In the gene-disrupted mice as the disease model were used to elucidate the pathogenesis and were found to express specifically some types of chemokine parallel to the accumulation of GM2-ganglioside in the brain. This phenomenon was considered to be applicable to develop novel cell replacement therapy.6. Novel apoptosis-inducing compounds were discovered from the synthetic key intermediates of the bioactive halichroline that has the inhibitory action on the induced-expression of vascular cell adhesion molecule (VCAM-1) on the human umbilical vascular endothelial cells (HUVEC). Less