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The tight skin (Tsk/+) mouse is a model for fibrotic disorders. The genetic defect in the Tsk/+ is an in-frame duplication between exons 17 and 40 of the fibrillin-1 gene which gives rise to a large transcript and protein. Mice homozygous for the mutation die in utero, whereas heterozygotes survive and spontaneously develop connective tissue disease. In this study, we generated hammerhead ribozymes directed against the mutant fibrillin-1 transcript. A partially mispairing ribozyme was the most effective vehicle to cleave the mutant transcript without undesired cleavage of wild type transcripts, as shown by cell-free RNA cleavage and cleavage in cell lines harboring the ribozyme, by RT-PCR, Northern and Western Blotting. Global gene expression profiling using oligonucleotide microarrays showed the expected reduction in fibrillin-1 mRNA, and down-regulation of several gene cohorts in ribozyme harboring TskR1 cells compared to Tsk/+ cells. Two of the functional clusters included genes regulating extracellular matrix such as connective tissue growth factor, serpine-1 (plasminogen activator inhibitor-1) and TIMP-1 and TIMP-3, and those involved in cytoskeletal organization and myofibroblast formation including calponins and transgelin. Ribozyme-mediated inhibition was confirmed by Western Blot and functional analysis using cell-reporter systems and remodeling of three dimensional collagen gels. Our results underline the therapeutic potential of hammerhead ribozymes in dominant negative defects and suggest that changes in microfibril architecture brought about by fibrillin-1 mutation lead to a complex disease phenotype.

In this study an amphotropic retrovirus has been used to efficiently transduce normal human (NF) and scleroderma (systemic sclerosis; SSc) dermal fibroblasts (SScF) with a sequence encoding a temperature-sensitive mutant of the SV40 large T antigen (tsA58-U19). From the primary outgrowths of skin explants, cultures were generated whose growth was stringently temperature-dependent. When grown at a low, permissive temperature (35 degrees C), both normal and SSc-transduced cells continuously divided with similar doubling times, whereas at a high, nonpermissive temperature (39.5 degrees C), division of both the NF and SScF cells was rapidly arrested. These cells have been passaged more than 50 times, have the typical morphological appearance of fibroblasts, and have retained an anchorage-dependent phenotype. The transduced normal cells (tsT-NF) synthesized the matrix molecules collagen and fibronectin and expressed phenotypic antigens characteristic of their nontransduced counterparts, including MHC Class I, VLA beta 1 (CD29), Hermes 1 (CD44), VLA-4 alpha (CD49d), ICAM-1 (CD54) and LFA-3 (CD58) and the cell surface ectoenzymes neutral endopeptidase (CD10), aminopeptidase N (CD13), and dipeptidyl peptidase IV (CD26). Analysis of the transduced SSc fibroblasts (tsT-SScF) showed that these cells exhibited certain major features of the SSc pathology, notably the abnormally high synthesis of type I collagen, increased expression of ICAM-1, and depressed levels of CD26. Moreover, these phenotypic characteristics were retained even after prolonged culture in vitro. The tsT-SScF cells also retained their responsiveness to cytokines, since interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) both produced a marked increase in ICAM-1 expression. Our findings show that infection of SScF with the SV40 tsT antigen extends the life span of these cells and does not ablate their abnormal phenotypic and functional characteristics.

The direct transfer of certain lysosomal enzymes during cell-to-cell contact between normal lymphocytes and enzyme-deficient recipient cells has previously been reported in vitro and may play an important role in the correction of lysosomal storage diseases by bone marrow transplantation in vivo. In the present study we have used a number of different T, B, and plasma cell lines to examine the expression and immunological specificity of the transfer of the lysosomal enzyme, beta-glucuronidase (Gus). Each of these groups of cell had differing intracellular and secreted levels of Gus activity, which were nevertheless similar within each group. Dermal fibroblasts deficient in the Gus enzyme acquired substantial amounts of additional activity when they were cultured together with the T cells, the B cells, or the plasma cells. This occurred by the direct transfer of Gus from all three types of cell. In addition, with plasma cells, which had very high intracellular enzyme activity and also secreted high levels of Gus into their culture medium, the secreted enzyme was readily internalized by the fibroblasts via the mannose 6-phosphate receptor (MPR). It was notable that the purified endogenous enzymes from plasma cells as well as from B cells, but not from T cells, were also endocytosed by the fibroblasts utilizing this receptor-mediated process. Although the Gus activity from all the cell lines examined had the same molecular size, polyacrylamide electrophoresis and isoelectric focusing patterns showed that the immunologically distinct types of lymphoid cell have characteristic, unique pathways of post-translational lysosomal enzyme processing. These results show that the transfer of lysosomal enzymes from lymphoid cells can occur by two distinct mechanisms, both likely to have important roles in enzyme replacement therapy.