Integration-free and stable expression of FVIII using a human artificial chromosome.

Human artificial chromosome (HAC) has several advantages as a gene therapy vector, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts. To examine the copy number effect on the gene expression levels and its stability for a long-term culture for a future application in gene therapy, we constructed a HAC vector carrying the human factor VIII (FVIII) complementary DNA, FVIII-HAC in Chinese hamster ovary (CHO) cells. One and more copies of FVIII gene on the HAC were expressed in the copy-number-dependent manner in the CHO cells. The HAC with 16 copies of FVIII, FVIII (16)-HAC, was transferred from CHO hybrids into a human immortalized mesenchymal stem cell using microcell-mediated chromosome transfer. The expression levels of HAC-derived FVIII transgene products were compared with transfected FVIII plasmids. The former showed expression levels consistent with those of the original clones, even after 50 population doublings, whereas the latter showed a remarkable decrease in expression despite unvarying DNA content, indicating that the gene on the HAC is resistant to gene silencing. These results suggest that the HAC-mediated therapeutic gene-expression system may be a powerful tool for stable expression of transgenes, and possibly for industrial production of gene products.

Gastrointestinal angiodysplasia in a patient with type 2 von Willebrand's disease and analysis of exon 28 of the von Willebrand factor gene.

Although the association between gastrointestinal angiodysplasia and von Willebrand's disease has been suggested, molecular mechanisms involved in the formation of angiodysplasia in patients with von Willebrand's disease remained undetermined. We examined exon 28 of the von Willebrand factor gene in a patient with both von Willebrand's disease and recurrent bleeding from angiodysplasia in the duodenum as well as his father's, and found a point mutation, C 3916-->T (amino acid substitution; Arg 543-->Trp), in the A1 domain of the von Willebrand factor gene. This mutation was identical with a previously reported mutation in a patient with von Willebrand's disease complicated with gastrointestinal angiodysplasia.

Effect of ureteral obstruction on matrix metalloproteinase-2 in rat renal cortex.

BACKGROUND: Urinary tract obstruction induces renal damage, including not only interstitial fibrosis but also total loss of tissue integrity in the obstructed kidney. These processes require degradation and remodeling of the extracellular matrix. In the present study, we examined changes in matrix metalloproteinase-2 (MMP-2), a proteolytic enzyme, in a unilateral ureteral obstruction rat kidney model. METHODS AND RESULTS: Gelatin zymography showed that the both mature and the immature forms of MMP-2 were increased in the obstructed renal cortex. We used real-time reverse transcription polymerase chain reaction (RT-PCR) for the quantification of mRNA expression. MMP-2 mRNA was upregulated in the ligated kidney and peaked on day 3 after obstruction, while, in contrast, MMP-9 mRNA was significantly decreased. Type 1- and type 2-tissue inhibitors of metalloproteinase were increased throughout the experimental period. RT-PCR showed a concomitant increase in the mRNA expression of membrane type-1 MMP (MT1-MMP) and extracellular MMP inducer (EMMPRIN). CONCLUSIONS: We conclude that MMP-2 production in the renal cortex is increased in the early phase of ureteral obstruction and could be involved in the damage induced by ureteral obstruction.