Transplacental delivery of factor IX Fc-fusion protein ameliorates bleeding phenotype of newborn hemophilia B mice.

Hemophilia B is an inherited hemorrhagic disorder characterized by a deficiency of blood coagulation factor IX (FIX) that results in abnormal blood coagulation. The blood coagulation is already evident in hemophiliacs at the fetal stage, and thus intracranial hemorrhage and other bleeding complications can occur at birth, leading to sequelae. Therefore, it is important to develop effective treatments for hemophiliacs in utero. In this study, in order to transplacentally deliver FIX from pregnant mice to their fetuses, an improved adenovirus (Ad) vector expressing human FIX fused with the IgG Fc domain (FIX Fc fusion protein), which plays a crucial role in neonatal Fc receptor (FcRn)-mediated transcytosis across the placenta, was intravenously administered to E13.5 pregnant mice. Significant levels of FIX Fc fusion protein were detected in 0-day-old newborn mice whose mothers were administered an Ad vector expressing FIX Fc fusion protein. Wild-type FIX overexpressed in the pregnant mice was not delivered to the fetuses. Plasma FIX levels in the newborn mice were relatively well correlated with those in their mothers, although transplacental delivery efficiencies of FIX Fc fusion protein were slightly reduced when the FIX Fc fusion protein was highly expressed in the mother mice. Plasma FIX levels in the newborn mice were about 3.6-6.4% of those in their mothers, Transplacental delivery of FIX Fc fusion protein to their fetuses successfully improved the blood clotting ability in the newborn mice.

Development of a novel adenovirus serotype 35 vector vaccine possessing an RGD peptide in the fiber knob and the E4 orf 4, 6, and 6/7 regions of adenovirus serotype 5.

Adenovirus (Ad) vectors based on human adenovirus serotype 5 (Ad5) have attracted significant attention as vaccine vectors for infectious diseases. However, the effectiveness of Ad5 vectors as vaccines is often inhibited by the anti-Ad5 neutralizing antibodies retained by many adults. To overcome this drawback, we focused on human adenovirus serotype 35 (Ad35) vectors with low seroprevalence in adults. Although Ad35 vectors can circumvent anti-Ad5 neutralizing antibodies, vector yields of Ad35 vectors are often inferior to those of Ad5 vectors. In this study, we developed novel Ad35 vectors containing the Ad5 E4 orf 4, 6, and 6/7 or the Ad5 E4 orf 6 and 6/7 for efficient vector production, and compared their properties. These E4-modified Ad35 vectors efficiently propagated to a similar extent at virus titers comparable to those of Ad5 vectors. An Ad35 vector containing the Ad5 E4 orf 4, 6, and 6/7 mediated more efficient transduction than that containing the Ad5 E4 orf 6 and 6/7 in human cultured cells. Furthermore, insertion of an arginine-glycine-aspartate (RGD) peptide in the fiber region of an Ad35 vector containing the Ad5 E4 orf 4, 6, and 6/7 significantly improved the transgene product-specific antibody production following intramuscular administration in mice. The Ad35 vector containing the RGD peptide mediated efficient vaccine effects even in the mice pre-immunized with an Ad5.

Adenovirus Vector With ADP Gene Induces Cytopathic Effects in HEK293 Cells Without Significant Elevation of Virus Titers.

BACKGROUND/AIM: Efficient production of adenovirus vectors is crucial for their clinical use. Adenovirus death protein (ADP), which is encoded in the E3 region of the adenovirus genome, is involved in host-cell lysis and the subsequent release of progeny virus; however, the ADP gene is often removed from the adenovirus vector genome. MATERIALS AND METHODS: We have developed adenovirus vectors that possess the ADP gene and maintain a relatively large insertion capacity for foreign genes by deleting the partial E3 region. Adenovirus vector-mediated transgene expression levels and virus titers were examined. RESULTS: The adenovirus vectors maintaining the ADP gene showed cytopathic effect earlier than conventional adenovirus vector without the ADP gene following treatment of HEK293 cells, although there were no significant differences in total virus titers. CONCLUSION: The adenovirus vectors possessing the ADP gene showed efficient spread of progeny virus infection following transduction in HEK293 cells.

Transduction Properties of an Adenovirus Vector Containing Sequences Complementary to a Liver-Specific microRNA, miR-122a, in the 3'-Untranslated Region of the E4 Gene in Human Hepatocytes from Chimeric Mice with Humanized Liver.

Replication-incompetent adenovirus (Ad) vectors are promising gene delivery vehicles, especially for hepatocytes, due to their superior hepatic tropism; however, in vivo application of an Ad vector often results in hepatotoxicity, mainly due to the leaky expression of Ad genes from the Ad vector genome. In order to reduce the Ad vector-induced hepatotoxicity, we previously developed an Ad vector containing the sequences perfectly complementary to a liver-specific microRNA (miRNA), miR-122a, in the 3'-untranslated region (UTR) of the E4 gene. This improved Ad vector showed a significant reduction in the leaky expression of Ad genes and hepatotoxicity in the mouse liver and primary mouse hepatocytes; however, the safety profiles and transduction properties of this improved Ad vector in human hepatocytes remained to be elucidated. In this study, we examined the transgene expression and safety profiles of Ad vectors with miR-122a-targeted sequences in the 3'-UTR of the E4 gene in human hepatocytes from chimeric mice with humanized liver. The transgene expression levels of Ad vectors with miR-122a-targeted sequences in the 3'-UTR of the E4 gene were significantly higher than those of the conventional Ad vectors. The leaky expression levels of Ad genes of Ad vectors with miR-122a-targeted sequences in the 3'-UTR of the E4 gene in the primary human hepatocytes were largely reduced, compared with the conventional Ad vectors, resulting in an improvement in Ad vector-induced cytotoxicity. These data indicated that this improved Ad vector was a superior gene delivery vehicle without severe cytotoxicity for not only mouse hepatocytes but also human hepatocytes.