Biomedical subjects
K Kurachi
Publications and source records attributed to K Kurachi.
Primary myoblast-mediated gene transfer: persistent expression of human factor IX in mice.
Primary myoblast-mediated gene transfer was tested for its ability to mediate a persistent expression of recombinant human factor IX in SCID mice. Mouse primary myoblasts were transduced with factor IX retroviral vectors, LIXSN, which uses the retroviral long-terminal repeats as the promoter, or dLMMBAIX, which uses muscle creatine kinase enhancer and beta-actin promoter to drive factor IX transcription. In vitro, myoblasts transduced with either LIXSN or dLMMBAIX expressed recombinant human factor IX with full biological activity. Upon implantation of transduced myoblasts into skeletal muscles of SCID mice, a sustained systemic expression of factor IX at a level of 10-30 ng/ml plasma was achieved. This was further supported by the presence of recombinant factor IX protein and mRNA in muscle tissues after 5 months of myoblast implantation. Intramuscular implantation of the transduced myoblasts resulted in a gene transfer which was confined locally to the region of injection, with no dissemination to other organs and tissues including testis. Additionally, basic fibroblast growth factor co-injected with primary myoblasts significantly improved the expression level of recombinant factor IX in vivo. These results demonstrate that primary myoblast-mediated gene therapy for hemophilia B is feasible and safe, and can be optimized by using cytokines or other conditions which augment myoblast survival and fusion with myofibers.
Hepsin, a putative cell-surface serine protease, is required for mammalian cell growth.
Hepsin was previously identified as a putative cell-surface serine protease. When hepatoma cells were treated with anti-hepsin antibodies, their growth was substantially arrested, suggesting the requirement of hepsin molecules present at the cell surface for normal cell growth. This was further supported by a gross inhibition of cell growth with hepsin-specific antisense oligonucleotides. Upon treatment of cells with antisense oligonucleotides, rapid reduction in cellular hepsin was observed. This reduction in cellular hepsin levels was accompanied by drastic morphological changes. Various tissues in the developing mouse embryo showed greatly elevated hepsin levels in regions of active proliferation. These results indicate that hepsin plays an essential role in cell growth and maintenance of cell morphology.
Implanted myoblasts not only fuse with myofibers but also survive as muscle precursor cells.
Intramuscular implanted myoblasts can fuse with existing myofibers. Here we report that implanted primary myoblasts marked with retroviral transgenes can also persist as muscle precursor cells. These cells can be recovered as viable myoblasts from muscles of recipient mice even months after myoblast implantation, and they can fully resume expression of the transgenes in culture. Upon re-implantation into muscles, they again not only fuse with existing myofibers, but also survive as muscle precursor cells in the tissue. These reserve myogenic cells should be able to contribute to host myofibers in muscle regeneration when the recombinant myofibers are damaged, providing an additional mechanism to maintain a persistent expression of transgenes delivered by myoblast-mediated gene transfer.
Biology of factor IX.
Within the past 20 years or so, factor IX has been at the centre of particularly intensive studies of its physiology, pathology and biochemistry as well as its molecular genetics and biology. With the complete nucleotide sequence of its human gene determined in 1985 and the molecular defects of over 600 abnormal human factor IX genes analysed to date, factor IX is among the few mammalian proteins which have been exhaustively studied in almost every aspect. The enormous amount of information we now have on this medium-sized plasma protein sheds light on how a gene and its protein evolve, how the protein carries out a highly regulated, specific and pivotal role in the delicately balanced blood coagulation reaction, and the correlation between clinical presentations and its highly diverse molecular mechanism of defects. This wealth of knowledge makes factor IX an excellent model for deeper study, such as truly quantitative analysis of its structure-function relationship and in vivo function and regulation. It will also provide a sound foundation which may lead to improved treatment of haemophilia B and perhaps to its cure. This paper attempts to review the recent progress in research on factor IX.
Expression of human factor IX in mice after injection of genetically modified myoblasts.
Hemophilia B is an X chromosome-linked recessive bleeding disorder. To develop a somatic gene therapy for this disease, we have examined whether mouse skeletal myoblasts can serve as efficient vehicles for systemic delivery of recombinant factor IX. When mouse myoblasts (C2C12) transduced with a Moloney murine leukemia virus-based vector containing the bacterial beta-galactosidase gene were injected into mouse skeletal muscles, they fused with the existing and regenerating myofibers and continued to express beta-galactosidase. C2C12 myoblasts that were infected with recombinant retroviruses containing a human factor IX cDNA secreted biologically active human factor IX cDNA secreted biologically active human factor IX into the culture medium at a rate of 2.6 micrograms per 10(6) cells per day. Myotubes derived from these cells in culture continued to express human factor IX (0.68 micrograms/day from myotubes derived from 10(6) C2C12 cells). After injection of the transduced C2C12 myoblasts into skeletal muscles of mice, the systemic level of recombinant human factor IX was found to be as high as approximately 1 microgram/ml of serum. These results provide the rationale for using skeletal myoblasts as an efficient gene delivery vehicle in the somatic gene therapy for hemophilia B.
Deficiencies in factors IX and VIII: what is now known.
Genetic defects both gross and subtle have recently been identified in about 900 patients with hemophilia A or B. The defects, which include deletions, insertions, and point mutations, reveal much about genetic structure-function relationships in hemophilia. These insights will lead to improved treatment of bleeding disorders and perhaps to their cure.
Phase 1 clinical tests of influenza MDP-virosome vaccine (KD-5382).
MDP-virosome vaccine, which contains haemagglutinin (HA) and neuraminidase (NA) antigens isolated from influenza virus combined with 6-O-(2-tetradecylhexadecanoyl)-N-acetylmuramoyl-L-alanyl-D-isoglutamine) (B30-MDP) and cholesterol was tested, in comparison with a currently used HA vaccine, for immunogenicity and adverse reactions on 77 adult male volunteers. The volunteers were divided into eight groups, and each individual was injected subcutaneously once, or twice at a 4 week interval, in their upper arm with one of seven different MDP-virosome vaccine preparations or with HA vaccine as control. Of the three virus strains used as vaccine antigens, MDP-virosome vaccines induced higher haemagglutination inhibiting (HI) antibody to A/Yamagata/120/86 (H1N1) and A/Fukuoka/C29/86 (H3N2) than did HA vaccine, whereas it induced lower HI antibody to B/Nagasaki/1/87, comparable with that induced by HA vaccine. Regarding local adverse reactions, MDP-virosome vaccinees frequently developed mild local pain, reddening and swelling, which disappeared within 5 days; as regards systemic no adverse reactions, leucocytosis developed among the MDP-virosome vaccines, but no other reactions were observed. The leucocytosis may have been caused by the pharmacoimmunological activity of B30-MDP derivatives.
Biology of factor IX.
Hemophilia B, one of two common hereditary bleeding disorders, is caused by a deficiency of factor IX in the circulation. Molecular mechanisms of hemophilia B are highly heterogeneous including gene deletions, insertions, complex rearrangements, and a large number of point mutations. Currently, hemophilia B is treated by plasma protein replacement therapy. This therapy is effective but exposes patients to possible side effects and complications such as infection of blood-borne pathogens including hepatitis viruses and HIV-1. Intensive efforts to develop alternative, safer therapies for hemophilia B, including somatic gene therapy, are now under way.
A simple treatment of serum for precise determination of recombinant factor IX in the culture media.
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Expression of human factor IX in rat capillary endothelial cells: toward somatic gene therapy for hemophilia B.
In aiming to develop a gene therapy approach for hemophilia B, we expressed and characterized human factor IX in rat capillary endothelial cells (CECs). Moloney murine leukemia virus-derived retrovirus vectors that contain human factor IX cDNA linked to heterologous promoters and the neomycin-resistant gene were constructed and employed to prepare recombinant retroviruses. Rat CECs and NIH 3T3 cells infected with these viruses were selected with the neomycin analogue, G418 sulfate, and tested for expression of factor IX. A construct with the factor IX cDNA under direct control by long terminal repeat gave the highest level of expression (0.84 and 3.6 micrograms per 10(6) cells per day for CECs and NIH 3T3 cells, respectively) as quantitated by immunoassays as well as clotting activity assays. A single RNA transcript of 4.4 kilobases predicted by the construct and a recombinant factor IX of 68 kilodaltons identical to purified plasma factor IX were found. The recombinant human factor IX produced showed full clotting activity, demonstrating that CECs have an efficient mechanism for posttranslational modifications, including gamma-carboxylation, essential for its biological activity. These results, in addition to other properties of the endothelium, including large number of cells, accessibility, and direct contact with the circulating blood, suggest that CECs can serve as an efficient drug delivery vehicle producing factor IX in a somatic gene therapy for hemophilia B.
Hepsin, a cell membrane-associated protease. Characterization, tissue distribution, and gene localization.
Hepsin, a putative membrane-bound serine protease, was originally identified as a human liver cDNA clone (Leytus, S.P., Loeb, K.R., Hagen, F.S., Kurachi, K., and Davie, E.W. (1988) Biochemistry 27, 1067-1074). In the present study the human hepsin gene was localized to chromosome 19 at q11-13.2. The messenger RNA of hepsin is 1.85 kilobases in size and present in most tissues, with the highest level in liver. Hepsin is synthesized as a single polypeptide chain, and its mature form of 51 kDa was found in various mammalian cells including HepG2 cells and baby hamster kidney cells. It is present in the plasma-membrane in a molecular orientation of type II membrane-associated proteins, with its catalytic subunit (carboxyl-terminal half) at the cell surface, and its amino terminus facing the cytosol. Hepsin is found neither in cytosol nor in culture media. The results obtained suggest that hepsin has an important role(s) in cell growth and function.
The major component of a large, intracellular proteinase accumulated by inhibitors is a complex of alpha 2-macroglobulin and thrombin.
A large, intracellular proteinase accumulated by inhibitors (PABI) was found in cultured mammalian cells as a large, multicatalytic proteinase with a greatly elevated concentration in the presence of small peptide proteinase inhibitors (Tsuji and Kurachi (1989) J. Biol. Chem. 264, 16093). Electron microscopic analysis showed that the tertiary structure of PABI highly resembled that of alpha 2-macroglobulin complexed with a proteinase(s). Isolation of the anti-PABI cross-reacting material from calf serum added to the culture media of baby hamster kidney cells further supported that the primary component of PABI was alpha 2-macroglobulin. Immunoblot analyses and the substrate specificity of PABI indicated that the major proteinase component contained in PABI was thrombin. When alpha 2-macroglobulin was added to the PABI-depleted serum, a significant accumulation or a degradation of the intracellular alpha 2-macroglobulin was observed in the presence or absence of leupeptin, respectively. Similarly, when thrombin was added to the PABI-depleted fetal calf serum supplemented with fresh alpha 2-macroglobulin, a significant amount of intracellular thrombin was found only in the presence of leupeptin. These results indicate that the major component of the intracellular PABI molecules is a complex of alpha 2-macroglobulin with thrombin which is internalized from the culture media. Intracellular accumulation of PABI, therefore, is a phenomenon primarily relevant to the culture cells. Whether or not PABI is also generated in certain physiological or pathological conditions requires further study.
Characterization of a mouse factor IX cDNA and developmental regulation of the factor IX gene expression in liver.
A mouse factor IX cDNA was isolated and characterized. The cDNA was 1,837 bp in length and contained the coding region as well as short 5' and 3' untranslated sequences. Northern blot analysis of liver RNA showed two mRNA species of 3.2 kb (major) and 2.2 kb (minor) for the mouse factor IX. An antisense RNA probe prepared from the mouse cDNA was employed to determine the steady state level of factor IX mRNA in mouse liver at various developmental stages. The factor IX mRNA level was very low (2-5% of the adult level) during the gestational period until day -3 (gestational day 17) followed by a rapid increase at day -2 through birth. This phase of rapid increase was followed by a gradual increase before it reached the adult level at around 20 to 24 days. At birth, the factor IX mRNA level was found to be at about 43% of that of the adult. The mRNA levels in mouse liver agreed well with the plasma factor IX activity levels. These results indicate that reduced factor IX activity in newborns is due to the low levels of factor IX mRNA available for translation.
Recombinant antihemophilic factors.
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Characterization of hepsin, a membrane bound protease.
Hepsin is a membrane bound protease of 51 kDa present in mammalian cells. It contains a stretch of hydrophobic sequence of 27 amino acid residues in its N-terminal region. By employing fluorescent immunostaining of cells and western blot analysis of the various cell subfractions, the catalytic subunit (carboxyl terminal half) of hepsin is found at the cell surface. The hepsin gene is expressed in most tissues of a young adult baboon, particularly in the liver at high levels.
Functional characterization of the 5'-regulatory region of human factor IX gene.
Functional characterization of the 5'-regulatory region of the human factor IX gene was carried out by a series of gene dissection analyses. The region extending from -175 to -274 of the 5' end flanking sequence is required for the expression of this gene. Within this region, sequence elements AGCCACT at -238 and TCAAAT at -187 were assigned as the functional CAAT box and TATA box, respectively. The transcription initiation site was found to be at -150 for the factor IX chloramphenicol acetyltransferase chimeric gene. A negative regulatory (silencer) activity was located in the region spanning from 1.4 to 1.7 kilobases upstream of the promoter region. This region contains a short sequence element (ATCCTCTCC) known to have such activity. A strong promoter on the opposite strand was also located about 500 base pairs upstream of the promoter. The expression of factor IX gene was highly liver specific, as assessed by Northern blot analysis. Short sequence elements (TGGACC and CTTTGGACT) homologous to the known liver-specific elements were located in the vicinity of the defined promoter region.