Complete nucleotide sequence, origin of isoform and functional characterization of the mouse hepsin gene

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66259/1/j.1432-1327.1999.00431.x.pd

Heterogeneous Nuclear Ribonucleoprotein A3 Is the Liver Nuclear Protein Binding to Age Related Increase Element RNA of the Factor IX Gene

Background: In the ASE/AIE-mediated genetic mechanism for age-related gene regulation, a recently identified age-related homeostasis mechanism, two genetic elements, ASE (age-related stability element) and AIE (age-related increase element as a stem-loop forming RNA), play critical roles in producing specific age-related expression patterns of genes. Principal Finding: We successfully identified heterogeneous nuclear ribonucleoprotein A3 (hnRNP A3) as a major mouse liver nuclear protein binding to the AIE-derived RNAs of human factor IX (hFIX) as well as mouse factor IX (mFIX) genes. HnRNP A3 bound to the AIE RNA was not phosphorylated at its Ser 359, while hnRNP A3 in the mouse liver nuclear extracts was a mixture of phosphorylated and unphosphorylated Ser 359. HepG2 cells engineered to express recombinant hFIX transduced with adenoviral vectors harboring an effective siRNA against hnRNP A3 resulted in a substantial reduction in hFIX expression only in the cells carrying a hFIX expression vector with AIE, but not in the cells carrying a hFIX expression vector without AIE. The nuclear hnRNP A3 protein level in the mouse liver gradually increased with age, while its mRNA level stayed age-stable. Conclusions: We identified hnRNP A3 as a major liver nuclear protein binding to FIX-AIE RNA. This protein plays a critical role in age-related gene expression, likely through an as yet unidentified epigenetic mechanism. The present study assigned a novel functional role to hnRNP A3 in age-related regulation of gene expression, opening up a new avenue for studyin

Sialic-acid-binding lectin from the slug Limax flavus

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66061/1/j.1432-1327.1998.2540217.x.pd

Effects of a second intron on recombinant MFG retroviral vector

 The retroviral vectors based on an MFG-type backbone have superior expression characteristics, in part, due to the presence of the retroviral chimeric intron (MFG intron). We tested the hypothesis that inclusion of a second intron in MFG vectors may influence packaging and/or LTR-driven transgene expression. We constructed two MFG retroviral vectors, MFG/hFIXc and MFG/hFIXm2, containing human factor IX (hFIX) cDNA without and with a 0.3-kb hFIX intron, respectively. When tested with primary mouse myoblasts or HepG2 cells in culture for transient expression activity, pMFG/hFIXm2 plasmid produced two-to-three fold higher hFIX than pMFG/hFIXc. These vectors produced equivalent retroviral titers from packaging cells. In transduced cells, the splicing of the MFG intron in the retroviral transcripts occured at a similar efficiency; however, MFG/hFIXc virus gave two-fold higher hFIX expression than that of the MFG/hFIXm2 viral infection. Analyses of MFG/hFIXm2 virion RNA and transduced cell genomic DNA suggested that, although the hFIX intron containing viral RNA are packaged, these viruses fail to integrate their transgenes into the genome of transduced cells, suggesting a block at the reverse transcription and/or integration steps. Similar results were also obtained with the prototype vectors, LIXcSN and LIXm2SN, lacking the MFG intron. Together, these results suggest that a hFIX cDNA sequence in the retroviral vectors performs better over hFIX intron-containing minigene.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42460/1/705-146-3-601_11460601.pd

Bone marrow stromal cells as a genetic platformfor systemic delivery of therapeutic proteins in vivo : human factor IX model

Background Hemophilia B is an X-linked bleeding disorder that results from a deficiency in functional coagulation factor IX (hFIX). In patients lacking FIX, the intrinsic coagulation pathway is disrupted leading to a lifelong, debilitating and sometimes fatal disease. Methods We have developed an ex vivo gene therapy system using genetically modified bone marrow stromal cells (BMSCs) as a platform for sustained delivery of therapeutic proteins into the general circulation. This model exploits the ability of BMSCs to form localized ectopic ossicles when transplanted in vivo . BMSCs were transduced with MFG-hFIX, a retroviral construct directing the expression of hFIX. The biological activity of hFIX expressed by these cells was assessed in vitro and in vivo . Results Transduced cells produced biologically active hFIX in vitro with a specific activity of 90% and expressed hFIX at levels of ∼497 ng/10 6 cells/24 h and 322 ng/10 6 cells/24 h for human and porcine cells, respectively. The secretion of hFIX was confirmed by Western blot analysis of the conditioned medium using a hFIX-specific antibody. Transduced BMSCs (8 × 10 6 cells per animal) were transplanted within scaffolds into subcutaneous sites in immunocompromised mice. At 1 week post-implantation, serum samples contained hFIX at levels greater than 25 ng/ml. Circulating levels of hFIX gradually decreased to 11.5 ng/ml at 1 month post-implantation and declined to a stable level at 6.1 ng/ml at 4 months. Conclusions These findings demonstrate that genetically modified BMSCs can continuously secrete biologically active hFIX from self-contained ectopic ossicles in vivo , and thus represent a novel delivery system for releasing therapeutic proteins into the circulation. Copyright © 2002 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35232/1/292_ftp.pd

Construction of Human Factor IX Expression Vectors in Retroviral Vector Frames Optimized for Muscle Cells

Overview summary Skeletal muscle cells may serve as an efficient medium for systemic production of transgene products in a gene therapy. Retroviral vector constructs were prepared using human factor IX minigenes, various muscle-specific or nonspecific promoters, and muscle creatine kinase enhancer. These vectors were tested systematically to determine an optimal basic structure for producing factor IX in mouse skeletal muscle cells. The basic retroviral vector structure for an optimal expression of factor IX in skeletal muscle cells may contain an internal factor IX expression cassette composed of a β-actin promoter, muscle creatine kinase enhancer, and human factor IX minigene in a reverse orientation relative to the 5′ long terminal repeat.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63364/1/hum.1996.7.14-1743.pd

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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29323/1/0000388.pd