Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Expression of human factor IX in mice after injection of genetically modified myoblasts. 156 26

We have used molecular conjugates containing combinations of DNA, adenovirus, polylysine, and transferrin to transfect primary cells derived from canines with hemophilia B (factor IX deficiency), as well as a canine epithelial cell line. Transfection of canine hemophilia B fibroblasts with molecular conjugates resulted in efficient transfection and expression of luciferase DNA-adenovirus-polylysine (AdpL) conjugates or luciferase DNA-adenovirus-polylysine-transferrin (hTfpL/AdpL) conjugates. No expression in canine hemophilia B fibroblasts was evident after exposure to DNA alone, or DNA conjugated with polylysine and transferrin. Transfection efficiencies of 50% or more could be demonstrated in cells transfected with a beta-galactosidase reporter gene as part of an hTfpL/AdpL molecular conjugate. Transfection with canine factor IX AdpL conjugates or canine factor IX hTfpL/AdpL conjugates resulted in factor IX expression for more than 2 weeks in vitro in hemophilia B canine fibroblasts. Maximum levels of expression of over 700 ng of canine factor IX/10(6) cells/24 hr were demonstrated in fibroblasts after transfection with canine factor IX hTfpL/AdpL conjugates. Similar conjugates were used to transfect hemophilia B canine bone marrow stromal cells and Madin-Darby canine kidney cells that also expressed canine factor IX. The use of molecular conjugates to transfect primary cells may be feasible as a means of in vitro or in vivo gene therapy for hemophilia B, and can be tested in the canine hemophilia B model.
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PMID:Efficient transfection of primary cells in a canine hemophilia B model using adenovirus-polylysine-DNA complexes. 801 46

Skeletal muscle is a privileged target for long-term rAAV-mediated gene transfer in mouse, rat, dog and non-human primates. Intramuscular injections of rAAV encoding human factor IX in hemophilia B patients have been initiated, based on promising results gathered in affected dogs. We found that intramuscular rAAV administration in rats resulted in restricted transduction essentially along the myofibers axis with poor lateral diffusion. This suggested that the transduction rate might be limited by the ability of the virus to reach sites distant from the injection point. We tested whether hyaluronidase, an enzyme which dissociates the extracellular matrix, could enhance vector diffusion when injected in the rat muscle before administration of rAAV encoding either nuclear-localized beta-galactosidase (rAAVCMVnlsLacZ) or the human alpha-1-antitrypsin (rAAVCMVhAAT) under the control of the cytomegalovirus immediate--early promoter (CMV). The results showed that pretreatment of the rat anterior tibialis muscle with hyaluronidase resulted in: (1) a larger diffusion of the virus indicated by an increase in the area containing LacZ-transduced fibers, and (2) a two- to three-fold increase of transduction efficiency measured by the number of LacZ-positive fibers or by the hAAT serum concentration. We also provide evidence that hyaluronidase was well tolerated and was not associated with short- or long-term toxicity evaluated by morphological studies. Finally, in our experimental conditions, hyaluronidase did not promote rAAV dissemination to other organs as assessed by PCR to detect vector sequences. We conclude that pretreatment of skeletal muscle by hyaluronidase, a clinically available reagent, was harmless and resulted in a consistent and significant increase in rAAV diffusion and transduction levels.
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PMID:Hyaluronidase enhances recombinant adeno-associated virus (rAAV)-mediated gene transfer in the rat skeletal muscle. 1098 69

Hemophilia is a particularly attractive model for developing a gene transfer approach for the treatment of disease. The protein is very well characterized, the genes are cloned and available, and there are large and small animal models of the disease. Moreover, in contrast to many diseases, there is no requirement for a specific target tissue for gene delivery, and the gene product itself does not require precise regulation of expression. Earlier efforts to establish a gene transfer approach to the treatment of hemophilia had failed to achieve the twin goals of long-term expression at levels that were adequate to result in phenotypic improvement of the disease. We have exploited advances in vector development that occurred in the mid-1990s to establish an experimental basis for an AAV (adeno-associated viral vector)-mediated gene transfer approach to the treatment of hemophilia B. Based on the observation that introduction of an AAV vector into skeletal muscle could result in sustained expression of beta-galactosidase, we engineered an AAV vector expressing human factor IX and demonstrated in immunodeficient mice that intramuscular injection of the vector resulted in long-term expression of the secreted transgene product factor IX. Subsequently, we generated an AAV vector expressing canine factor IX; intramuscular injection into dogs with severe hemophilia B resulted in a dose-dependent increase in circulating levels of factor IX. The animal treated at the highest dose showed prolonged expression (>3 years and still under observation) at a level (70 ng/ml, 1.4% of normal circulating levels of factor IX) likely to result in phenotypic improvement in humans. Detailed studies in tissue culture using human myotubes have shown that muscle cells are capable of executing the posttranslational modifications required for activity of factor IX, and that the specific activity of myotube-synthesized factor IX is similar to that of hepatocyte-synthesized material, although some details of posttranslational processing differ. Based on these and other safety and efficacy studies, a clinical trial of AAV-mediated, muscle-directed gene transfer for hemophilia B has been initiated. The study has a dose-escalation design, with three subjects to be enrolled in three dose cohorts beginning with a dose of 2 x 10(11) vg/kg. Results in the initial dose cohort showed no evidence of toxicity associated with vector administration or transgene expression. Analysis of muscle biopsies done on injected tissue showed clear evidence of gene transfer by PCR and Southern blot and of gene expression by immunocytochemistry. The general characteristics of muscle transduction appear similar in humans and in other animal models. The goal of dose escalation is to find a dose that is nontoxic but that results in circulating levels of factor IX >1% in all patients.
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PMID:AAV-mediated gene transfer for hemophilia. 1179 24