Document Type : Original Research
Abstract
Background and Objectives: Significant progress has been made in treatment of hemophilia. Ex-vivo gene therapy is going popular due to the capability of this method in using isogenic cells for genetic manipulation and reintroducing them into same host after proliferation. Most gene therapy techniques use viral vectors, which usually harbor a strong and non-specific promoter (e.g. CMV early promoter) for driving the downstream gene. This may be a disadvantage due to uncontrollable nature of gene expression. In addition, considering the potentials of recently introduced stem cells as reservoirs and their potential to differentiate to other cell lines, uncontrolled expression may have unknown outcomes. To make gene therapy of hemophilia more resembling to the nature, we supposed F8 promoter might be a good candidate for driving downstream F8 coding sequence.
Materials and Methods: To test our hypothesis, we designed and constructed a DNA construct by PCR, which harbors EGFP coding sequence downstream to mouse F8 promoter and transfected it to a mouse hepatoma cell line. Transfection was assayed qualitatively by fluorescent microscopy.
Results: Fluorescence was detected in transfected cells a sign of presence of EGFP.
Conclusion: F8 promoter can drive expression of downstream genes, a capability which and may have potential to be used in gene therapy of hemophilia. A conclusion that should be examined by further studies.
Keywords
- Kaufman RJ. Advances toward gene therapy for hemophilia at the millennium. Hum Gene Ther 1999; 1;10(13):2091-107.
- Lu H, Chen L, Wang J, Huack B, Sarkar R, Zhou S, et al. Complete correction of hemophilia A with adeno-associated viral vectors containing a full-size expression cassette. Hum Gene Ther 2008;19(6):648-54.
- Kay MA, High K. Gene therapy for the hemophilias. Proc Natl Acad Sci USA 1999; 31;96(18):9973-5.
- Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S, et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 2005;2;435(7042):646-51.
- Roehl HH, Leibbrandt ME, Greengard JS, Kamantigue E, Glass WG, Giedlin M, et al. Analysis of testes and semen from rabbits treated by intravenous injection with a retroviral vector encoding the human factor VIII gene: no evidence of germ line transduction. Hum Gene Ther 2000;10;11(18):2529-40.
- Chuah MK, Schiedner G, Thorrez L, Brown B, Johnston M, Gillijns V, et al. Therapeutic factor VIII levels and negligible toxicity in mouse and dog models of hemophilia A following gene therapy with high-capacity adenoviral vectors. Blood 2003; 1;101(5):1734-43.
- Smith TA, Mehaffey MG, Kayda DB, Saunders JM, Yei S, Trapnell BC, et al. Adenovirus mediated expression of therapeutic plasma levels of human factor IX in mice. Nat Genet 1993;5(4):397-402.
- Kay MA, Manno CS, Ragni MV, Larson PJ, Couto LB, McClelland A, et al. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat Genet 2000; 24(3):257-61.
- Lai Y, Yue Y, Liu M, Ghosh A, Engelhardt JF, Chamberlain JS, et al. Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors. Nat Biotechnol 2005;23(11):1435-9.
- Roth DA, Tawa NE, Jr., O'Brien JM, Treco DA, Selden RF. Nonviral transfer of the gene encoding coagulation factor VIII in patients with severe hemophilia A. N Engl J Med 2001;344(23):1735-42.
- Tang MX, Szoka FC. The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes. Gene Ther 1997;4(8):823-32.
- Herzog RW, Cao O, Hagstrom JN, Wang L. Gene therapy for treatment of inherited haematological disorders. Expert Opin Biol Ther 2006;6(5):509-22.
- Saenko EL, Ananyeva NM, Moayeri M, Ramezani A, Hawley RG. Development of improved factor VIII molecules and new gene transfer approaches for hemophilia A. Curr Gene Ther 2003;3(1):27-41.
- Zhang Y, Fan Y, Zhao L, Tang H. [Differentiation of mouse bone marrow mesenchymal stem cells into hepatocyte in vivo]. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi 2005;22(3):521-4.
- Ong K, Horsfall W, Conway EM, Schuh AC. Early embryonic expression of murine coagulation system components. Thromb Haemost 2000;84(6):1023-30.
- Mus musculus Promoter Database (MmPD) http://rulai.cshl.edu/cgi-bin/CSHLmpd2/mmpd.pl. (Version 2.33, May 2005, NCBI build 33)
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