Expression of Coagulation Factor IX in a Haematopoietic Cell Line^[*]

Summary

We have developed a gene therapy project for haemophilia B which aims to express factor IX (FIX) in haematopoietic lineage. Haematopoietic stem cells and subsequent megakaryocyte-derived cells represent the target cells of this approach. Our speculation is that platelets can deliver the coagulation factor at the site of injury, and subsequently correct the haemostasis defect. In order to direct FIX expression in cells from the megakaryocytic lineage, we designed a FIX cassette where the FIX cDNA was placed under the control of the tissue-specific glycoprotein IIb (GPIIb) promoter. In stably transfected HEL cells, FIX production was higher when driven by the GPIIb promoter compared to the CMV promoter. Using a cassette containing both the GPIIb promoter and a truncated FIX intron 1, FIX synthesis was dramatically increased in HEL cells. Northern blot analysis demonstrated an increase in FIX mRNA amounts, which paralleled with an increase of FIX antigen in the culture supernatants. Using a one-stage clotting assay and an activation by FXIa and FVIIa/TF, the HEL-derived recombinant FIX was shown to be a biologically active protein. This recombinant protein exhibited a 60-kDa molecular mass and was more heterogeneous than plasma immunopurified FIX (Mononine®). The molecular mass difference could be partly explained by a different glycosylation pattern. The GPIIb promoter appears therefore to be a very attractive sequence to specifically direct FIX production in the megakaryocytic compartment of hematopoietic cells. These data also demonstrate that hematopoietic cells may represent potential target cells in an approach to gene therapy of haemophilia B.

Keywords

Factor IX - haemophilia - glycoprotein IIb promoter - haematopoietic cell line

• References

• 1 Salier JP, Hirosawa S, Kurachi K. Functional characterization of the 5’-regulatory region of human factor IX gene. J Biol Chem 1990; 265: 7062-8.
• 2 Choo KH, Gould KG, Rees DJ, Brownlee GG. Molecular cloning of the gene for human anti-haemophilic factor IX. Nature 1982; 299: 178-80.
• 3 Chance PF, Dyer KA, Kurachi K, Yoshitake S, Ropers HH, Wieacker P, Gartler SM. Regional localization of the human factor IX gene by molecular hybridization. Hum Genet 1983; 65: 207-8.
• 4 Boyd Y, Buckle VJ, Munro EA, Choo KH, Migeon BR, Craig IW. Assignment of the haemophilia B (factor IX) locus to the q26-qter region of the X chromosome. Ann Hum Genet 1984; 48: 145-52.
• 5 Camerino G, Grzeschik KH, Jaye M, De La Salle H, Tolstoshev P, Lecocq JP, Heilig R, Mandel JL. Regional localization on the human X chromosome and polymorphism of the coagulation factor IX gene (hemophilia B locus). Proc Natl Acad Sci U S A 1984; 81: 498-502.
• 6 Furie BC, Furie B. Biosynthesis of factor IX: implications for gene therapy. Thromb Haemost 1995; 74: 274-7.
• 7 Kaufman RJ. Post-translational modifications required for coagulation factor secretion and function. Thromb Haemost 1998; 79: 1068-79.
• 8 Furie B, Furie BC. The molecular basis of blood coagulation. Cell 1988; 53: 505-18.
• 9 Jallat S, Perraud F, Dalemans W, Balland A, Dieterle A, Faure T, Meulien P, Pavirani A. Characterization of recombinant human factor IX expressed in transgenic mice and in derived trans-immortalized hepatic cell lines. EMBO J 1990; 09: 3295-301.
• 10 Axelrod JH, Read MS, Brinkhous KM, Verma IM. Phenotypic correction of factor IX deficiency in skin fibroblasts of hemophilic dogs. Proc Natl Acad Sci U S A 1990; 87: 5173-7.
• 11 Dai Y, Roman M, Naviaux RK, Verma IM. Gene therapy via primary myoblasts: long-term expression of factor IX protein following transplantation in vivo. Proc Natl Acad Sci U S A 1992; 89: 10892-5.
• 12 Yao SN, Kurachi K. Expression of human factor IX in mice after injection of genetically modified myoblasts. Proc Natl Acad Sci U S A 1992; 89: 3357-61.
• 13 Gerrard AJ, Austen D, Brownlee G. Recombinant factor IX secreted by transduced human keratinocytes is biologically active. Br J Haematol 1996; 95: 561-3.
• 14 Hurwitz DR, Kirchgesser M, Merrill W, Galanopoulos T, McGrath CA, Emami S, Hansen M, Cherington V, Appel JM, Bizinkauskas CB, Brackmann HH, Levine PH, Greenberger JS. Systemic delivery of human growth hormone or human factor IX in dogs by reintroduced genetically modified autologous bone marrow stromal cells. Hum Gene Ther 1997; 08: 137-56.
• 15 Phillips DR, Charo if, Parise LV, Fitzgerald LA. The platelet membrane glycoprotein IIb-IIIa complex. Blood 1988; 71: 831-43.
• 16 Uzan G, Frachet P, Lajmanovich A, Prandini MH, Denarier E, Duperray A, Loftus J, Ginsberg M, Plow E, Marguerie G. cDNA clones for human platelet GPIIb corresponding to mRNA from megakaryocytes and HEL cells. Evidence for an extensive homology to other Arg-Gly-Asp adhesion receptors. Eur J Biochem 1988; 171: 87-93.
• 17 Frachet P, Uzan G, Thevenon D, Denarier E, Prandini M, Marguerie G. GPIIb and GPIIIa amino acid sequences deduced from human megakaryocyte cDNAs. Mol Biol Rep 1990; 14 (01) 27-33.
• 18 Berridge MV, Ralph SJ, Tan AS. Cell-lineage antigens of the stem cellmegakaryocyte-platelet lineage are associated with the platelet IIb-IIIa glycoprotein complex. Blood 1985; 66: 76-85.
• 19 Fraser JK, Leahy MF, Berridge MV. Expression of antigens of the platelet glycoprotein IIb/IIIa complex on human hematopoietic stem cells. Blood 1986; 68: 762-9.
• 20 Okumura N, Tsuji K, Nakahata T. Changes in cell surface antigen expressions during proliferation and differentiation of human erythroid progenitors. Blood 1992; 80: 642-50.
• 21 Uzan G, Prenant M, Prandini MH, Martin F, Marguerie G. Tissue-specific expression of the platelet GPIIb gene. J Biol Chem 1991; 266: 8932-9.
• 22 Prandini MH, Uzan G, Martin F, Thevenon D, Marguerie G. Characterization of a specific erythromegakaryocytic enhancer within the glycoprotein IIb promoter. J Biol Chem 1992; 267: 10370-4.
• 23 Prandini MH, Martin F, Thevenon D, Uzan G. The tissue-specific transcriptional regulation of the megakaryocytic glycoprotein IIb gene is controlled by interactions between a repressor and positive cis-acting elements. Blood 1996; 88: 2062-70.
• 24 Fong AM, Santoro SA. Transcriptional regulation of ex_IIb integrin gene expression during megakaryocytic differentiation of K562 cells. Role of a silencer element. J Biol Chem 1994; 269: 18441-7.
• 25 Enjolras N, Rodriguez MH, Plantier JL, Maurice M, Attali O, Negrier C. The three in-frame ATG, clustered in the translation initiation sequence of human factor IX gene, are required for an optimal protein production. Thromb Haemost 1999; 82: 1264-9.
• 26 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-5.
• 27 Long MW, Heffner CH, Williams JL, Peters C, Prochownik EV. Regulation of megakaryocyte phenotype in human erythroleukemia cells. J Clin Invest 1990; 85: 1072-84.
• 28 de la Salle H, Altenburger W, Elkaim R, Dott K, Dieterle A, Drillien R, Cazenave JP, Tolstoshev P, Lecocq JP. Active -y-carboxylated human factor IX expressed using recombinant DNA techniques. Nature 1985; 316: 268-70.
• 29 Gordon EM, Tang H, Salazar RL, Kohn DB. Expression of coagulation factor IX (Christmas factor) in human hepatoma (HepG2) cell cultures after retroviral vector-mediated transfer. Am J Pediatr Hematol Oncol 1993; 15: 196-203.
• 30 Bond M, Jankowski M, Patel H, Karnik S, Strang A, Xu B, Rouse J, Koza S, Letwin B, Steckert J, Amphlett G, Scoble H. Biochemical characterization of recombinant factor IX. Semin Hematol 1998; 35: 11-7.
• 31 Herzog RW, Hagstrom JN, Kung SH, Tai SJ, Wilson JM, Fisher KJ, High KA. Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno-associated virus. Proc Natl Acad Sci U S A 1997; 94: 5804-9.
• 32 Herzog RW, Yang EY, Couto LB, Hagstrom JN, Elwell D, Fields PA, Burton M, Bellinger DA, Read MS, Brinkhous KM, Podsakoff GM, Nichols TC, Kurtzman GJ, High KA. Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector. Nat Med 1999; 05: 56-63.
• 33 Snyder RO, Miao CH, Patijn GA, Spratt SK, Danos O, Nagy D, Gown AM, Winther B, Meuse L, Cohen LK, Thompson AR, Kay MA. Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors. Nat Genet 1997; 16: 270-6.
• 34 Snyder RO, Miao C, Meuse L, Tubb J, Donahue BA, Lin HF, Stafford DW, Patel S, Thompson AR, Nichols T, Read MS, Bellinger DA, Brinkhous KM, Kay MA. Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors. Nat Med 1999; 05: 64-70.
• 35 Kay MA, Manno CS, Ragni MV, Larson PJ, Couto LB, McClelland A, Glader B, Chew AJ, Tai SJ, Herzog RW, Arruda V, Johnson F, Scallan C, Skarsgard E, Flake AW, High KA. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat Genet 2000; 24: 257-61.
• 36 White SJ, Page SM, Margaritis P, Brownlee GG. Long-term expression of human clotting factor IX from retrovirally transduced primary human keratinocytes in vivo. Hum Gene Ther 1998; 09: 1187-95.
• 37 Hoeben RC, Einerhand MP, Briet E, van Ormondt H, Valerio D, van der Eb AJ. Toward gene therapy in haemophilia A: retrovirus-mediated transfer of a factor VIII gene into murine haematopoietic progenitor cells. Thromb Haemost 1992; 67: 341-5.
• 38 Challita PM, Kohn DB. Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo. Proc Natl Acad Sci USA 1994; 91: 2567-71.
• 39 Nakai H, Herzog RW, Hagstrom JN, Walter J, Kung SH, Yang EY, Tai SJ, Iwaki Y, Kurtzman GJ, Fisher KJ, Colosi P, Couto LB, High KA. Adeno-associated viral vector-mediated gene transfer of human blood coagulation factor IX into mouse liver. Blood 1998; 91: 4600-7.
• 40 Baskar JF, Smith PP, Nilaver G, Jupp RA, Hoffmann S, Peffer NJ, Tenney DJ, Colberg-Poley AM, Ghazal P, Nelson JA. The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice. J Virol 1996; 70: 3207-14.
• 41 Wang JM, Zheng H, Sugahara Y, Tan J, Yao SN, Olson E, Kurachi K. Construction of human factor IX expression vectors in retroviral vector frames optimized for muscle cells. Hum Gene Ther 1996; 07: 1743-56.
• 42 Hagstrom JN, Couto LB, Scallan C, Burton M, McCleland ML, Fields PA, Arruda VR, Herzog RW, High KA. Improved muscle-derived expression of human coagulation factor IX from a skeletal actin/CMV hybrid enhancer/ promoter. Blood 2000; 95: 2536-42.
• 43 Miao CH, Ohashi K, Patijn GA, Meuse L, Ye X, Thompson AR, Kay MA. Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro. Mol Ther 2000; 01: 522-32.
• 44 Kurachi S, Hitomi Y, Furukawa M, Kurachi K. Role of intron I in expression of the human factor IX gene. J Biol Chem 1995; 270: 5276-81.
• 45 Monahan PE, Samulski RJ, Tazelaar J, Xiao X, Nichols TC, Bellinger DA, Read MS, Walsh CE. Direct intramuscular injection with recombinant AAV vectors results in sustained expression in a dog model of hemophilia. Gene Ther 1998; 05: 40-9.
• 46 Bristol JA, Freedman SJ, Furie BC, Furie B. Profactor IX: the propeptide inhibits binding to membrane surfaces and activation by factor XIa. Biochemistry 1994; 33: 14136-43.
• 47 Berkner KL. Expression of recombinant vitamin K-dependent proteins in mammalian cells: factors IX and VII. Methods Enzymol 1993; 222: 450-77.
• 48 Wilcox DA, Olsen JC, Ishizawa L, Bray PF, French DL, Steeber DA, Bell WR, Griffith M, White GCn. Megakaryocyte-targeted synthesis of the integrin ?₃-subunit results in the phenotypic correction of Glanzmann thrombasthenia. Blood 2000; 95: 3645-51.
• 49 Cavazzana-Calvo M, Hacein-Bey S, de Saint GBasile, Gross F, Yvon E, Nusbaum P, Selz F, Hue C, Certain S, Casanova JL, Bousso P, Deist FL, Fischer A. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000; 288: 669-72.
• 50 Wilcox DA, Rosenberg JB, White GCn, Montgomery RR. Factor VIII (FVIII) trafficks to megakaryocyte ex-granules following retroviral transduction of human CD34+ cells. Blood 1999; 94: 1962 (abstract).
• 51 Plantier JL, Enjolras N, Rodriguez MH, Négrier C. P selectin tail targets the Factor IX in secretory granules of AtT20 cells. Thromb Haemost 1999; supplement: 1505 (abstract).