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Thromb Haemost 2004; 92(02): 317-327
DOI: 10.1160/TH04-02-0068
Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH

Expression of therapeutic levels of factor VIII in hemophilia A mice using a novel adeno/adeno-associated hybrid virus

Dmitri V. Gnatenko
1   Department of Medicine, State University of New York, Stony Brook, New York, USA
,
Yong Wu
1   Department of Medicine, State University of New York, Stony Brook, New York, USA
,
Jolyon Jesty
1   Department of Medicine, State University of New York, Stony Brook, New York, USA
,
Andrea L. Damon
1   Department of Medicine, State University of New York, Stony Brook, New York, USA
3   Program in Genetics, State University of New York at Stony Brook, New York, USA
,
Patrick Hearing
2   Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, New York, USA
3   Program in Genetics, State University of New York at Stony Brook, New York, USA
,
Wadie F. Bahou
1   Department of Medicine, State University of New York, Stony Brook, New York, USA
2   Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook, New York, USA
3   Program in Genetics, State University of New York at Stony Brook, New York, USA
› Author AffiliationsFinancial support: This work has been supported by grants HL53665 (WFB) and AI41636 (PH) from the National Institutes of Health
Further Information

Publication History

Received 04 February 2004

Accepted after resubmission 01 June 2004

Publication Date:
30 November 2017 (online)

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Summary

We have generated an E1a/E1b/E3-deleted adeno/adeno-associated (Ad/AAV) hybrid virus driven by a small nuclear RNA (pHU1-1) promoter for expression of a B domain-deleted (Thr761-Asn1639) factor VIII transgene (FVIIIΔ761-1639). Productive replication of Ad/AAV/FVIIIΔ761-1639 in AAV repexpressing cells resulted in generation of monomeric and dimeric mini-adenoviral (mAd) replicative forms that retained the AAV integration elements (mAd/FVIIIΔ761-1639). In vitro studies using Ad/AAV/FVIIIΔ761-1639 generated ∼2-logs greater FVIII activity than mAd/FVIIIΔ761-1639. To determine its capacity for in vivo excision and/or genomic integration, Ad/AAV/FVIIIΔ761-1639 was injected by tail vein into three groups of hemophilia A mice (2 X 1011 vp [n = 3]; 4 X 1011 vp [n = 3]; 8 X 1011 vp [n = 3]), with clear concentration-dependent increase in FVIII activity (range 160-510 mU/ml; plasma activity 16% – 51% of normal). Peak activity was seen by Day (D) 5, with slow return to baseline by D28 (0.1 – 0.9% activity); in only 3/9 mice was loss of FVIII activity associated with development of anti-FVIII antibodies. Quantitative-PCR using genomic DNA isolated from D28 liver, spleen, heart, lungs, and kidney demonstrated the highest concentration in liver (∼10 genomes/ cell), with little to no organ toxicity at early (D5 or 6) or late (D28) post-infusion time points. There was no evidence for spontaneous transgene excision or genomic integration in vivo as evaluated by quantitative PCR and genomic blotting. These data establish (i) the feasibility and applicability of developing high-titer Ad/AAV hybrid viruses for FVIII delivery using a small cellular promoter, (ii) the potential utility of this virus for generation of “gutted” monomeric and dimeric mAD/FVIII retaining AAV integration elements, and (iii) that the development of strategies for regulated Rep68/78 co-expression may provide a novel approach for excision, integration, and long-term FVIII transgene expression.

Keywords

Gene therapy - haemophilia A - hybrid vector
 

  • References

  • 1 Hoyer LW. Hemophilia A. N Engl J Med 1994; 330: 38-47.
    CrossrefPubMedGoogle Scholar
  • 2 Vandendriessche T, Collen D, Chuah MK. Gene therapy for the hemophilias. J Thromb Haemost 2003; 1: 1550-1558.
    CrossrefPubMedGoogle Scholar
  • 3 Balague C, Zhou J, Dai Y. et al. Sustained high-level expression of full-length human factor VIII and restoration of clotting activity in hemophilic mice using a minimal adenovirus vector. Blood 2000; 95: 820-828.
    PubMedGoogle Scholar
  • 4 Chuah MK. 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; 101: 1734-1743.
    CrossrefPubMedGoogle Scholar
  • 5 Reddy PS. et al. Sustained human factor VIII expression in hemophilia A mice following systemic delivery of a gutless adenoviral vector. Mol Ther 2002; 5: 63-73.
    CrossrefPubMedGoogle Scholar
  • 6 Sandalon Z, Gnatenko DV, Bahou WF. et al. Adeno-associated virus (AAV) Rep protein enhances the generation of a recombinant mini-adenovirus (Ad) utilizing an Ad/AAV hybrid virus. J Virol 2000; 74: 10381-9.
    CrossrefPubMedGoogle Scholar
  • 7 Lieber A, Steinwaerder DS, Carlson CA. et al. Integrating adenovirus-adeno-associated virus hybrid vectors devoid of all viral genes. J Virol 1999; 73: 9314-9324.
    PubMedGoogle Scholar
  • 8 Kotin RM. et al. Site-specific integration by adeno-associated virus. Proc Nat Acad Sci USA 1990; 87: 2211-2215.
    CrossrefPubMedGoogle Scholar
  • 9 Philpott NJ, Giraud-Wali C, Dupuis C. et al. Efficient integration of recombinant adenoassociated virus DNA vectors requires a p5-rep sequence in cis. J Virol 2002; 76: 5411-5421.
    CrossrefPubMedGoogle Scholar
  • 10 Philpott NJ. et al. A p5 integration efficiency element mediates Rep-dependent integration into AAVS1 at chromosome 19. Proc Natl Acad Sci USA 2002; 99: 12381-5.
    CrossrefPubMedGoogle Scholar
  • 11 Gnatenko DV, Saenko E, Jesty J. et al. Human factor VIII can be packaged and functionally expressed in an adeno-associated virus background: applicability to hemophilia A gene therapy. Br J Haematol 1999; 104: 27-36.
    CrossrefPubMedGoogle Scholar
  • 12 Chao H. et al. Sustained expression of human factor VIII in mice using a parvovirus-based vector. Blood 2000; 95: 1594-1599.
    CrossrefPubMedGoogle Scholar
  • 13 Clark KR, Voulgaropoulou F, Johnson PR. A stable cell line carrying adenovirus-inducible rep and cap genes allows for infectivity titration of adeno-associated virus vectors. Gene Ther 1996; 3: 1124-1132.
    PubMedGoogle Scholar
  • 14 Connelly S, Gardner JM, McClelland A. et al. High-level tissue-specific expression of functional human factor VIII in mice. Hum Gene Ther 1996; 7: 183-195.
    CrossrefPubMedGoogle Scholar
  • 15 Ryan JH, Zolotukhin S, Muzyczka N. Sequence requirements for binding of Rep68 to the adeno-associated virus terminal repeats. J Virol 1996; 70: 1542-1553.
    PubMedGoogle Scholar
  • 16 Xiao X, Xiao W, Li J. et al. A novel 165-basepair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle. J Virol 1997; 71: 941-948.
    PubMedGoogle Scholar
  • 17 Wang XS, Ponnazhagan S, Srivastava A. Rescue and replication signals of the adenoassociated virus 2 genome. J Mol Biol 1995; 250: 573-580.
    CrossrefPubMedGoogle Scholar
  • 18 Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol 1967; 26: 365-369.
    CrossrefPubMedGoogle Scholar
  • 19 Bi L, Lawler AM, Antonarakis E. et al. Targeted disruption of the mouse factor VIII gene produces a model of haemophilia. Nat Genet 1995; 10: 119-121.
    CrossrefPubMedGoogle Scholar
  • 20 Schmidt VA, Nierman WC, Maglott DR. et al. The human proteinase-activated receptor-3 (PAR-3) gene. Identification within a Par gene cluster and characterization in vascular endothelial cells and platelets. J Biol Chem 1998; 273: 15061-8.
    CrossrefPubMedGoogle Scholar
  • 21 Gnatenko DV, Dunn JJ, McCorkle SR. et al. Transcript profiling of human platelets using microarray and serial analysis of gene expression. Blood 2003; 101: 2285-2293.
    CrossrefPubMedGoogle Scholar
  • 22 Wang XS, Ponnazhagan S, Srivastava A. Rescue and replication of adeno-associated virus type 2 as well as vector DNA sequences from recombinant plasmids containing deletions in the viral inverted terminal repeats: selective encapsidation of viral genomes in progeny virions. J Virol 1996; 70: 1668-1677.
    PubMedGoogle Scholar
  • 23 Im DS, Muzyczka N. The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity. Cell 1990; 61: 447-457.
    CrossrefPubMedGoogle Scholar
  • 24 Gottlieb J, Muzyczka N. In vitro excision of adeno-associated virus DNA from recombinant plasmids: isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences. Mol Cell Biol 1988; 8: 2513-2522.
    CrossrefPubMedGoogle Scholar
  • 25 Berns KI, Giraud C. Biology of adeno-associated virus. Curr Top Microbiol Immunol 1996; 218: 1-23.
    PubMedGoogle Scholar
  • 26 Greber UF, Webster P, Weber J. et al. The role of the adenovirus protease on virus entry into cells. EMBO J 1996; 15: 1766-1777.
    PubMedGoogle Scholar
  • 27 Connelly S, Andrews JL, Gallo AM. et al. Sustained phenotypic correction of murine hemophilia A by in vivo gene therapy. Blood 1998; 91: 3273-3281.
    PubMedGoogle Scholar
  • 28 Nakai H, Yant SR, Storm TA. et al. Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo. J Virol 2001; 75: 6969-6976.
    CrossrefPubMedGoogle Scholar
  • 29 Gallo-Penn AM, Shirley PS, Andrews JL. et al. Systemic delivery of an adenoviral vector encoding canine factor VIII results in shortterm phenotypic correction, inhibitor development, and biphasic liver toxicity in hemophilia A dogs. Blood 2001; 97: 107-113.
    CrossrefPubMedGoogle Scholar
  • 30 Chirmule N. et al. Immune responses to adenovirus and adeno-associated virus in humans. Gene Ther 1999; 6: 1574-1583.
    CrossrefPubMedGoogle Scholar
  • 31 Halbert CL, Rutledge EA, Allen JM. et al. Repeat transduction in the mouse lung by using adeno-associated virus vectors with different serotypes. J Virol 2000; 74: 1524-1532.
    CrossrefPubMedGoogle Scholar
  • 32 Chirmule N. et al. Humoral immunity to adeno-associated virus type 2 vectors following administration to murine and nonhuman primate muscle. J Virol 2000; 74: 2420-2425.
    CrossrefPubMedGoogle Scholar
  • 33 Scallan CD, Lillicrap D, Jiang H. et al. Sustained phenotypic correction of canine hemophilia A using an adeno-associated viral vector. Blood 2003; 102: 2031-2037.
    CrossrefPubMedGoogle Scholar
  • 34 Recchia A, Parks RJ, Lamartina S. et al. Sitespecific integration mediated by a hybrid adenovirus/adeno-associated virus vector. Proc Natl Acad Sci U S A 1999; 96: 2615-2620.
    CrossrefPubMedGoogle Scholar
  • 35 http://www.criver.com/techdocs/baseline.html
    PubMed
  • 36 Jesty J, Godfrey HP. Parlin, a general microcomputer program for parallel-line analysis of bioassays. Am J Clin Pathol 1986; 85: 485-489.
    CrossrefPubMedGoogle Scholar