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Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector

Nature Medicine volume 5, pages 56–63 (1999)Cite this article

Abstract

Hemophilia B is a severe X-linked bleeding diathesis caused by the absence of functional blood coagulation factor IX, and is an excellent candidate for treatment of a genetic disease by gene therapy. Using an adeno-associated viral vector, we demonstrate sustained expression (>17 months) of factor IX in a large-animal model at levels that would have a therapeutic effect in humans (up to 70 ng/ml, adequate to achieve phenotypic correction, in an animal injected with 8.5 × 10 12 vector particles/kg). The five hemophilia B dogs treated showed stable, vector dose-dependent partial correction of the whole blood clotting time and, at higher doses, of the activated partial thromboplastin time. In contrast to other viral gene delivery systems, this minimally invasive procedure, consisting of a series of percutaneous intramuscular injections at a single timepoint, was not associated with local or systemic toxicity. Efficient gene transfer to muscle was shown by immunofluorescence staining and DNA analysis of biopsied tissue. Immune responses against factor IX were either absent or transient. These data provide strong support for the feasibility of the approach for therapy of human subjects.

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Figure 1: a–c, Hematoxylin and eosin staining of cross-sections of tibialis anterior muscle of a normal dog injected with 2.8 × 1011 particles of AAV-CMV-cF.IX.
Figure 2: a–d, WBCT of hemophilia B dogs as a function of time after intramuscular injection with AAV-CMV-cF.IX.
Figure 3: Canine F.IX levels in plasma samples of hemophilia B dogs (in ng/ml) as a function of time after intramuscular injection of AAV-CMV-cF.IX vector.
Figure 4: a and b, Western blot analysis of anti-cF.IX in serum samples of hemophilia B dogs 3 (a) and 5 (b).
Figure 5: Immunofluorescence staining of canine F.IX produced by muscle fibers 46 days after intramuscular injection of 4.4 × 1010 particles of AAV-CMV-cF.IX (per site) into the tibialis anterior muscle of hemophilia B dog 1. a, Negative control (muscle from uninjected dog). b , Injected site of dog 1.
Figure 6: Southern blot hybridization of genomic DNA isolated from biopsied muscle tissue 6 weeks after injection of AAV-CMV-cF.IX into the tibialis anterior of a normal or hemophilic dog.

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Acknowledgements

The authors thank Acuson for supplying the ultrasound equipment, M. Haskins for making the normal dog experiment possible, and J.H. Liu, S.J. Tai, and M.L. McCleland, as well as the Cell Morphology Core of the Institute for Human Gene Therapy at the University of Pennsylvania, for technical assistance. We also acknowledge the work of the staff at the vector production facility at Avigen, and the staff of the Francis Owen Blood Research Laboratory at the University of North Carolina-Chapel Hill. This work was supported by National Institutes of Health Grants R01 HL53668 and P50 HL54500 to K.A.H., and Avigen, a company in which K.A.H. holds equity. P.A.F. is supported by the Katherine Dormandy Trust for Hemophilia, and J.H.H. was supported by NIH grant F32 HL09397

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Authors and Affiliations

  1. Departments of Pediatrics and Pathology, University of Pennsylvania Medical Center and The Children's Hospital of Philadelphia, Philadelphia, 19104, Pennsylvania, USA

    Roland W. Herzog, J. Nathan Hagstrom, Paul A. Fields & Katherine A. High

  2. Department of Surgery, The Children's Hospital of Philadelphia, Philadelphia, 19104, Pennsylvania, USA

    Edmund Y. Yang

  3. Avigen Inc., 1201 Harbor Bay Parkway #1000, Alameda, 94502, California, USA

    Linda B. Couto, Melissa Burton, Gregory M. Podsakoff & Gary J. Kurtzman

  4. Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, 27599, North Carolina, USA

    Dan Elwell, Dwight A. Bellinger, Marjorie S. Read, Kenneth M. Brinkhous & Timothy C. Nichols

  5. Institute for Human Gene Therapy, University of Pennsylvania , Philadelphia, 19104, Pennsylvania, USA

    Katherine A. High

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Correspondence to Katherine A. High.

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Herzog, R., Yang, E., Couto, L. et al. Long-term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector. Nat Med 5, 56–63 (1999). https://doi.org/10.1038/4743

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