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A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells

Nature Biotechnology volume 39, pages 47–55 (2021)Cite this article

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Abstract

Nine dogs with hemophilia A were treated with adeno-associated viral (AAV) gene therapy and followed for up to 10 years. Administration of AAV8 or AAV9 vectors expressing canine factor VIII (AAV-cFVIII) corrected the FVIII deficiency to 1.9–11.3% of normal FVIII levels. In two of nine dogs, levels of FVIII activity increased gradually starting about 4 years after treatment. None of the dogs showed evidence of tumors or altered liver function. Analysis of integration sites in liver samples from six treated dogs identified 1,741 unique AAV integration events in genomic DNA and expanded cell clones in five dogs, with 44% of the integrations near genes involved in cell growth. All recovered integrated vectors were partially deleted and/or rearranged. Our data suggest that the increase in FVIII protein expression in two dogs may have been due to clonal expansion of cells harboring integrated vectors. These results support the clinical development of liver-directed AAV gene therapy for hemophilia A, while emphasizing the importance of long-term monitoring for potential genotoxicity.

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Fig. 1: Long-term expression of AAV-cFVIII in dogs with hemophilia A.
Fig. 2: Assessment of liver function after AAV-cFVIII administration.
Fig. 3: Distributions of AAV vector integration sites in the treated dogs.
Fig. 4: Clonal expansion of cells harboring AAV vectors.
Fig. 5: Three examples of clusters of AAV vector integration sites found in the dog genome.

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Data availability

The raw sequencing data supporting this study are available at the Zenodo data server (https://doi.org/10.5281/zenodo.3666122), while demultiplexed sample reads generated during the analysis are available at the NIH SRA (BioProject ID: PRJNA606282). Source data are provided with this paper.

Code availability

The AAVenger software and analysis software supporting this study are available at the Zenodo data server (https://doi.org/10.5281/zenodo.3666122). Source data are provided with this paper.

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Acknowledgements

We are grateful to members of the Sabatino and Bushman laboratories for help and suggestions. We acknowledge the Research Vector Core at the Children’s Hospital of Philadelphia for production of the SC AAV vectors and the Penn Vector Core at the University of Pennsylvania for preparing the TC AAV vectors. We thank M. Keiser for assistance with immunohistochemistry and A. Messer for assisting with the analysis of the canine samples. We also thank N. Hoepp for discussions on canine liver clinical pathology. We thank S. Sherrill-Mix for help with statistical analysis. This work was supported by grants from the National Institutes of Health (RO1HL083017 (H.H.K.), R24HL63098 and N0175N92019D00041 (T.C.N.), RO1HL126850 (D.E.S.) and RO1AI082020, RO1CA241762, RO1HL142791 and U19AI149680 (F.D.B)). We also acknowledge support from the Penn Center for AIDS Research (P30AI045008) and the PennCHOP Microbiome Program (F.D.B.).

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Author notes

  1. These authors contributed equally: Giang N. Nguyen, John K. Everett.

Authors and Affiliations

  1. The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Giang N. Nguyen, Samita Kafle, Christian Wood & Denise E. Sabatino

  2. Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    John K. Everett, Aoife M. Roche, Hayley E. Raymond, Jacob Leiby & Frederic D. Bushman

  3. Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Charles-Antoine Assenmacher

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

    Elizabeth P. Merricks, C. Tyler Long & Timothy C. Nichols

  5. UNC Blood Research Center, University of North Carolina School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Elizabeth P. Merricks, C. Tyler Long & Timothy C. Nichols

  6. Department of Genetic Medicine, Johns Hopkins School of Medicine, Baltimore, MD, USA

    Haig H. Kazazian

  7. Division of Hematology, Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Denise E. Sabatino

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Contributions

G.N.N., J.K.E., S.K., H.E.R., A.M.R., J.L. and C.W. performed the experiments. E.P.M., C.T.L. and T.C.N. performed the vector administration, sample collection and follow-up with the dogs. C.A.-A. performed the dog liver histopathology analysis. D.E.S., H.H.K., T.C.N. and F.D.B. designed the experiments. D.E.S. and F.D.B. wrote the manuscript.

Corresponding author

Correspondence to Denise E. Sabatino.

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Competing interests

D.E.S. receives royalties from a licensing agreement with Spark Therapeutics. D.E.S. and G.N.N. are inventors on a patent on FVIII and hemophilia A gene therapy.

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Extended data

Extended Data Fig. 1 Immunohistochemical detection of FVIII in liver after AAV administration.

Immunohistochemical detection of FVIII in the liver from untreated (Hem A) (a) and treated (b,c,d,e,f) hemophilia A dogs. Locations of FVIII production are indicated by the brown stain. Most of the cFVIII staining was pan-lobular in distribution (b,c,d,e) while some areas had what appeared to be small clonal populations of cells that express cFVIII (b,f). Liver sections from multiple lobes were stained in n = 4 independent experiments. Images are representative of each dog. Scale bar representing 50μm applies to all images.

Supplementary information

Supplementary Information

Supplementary Figs. 1–12, Tables 1–12 and AAV Vector Sequences.

Reporting Summary

Uncropped western blot image for Supplementary Fig. 4

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Nguyen, G.N., Everett, J.K., Kafle, S. et al. A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells. Nat Biotechnol 39, 47–55 (2021). https://doi.org/10.1038/s41587-020-0741-7

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