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Effective gene therapy with nonintegrating lentiviral vectors

Nature Medicine volume 12, pages 348–353 (2006)Cite this article

Abstract

Retroviral and lentiviral vector integration into host-cell chromosomes carries with it a finite chance of causing insertional mutagenesis1. This risk has been highlighted by the induction of malignancy in mouse models, and development of lymphoproliferative disease in three individuals with severe combined immunodeficiency–X1 (refs. 2,3). Therefore, a key challenge for clinical therapies based on retroviral vectors is to achieve stable transgene expression while minimizing insertional mutagenesis. Recent in vitro studies have shown that integration-deficient lentiviral vectors can mediate stable transduction4,5,6. With similar vectors, we now show efficient and sustained transgene expression in vivo in rodent ocular and brain tissues. We also show substantial rescue of clinically relevant rodent models of retinal degeneration. Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. For therapeutic application to postmitotic tissues, this system substantially reduces the risk of insertional mutagenesis.

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Figure 1: Efficient in vivo expression of eGFP from integration-deficient vectors in mouse ocular tissues.
Figure 2: Efficient expression of eGFP from integration-deficient vectors in mouse brain.
Figure 3: Rescue of ocular function in Rpe65rd12/rd12 mice.
Figure 4: Analyses of HIV vector integration in vivo.

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Acknowledgements

The authors thank L. Naldini for lentiviral plasmids, M. Balda for ARPE-19 cells, S. Wilkie for human RPE65 cDNA, D. Thompson for RPE65-specific antibody and D. King for human actin plasmid. This work was supported by the Wellcome Trust (A.J.T. and R.J.Y.-M.), the Sir Jules Thorn Charitable Trust (A.J.T. and R.R.A.), the Special Trustees of Moorfields Eye Hospital (R.R.A.) and the European Union Integrated Project CONSERT (Concerted Safety and Efficiency of Retroviral Transgenesis in Gene Therapy of Inherited Diseases) 005242 (C.v.K., A.J.T. and S.J.H.).

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Author notes

  1. Rafael J Yáñez-Muñoz

    Present address: Nuclear Biology Group, Department of Medical and Molecular Genetics, King's College London School of Medicine, Guy's Tower, Guy's Hospital, London, SE1 9RT, UK

  2. Rafael J Yáñez-Muñoz and Kamaljit S Balaggan: These authors contributed equally to this work.

Authors and Affiliations

  1. Molecular Immunology Unit, Institute of Child Health, University College London, 30 Guilford Street, London, WC1N 1EH, UK

    Rafael J Yáñez-Muñoz, Steven J Howe, Christine Kinnon, Robin R Ali & Adrian J Thrasher

  2. Centre for Medical Oncology, Institute of Cancer and the CR-UK Clinical Centre, Barts and The London, Queen Mary's School of Medicine and Dentistry, John Vane Science Centre, Charterhouse Square, London, EC1M 6BQ, UK

    Rafael J Yáñez-Muñoz

  3. Division of Molecular Therapy, Institute of Ophthalmology, University College London, 11-43 Bath Street, London, EC1V 9EL, UK

    Kamaljit S Balaggan, Angus MacNeil, Alexander J Smith, Prateek Buch, Robert E MacLaren, Susie E Barker, Yanai Duran & Robin R Ali

  4. Department of Internal Medicine I, University Hospital, Hugstetter Strasse 55, 79106 Freiburg and Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University, Stefan-Meier-Strasse 17, Freiburg, 79104, Germany

    Manfred Schmidt, Cynthia Bartholomae & Christof von Kalle

  5. National Center for Tumor Diseases (NCT), Im Neuenheimer Feld 350, Heidelberg, 69120, Germany

    Manfred Schmidt, Cynthia Bartholomae & Christof von Kalle

  6. Department of Anatomy and Developmental Biology, University College London, Gower Street, London, WC1E 6BT, UK

    Patrick N Anderson

  7. Cincinnati Children's Research Foundation, Molecular and Gene Therapy Program, 3333 Burnet Avenue, Cincinnati, 45229-3039, Ohio, USA

    Christof von Kalle

  8. Kellogg Eye Center, 1000 Wall Street, Room 541, Ann Arbor, 48105, Michigan, USA

    John R Heckenlively

  9. Department of Immunology, Great Ormond Street Hospital for Children NHS Trust, London, WC1N 3JH, UK

    Adrian J Thrasher

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  1. Rafael J Yáñez-Muñoz
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Corresponding authors

Correspondence to Rafael J Yáñez-Muñoz or Robin R Ali.

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Competing interests

We are applying for a patent based on the results described in the manuscript.

Supplementary information

Supplementary Fig. 1

Integration-deficient lentiviral vectors mediate transient transduction in cultured proliferating cells. (PDF 204 kb)

Supplementary Fig. 2

hrGFP expression from integration-deficient vectors in rat RPE. (PDF 189 kb)

Supplementary Fig. 3

Transduction of human RPE with integration-deficient vectors. (PDF 325 kb)

Supplementary Fig. 4

Rescue of ocular function in RCS rats. (PDF 154 kb)

Supplementary Table 1

Characterization of paired integration-deficient and integration-proficient HIV-1 vector stocks. (PDF 24 kb)

Supplementary Table 2

Summary of integration events detected by LAM-PCR in eyecups injected subretinally with HIV vectors. (PDF 24 kb)

Supplementary Table 3

Summary of ocular injections for GFP imaging studies in rodents. (PDF 24 kb)

Supplementary Methods (PDF 51 kb)

Supplementary Note (PDF 39 kb)

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Yáñez-Muñoz, R., Balaggan, K., MacNeil, A. et al. Effective gene therapy with nonintegrating lentiviral vectors. Nat Med 12, 348–353 (2006). https://doi.org/10.1038/nm1365

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