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Overcoming adeno-associated virus vector size limitation through viral DNA heterodimerization

Nature Medicine volume 6, pages 599–602 (2000)Cite this article

Adeno-associated virus (AAV) vectors represent a class of small, non-enveloped, replication-defective DNA viruses that are being developed for human gene therapy. AAV is the only non-pathogenic viral vector now available and has been used successfully in establishing long-term gene expression without substantial immune response or toxicity in both dividing and non-dividing cells in vivo1. Use of this vector system for efficient gene transfer into the central nervous system1,2, muscle3,4,5, lung6, gut7, liver8,9 and eye10 has established preclinical paradigms that now support testing in clinical trials11. In studies in vivo, AAV vectors have met the main objectives required to treat both genetic and acquired disorders successfully (that is, long-term gene expression and lack of immune response and toxicity). However, an intrinsic limitation of this gene delivery system has always been its small packaging capacity of 5 kilobases (kb) (ref. 12). For this reason, the current AAV vectors preclude the use of large genes such as dystrophin13 (Duchenne muscular dystrophy), dysferlin14 (limb girdle muscular dystrophy 2B) and Factor VIII (hemophilia A).

Previous efforts to circumvent the problem of the size limitation imposed by the AAV vector have focused mainly on altering the candidate genes into ‘mini-expression cassettes’ suitable for AAV packaging. These strategies have included decreasing the size of the candidate gene by deleting non-essential sequences, as well as using minimal promoters and small polyadenylation signals15. Although this approach has met with limited success for such genes as CFTR, which encodes the cystic fibrosis transmembrane conductance regulator protein, most large genes are not suited to this strategy. Moreover, the use of minimal promoters usually restricts the ability to regulate gene expression properly, therefore compounding the problem of controlling the therapeutic protein.

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Figure 1: Strategy for packaging a large gene by the split AAV vectors through heterodimerization.
Figure 2: LacZ transgene expression from split AAV vectors in vitro and in vivo.
Figure 3: Characterization of the split AAV vector heterodimerization by RT–PCR and DNA-PCR.

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Acknowledgements

We thank T. Van Dyke and J. Samulski for their critical reading and suggestions for the manuscript. This work is supported in part by National Institutes of Health grants R21 DK55966 from the National Institute of Diabetes and Digestive and Kidney Diseases, AR45967 and AR 45925 from the National Institute of Arthritis, Musculoskeletal and Skin Diseases, and an equipment grant from the Parent Project of USA.

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Authors and Affiliations

  1. Department of Molecular Genetics and Biochemistry & Gene Therapy Center & Duchenne Muscular Dystrophy Research Center, University of Pittsburgh, Room W1213, BST, Pittsburgh, 15261, Pennsylvania, USA

    Liangwu Sun, Juan Li & Xiao Xiao

  2. Department of Orthopedic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Xiao Xiao

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Correspondence to Xiao Xiao.

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Sun, L., Li, J. & Xiao, X. Overcoming adeno-associated virus vector size limitation through viral DNA heterodimerization. Nat Med 6, 599–602 (2000). https://doi.org/10.1038/75087

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