1. ¹Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
2. ²Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
3. ³Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
4. ⁴Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida, USA
5. ⁵Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut, USA
6. ⁶Department of Psychiatry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Correspondence: R. Jude Samulski, CB # 7352, Gene Therapy Center, 7113 Thurston Building, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7352, USA. E-mail: rjs@med.unc.edu

The first two authors contributed equally to this work.

Received 30 November 2007; Accepted 15 April 2008; Published online 20 May 2008.

Abstract

We report a DNA shuffling–based approach for developing cell type–specific vectors through directed evolution. Capsid genomes of adeno-associated virus (AAV) serotypes 1–9 were randomly fragmented and reassembled using PCR to generate a chimeric capsid library. A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Molecular modeling studies suggest that AAV2 contributes to surface loops at the icosahedral threefold axis of symmetry, while AAV1 and 9 contribute to two- and fivefold symmetry interactions, respectively. The C-terminal domain (AAV9) was identified as a critical structural determinant of melanoma tropism through rational mutagenesis. Chimeric-1829 utilizes heparan sulfate as a primary receptor and transduces melanoma cells more efficiently than all serotypes. Further, chimeric-1829 demonstrates altered tropism in rodent skeletal muscle, liver, and brain including nonhuman primates. We determined a unique immunological profile based on neutralizing antibody (NAb) titer and crossreactivity studies strongly supporting isolation of a synthetic laboratory-derived capsid variant. Application of this technology to alternative cell/tissue types using AAV or other viral capsid sequences is likely to yield a new class of biological nanoparticles as vectors for human gene transfer.