Article

Engineering and selection of shuffled AAV genomes: A new strategy for producing targeted biological nanoparticles

Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7352, USA.
Molecular Therapy (Impact Factor: 6.43). 08/2008; 16(7):1252-60. DOI: 10.1038/mt.2008.100
Source: PubMed

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.

Download full-text

Full-text

Available from: Stefan Leichtle, Feb 09, 2014
1 Follower
 · 
115 Views
 · 
25 Downloads
    • "A wealth of naturally occurring AAV variants have been studied extensively and yielded large descriptive datasets (Gao et al., 2003, 2004, 2005). Forward genetic studies have resulted in important findings (Adachi et al., 2014; Asuri et al., 2012; Bartel et al., 2012; Dalkara et al., 2013; Huttner et al., 2003; Koerber et al., 2008; Li et al., 2008; Lisowski et al., 2014; Maheshri et al., 2006; Mays et al., 2013; Perabo et al., 2003; Shen et al., 2007; Wu et al., 2000) but often remain constrained by the limited tolerance for structural change of the rigid architecture of AAVs (Adachi et al., 2014; Huttner et al., 2003; Lochrie et al., 2006; Mays et al., 2013; Shen et al., 2007; Vandenberghe et al., 2009; Wu et al., 2000). Rational AAV design has therefore been difficult in structurally isolating and modulating vector phenotypes while retaining integrity and the desirable aspects of its innate function and biology of the particle. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Adeno-associated virus (AAV) vectors have emerged as a gene-delivery platform with demonstrated safety and efficacy in a handful of clinical trials for monogenic disorders. However, limitations of the current generation vectors often prevent broader application of AAV gene therapy. Efforts to engineer AAV vectors have been hampered by a limited understanding of the structure-function relationship of the complex multimeric icosahedral architecture of the particle. To develop additional reagents pertinent to further our insight into AAVs, we inferred evolutionary intermediates of the viral capsid using ancestral sequence reconstruction. In-silico-derived sequences were synthesized de novo and characterized for biological properties relevant to clinical applications. This effort led to the generation of nine functional putative ancestral AAVs and the identification of Anc80, the predicted ancestor of the widely studied AAV serotypes 1, 2, 8, and 9, as a highly potent in vivo gene therapy vector for targeting liver, muscle, and retina. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 07/2015; DOI:10.1016/j.celrep.2015.07.019 · 8.36 Impact Factor
  • Source
    • "Other examples of chimeras selected for infectivity without antibody selection which display increased antibody neutralization resistance have been reported. Li et al. (70) used iterative cycles of infection to select variants with hamster melanoma cell tropism started from a shuffled library constructed from AAV serotypes 1–9, except serotype 7. In this study, a specific chimeric AAV variant was isolated, containing residues 1–409 from AAV1, 410–450 from AAV8, 451–704 from AAV2, and 705–736 from AAV9. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The recombinant adeno-associated virus (rAAV) gene delivery system is entering a crucial and exciting phase with the promise of more than 20 years of intense research now realized in a number of successful human clinical trials. However, as a natural host to AAV infection, anti-AAV antibodies are prevalent in the human population. For example, ~70% of human sera samples are positive for AAV serotype 2 (AAV2). Furthermore, low levels of pre-existing neutralizing antibodies in the circulation are detrimental to the efficacy of corrective therapeutic AAV gene delivery. A key component to overcoming this obstacle is the identification of regions of the AAV capsid that participate in interactions with host immunity, especially neutralizing antibodies, to be modified for neutralization escape. Three main approaches have been utilized to map antigenic epitopes on AAV capsids. The first is directed evolution in which AAV variants are selected in the presence of monoclonal antibodies (MAbs) or pooled human sera. This results in AAV variants with mutations on important neutralizing epitopes. The second is epitope searching, achieved by peptide scanning, peptide insertion, or site-directed mutagenesis. The third, a structure biology-based approach, utilizes cryo-electron microscopy and image reconstruction of AAV capsids complexed to fragment antibodies, which are generated from MAbs, to directly visualize the epitopes. In this review, the contribution of these three approaches to the current knowledge of AAV epitopes and success in their use to create second generation vectors will be discussed.
    Frontiers in Immunology 01/2014; 5:9. DOI:10.3389/fimmu.2014.00009
  • Source
    • "In contrast to primary human melanocytes, the transduction of melanoma cell lines by rAAV has previously been reported. Li et al developed a chimeric rAAV by DNA shuffling that had increased ability to transduce a range of human and mouse melanoma cell lines compared with the parental serotypes (1, 2, 8, and 9), however it failed to transduce two melanoma cell lines that had been in culture for less than 14 days [43]. Indeed, we found three melanoma cell lines (Trombelli, SKMel23, and SKMel29) to be readily transduced by both rAAV2 and rAAV6, although higher rates of transduction were seen with rAAV2 than with rAAV6. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The study of melanocyte biology is important to understand their role in health and disease. However, current methods of gene transfer into melanocytes are limited by safety or efficacy. Recombinant adeno-associated virus (rAAV) has been extensively investigated as a gene therapy vector, is safe and is associated with persistent transgene expression without genome integration. There are twelve serotypes and many capsid variants of rAAV. However, a comparative study to determine which rAAV is most efficient at transducing primary human melanocytes has not been conducted. We therefore sought to determine the optimum rAAV variant for use in the in vitro transduction of primary human melanocytes, which could also be informative to future in vivo studies. We have screened eight variants of rAAV for their ability to transduce primary human melanocytes and identified rAAV6 as the optimal serotype, transducing 7-78% of cells. No increase in transduction was seen with rAAV6 tyrosine capsid mutants. The number of cells expressing the transgene peaked at 6-12 days post-infection, and transduced cells were still detectable at day 28. Therefore rAAV6 should be considered as a non-integrating vector for the transduction of primary human melanocytes.
    PLoS ONE 04/2013; 8(4):e62753. DOI:10.1371/journal.pone.0062753 · 3.23 Impact Factor
Show more