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. 2008 Oct;16(10):1703-9.
doi: 10.1038/mt.2008.167. Epub 2008 Aug 26.

DNA shuffling of adeno-associated virus yields functionally diverse viral progeny

James T Koerber  1 , Jae-Hyung Jang, David V Schaffer
Affiliations

DNA shuffling of adeno-associated virus yields functionally diverse viral progeny

James T Koerber et al. Mol Ther. 2008 Oct.

Abstract

Adeno-associated virus (AAV) vectors are extremely effective gene-delivery vehicles for a broad range of applications. However, the therapeutic efficacy of these and other vectors is currently limited by barriers to safe, efficient gene delivery, including pre-existing antiviral immunity, and infection of off-target cells. Recently, we have implemented directed evolution of AAV, involving the generation of randomly mutagenized viral libraries based on serotype 2 and high-throughput selection, to engineer enhanced viral vectors. Here, we significantly extend this capability by performing high-efficiency in vitro recombination to create a large (10(7)), diverse library of random chimeras of numerous parent AAV serotypes (AAV1, 2, 4-6, 8, and 9). In order to analyze the extent to which such highly chimeric viruses can be viable, we selected the library for efficient viral packaging and infection, and successfully recovered numerous novel chimeras. These new viruses exhibited a broad range of cell tropism both in vitro and in vivo and enhanced resistance to human intravenous immunoglobulin (IVIG), highlighting numerous functional differences between these chimeras and their parent serotypes. Thus, directed evolution can potentially yield unlimited numbers of new AAV variants with novel gene-delivery properties, and subsequent analysis of these variants can further extend basic knowledge of AAV biology.

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Figures

Figure 1
Figure 1. Amino acid sequences of shuffled clones
Graphical representations of the primary protein sequences of each chimera are shown, with the protein segments colored to reflect the parent serotypes from which they were derived. Point mutations present within each sequence are shown next to each clone. Graphical representation of several key viral regions, including surface loops,,, hypervariable regions (HVRs), β-barrel core, CD8+ T-cell epitopes, and antibody (Ab) epitopes,, are also depicted to highlight the diversity within these regions. AAV, adeno-associated virus.
Figure 2
Figure 2. Structural comparison of selected viral chimeras
Molecular models of the adeno-associated virus (AAV) capsid, based on the AAV2 structure, are shown for each chimera along with a model depicting the location of each loop region. Each region is shaded according to the parent serotype from which it was derived, highlighting the substantial sequence variability on exterior regions of the capsid. White arrows indicate important structural regions discussed in text.
Figure 3
Figure 3. PCR screen of plasmid and viral library
PCRs were performed with serotype-specific primers annealing ~1,400 base pairs (bp) and ~1,700 bp downstream from the cap start codon for the forward and reverse primers, respectively. Positive PCRs yield a band of 200–300 bp in size, depending on the serotype. The plasmid library and vector genome DNA from the packaged virus and passaged virus were analyzed along with the individual parent pSub2 plasmids (labeled 1, 2, 4, 5, 6, 8, and 9). AAV, adeno-associated virus.
Figure 4
Figure 4. Transduction efficiencies of chimeras and viral parents
rAAV-Luc vectors were used to transduce a panel of cell lines: CHO, pgsA (lacking all GAGs), Pro5, Lec2 (lacking sialic acid), a breast cancer cell line (MDA-MB231), and a neuroblastoma cell line (SHSY-5Y). (a) Relative luciferase signals were normalized to total protein (RLU/mg) for AAV2-related chimeras along with AAV2, 8, and 9 controls (n = 3). (Note: cA1 packaged but was noninfectious.) (b) Normalized relative luciferase values for AAV1/6-related chimeras along with AAV1 and 6 controls are shown (n = 3). AAV, adeno-associated virus; RLU, relative luciferase unit.
Figure 5
Figure 5. In vivo biodistribution transduction efficiencies of chimera and viral parents
rAAV-Luc vectors (1011 vector genomes) were delivered via tail vein injections to BALB/c mice (n = 3). For each vector and each tissue analyzed, levels of luciferase expression were normalized to total protein (RLU/mg). AAV, adeno-associated virus; RLU, relative luciferase unit.
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