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

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.

Figure 1

(a) Strategy for generating a combinatorial adeno-associated virus (AAV) library that will be subjected to directed evolution to isolate cell type/receptor-specific AAV mutants. AAV Cap genes were randomly fragmented and reassembled using cycles of denaturation, annealing, and extension using PCR. Following this reassembly reaction, PCR amplification with specific primers was used to generate full-length Cap chimeras for cloning into wild-type AAV2 backbone to generate shuffled plasmid library. AAV particle library was generated by plasmid transfection protocol. Specific cell line (hamster melanoma CS1 cells) previously shown to be restricted to transduction of AAVs will be tested with the chimeric AAV library for a premissive clone. Subsequent steps will involve development of novel AAV variants as targeted vectors for in vivo gene delivery applications. (b) Dot blot demonstrating the directed evolution of cell type–specific AAV variant from CS1 cells after five infectious cycles (n = 5). Data are shown in duplicate. ITR, inverted terminal repeat.

Figure 2. Physical characterization of chimeric-1829

(a) Primary structure of novel adeno-associated virus (AAV) variant selected from CS1cell. (b) Three-dimensional model showing VP3 subunits of AAV1829 in relation to the fivefold (white pentagon); threefold (white triangle) and twofold (white oval) axes of symmetry. AAV1-derived residues are colored salmon, AAV2 in blue, AAV8 in dark pink, and AAV9 in yellow. (c) Black box region in 2. Panel b shown at higher magnification. Key residues in the C-terminal AAV9 region that differ from AAV2 are colored gray (706); cyan (709); orange (712); green (714); white (716); and black (718) using Vp1 numbering. (d) Heparin binding profiles of AAV1829 and AAV2. L, load; FT, flow through; W, wash; e, elution.

Figure 3. Biological characterization of chimeric-1829 compared with some parental serotypes

(a) Fluorescence micrographs of green fluorescent protein (GFP) transgene expression in CS1 cells transduced with AAV serotypes 1–9 (except 7) and the novel variant chimeric-1829 at a multiplicity of infection (MOI) of 1,000 for 48 hours. (b) Representative fluorescence micrographs of GFP transgene expression in different human and mouse melanoma cell lines infected with chimeric-1829 and parental serotypes at an MOI of 1,000 for 48 hours. (c) Heparin-inhibition assay. Top panels show CS1 cells, and bottom panels show 293 cells. Left and right columns are transduction profiles in the absence and presence of heparin, respectively.

Figure 4. In vivo transduction ability compared with some parental serotypes

(a) Luciferase transgene expression in the hindlimbs of Balb/C mouse injected with 10¹⁰ vector genomes of the novel adeno-associated virus (AAV) variant and parental AAV1, 2. Bioluminescent images were obtained at the end of 1 month using a Xenogen IVIS system. (b) Luciferase transgene expression in the liver through tail vein injecton of 10¹¹ vector genomes of the novel AAV variant and parental AAV1, 2. Bioluminescent images were obtained at the end of 1 month using a Xenogen IVIS system. (c) Patterns of transduction in the rat striatum 1 month after a 1 μl infusion of either AAV1829 or AAV2. (d) Comparison of the transduction efficiency of AAV2 and chimeric-1829 after injections into the (a and c) caudate nucleus and (b and d) substantia nigra of monkeys after immunostaining for green fluorescent protein (GFP). Scale bar = 50 μm. (e,f) Stereological counts of GFP-labeled cells and area of spread after each vector show that AAV2 shows greater transduction efficiency over a larger volume than chimeric-1829 in these areas.

Figure 5. Neutralizing antibody (Nab) titer and crossreactivity

(a) Enzyme-linked immunosorbent semi-quantitative assessment of the affinity of chimeric-1829 and adeno-associated virus-2 (AAV2) to A20 (AAV2 molyclonal antibody), for details see Materials and Methods. (b) NAb titer of parental serotypes sera and their crossreactivity against AAV1829. (c) NAb titer of chimeric-1829 sera and it’s crossreactivity against parental serotypes.

Figure 6. The transduction efficiency of chimeric-1829 and its mutants

(a) Diagram of the chimeric capsid constructs and site point mutants. (b) Fluorescence micrographs of green fluorescent protein transgene expression in CS1 cells transduced with AAV1829 and its mutants at a multiplicity of infection (MOI) of 1,000 for 48 hours. (c) Luciferase transgene expression in CS1 cells transduced with AAV1829 and its mutants at an MOI of 1,000 for 48 hours. 1, Chimeric-1829; 2, AAV182; 3, AAV2–9; 4, chimeric-1829 Y706N; 5, Chimeric-1829 A714T; 6, Chimeric-1829 N716D; 7, Chimeric-1829 E718N; 8, AAV2; 9, AAV129. AAV, adeno-associated virus; RLU, relative luciferase unit.