Reengineering a receptor footprint of adeno-associated virus enables selective and systemic gene transfer to muscle

Abstract

Reengineering the receptor footprints of adeno-associated virus (AAV) isolates may yield variants with improved properties for clinical applications. We generated a panel of synthetic AAV2 vectors by replacing a hexapeptide sequence in a previously identified heparan sulfate receptor footprint with corresponding residues from other AAV strains. This approach yielded several chimeric capsids displaying systemic tropism after intravenous administration in mice. Of particular interest, an AAV2/AAV8 chimera designated AAV2i8 displayed an altered antigenic profile, readily traversed the blood vasculature, and selectively transduced cardiac and whole-body skeletal muscle tissues with high efficiency. Unlike other AAV serotypes, which are preferentially sequestered in the liver, AAV2i8 showed markedly reduced hepatic tropism. These features of AAV2i8 suggest that it is well suited to translational studies in gene therapy of musculoskeletal disorders.

Figure 1

Structure-function correlates of AAV2i vectors with reengineered receptor footprints. (a) Three-dimensional structural model of the AAV2 capsid highlighting the 585–590 region containing basic residues implicated in heparan sulfate binding. Inset shows VP3 trimer, with residues 585-RGNRQA-590 located on the innermost surface loop highlighted in red. VP3 monomers are colored salmon, blue, and gray. Images were rendered using Pymol. (b) Representative live animal bioluminescent images of luciferase transgene expression profiles in BALB/c mice (n = 3) injected intravenously (tail vein) with AAV2i CMV-Luc vectors (dose 1 × 10¹⁰ vg in 200 µl PBS). Photographs and bioluminescent images were obtained at 1 week after injection. The overlay demonstrates decreased transduction efficiency for most AAV2i mutants with the exception of AAV2i8. Bioluminescence scale ranges from 0–4 × 10⁵ relative light units (photons/sec/cm²). Residues within the 585–590 region in each AAV2i mutant are indicated below corresponding mouse image data. (c) Representative live animal bioluminescent images of luciferase transgene expression profiles in BALB/c mice (n = 3) injected intravenously (tail vein) with AAV2, AAV8, AAV2i8 and structurally related AAV2i mutants (dose 1 × 10¹¹ vg in 200 µl PBS) packaging the CBA (chicken beta actin)-Luc cassette. All AAV2i mutants show a systemic transduction profile similar to that of AAV8, with AAV2i8 showing enhanced transduction efficiency. Bioluminescence scale ranges from 0–3 × 10⁶ relative light units (photons/sec/cm²). Residues within 585–590 region in each AAV2i mutant is indicated below corresponding mouse image data. (d) Comparison of AAV2, AAV2i8 and AAV8 capsid surface residues based on schematic “Roadmap” projections. A section of the asymmetric unit surface residues on the capsid crystal structures of AAV2 and AAV8, as well as a model of AAV2i8, are shown. Close-up views of the heparan sulfate binding region and residues 585–590 reveal a chimeric footprint on the AAV2i8 capsid surface. Red, acidic residues; blue, basic residues; yellow, polar residues; green, hydrophobic residues. Each residue is shown with a black boundary and labeled with VP1 numbering based on the AAV2 capsid protein sequence.

Figure 2

Selective muscle tropism of AAV2i8. (a) Quantification of luciferase transgene expression in three major tissues: cardiac (black bars), skeletal muscle (pooled hind limb and abdominal; gray bars) and liver (white bars). Tissue lysates were obtained from BALB/c mice (n = 3) at 2 weeks after administration of AAV2, AAV2i8 and AAV8 (dose 1 × 10¹¹ vg, tail vein) and subjected to luminometric analysis. AAV2i8 shows high transduction in cardiac and skeletal muscle and low transduction in liver. Luciferase levels are shown as relative light units normalized to protein levels determined using a Bradford assay. Error bars indicate s.d. (b) Vector genome copy numbers (luciferase transgene) in three major tissues: cardiac (black bars), skeletal muscle (pooled hind limb and abdominal; gray bars) and liver (white bars). Host genomic as well vector DNA was extracted from tissue lysates obtained from BALB/c mice (n = 3) at 2 weeks after administration of AAV2, AAV2i8 and AAV8 (dose 1 × 10¹¹ vg, tail vein). Host and vector genome copy number were determined by Q-PCR with specific primer sets against the lamin gene and luciferase transgene, respectively. AAV2i8 shows enhanced muscle sequestration and decreased accumulation in liver tissue compared with AAV2 and AAV8. (c) Luciferase transgene expression in major muscle sub-groups obtained from BALB/c mice (n = 3) at 2 weeks after intravenous administration of AAV2i8 (dose 1 × 10¹¹ vg, tail vein) packaging the CBA (chicken beta actin)-Luc cassette. Tissue lysates from five different muscle groups from the hind limb skeletal muscle (alternating black and white bars), three groups from the forelimb (alternating black and white bars), intercostals, cardiac, facial, diaphragm, tongue, abdominal and vertebral muscle types (black bars) were subjected to luminometric analysis. Luciferase levels are shown as relative light units normalized to protein levels determined by a Bradford assay. Error bars indicate s.d.

Figure 3

Blood transport profile of AAV2i8. (a–c) Luciferase transgene expression in pooled skeletal muscle subgroups from right and left hind limb of BALB/c mice (n = 4) after isolated perfusion of AAV2i8 (black bars) or AAV8 (gray bars) into each saphenous vein. Tissue lysates prepared after administration of three different doses (1 × 10⁹ (a), 1 × 10¹⁰ (b), 1 × 10¹¹ (c) vg) in low (200 µl), medium (500 µl) or high (1 ml) volume of injection were subjected to luminometric analysis. Luciferase levels are shown as relative light units ormalized to protein levels determined using a Bradford assay. (d) Vector genome copy numbers recovered from blood at different time intervals after administration through the tail vein (n = 3). Whole blood DNA was extracted and analyzed by Q-PCR with primers against the luciferase transgene. AAV2i8 shows prolonged circulation compared with AAV8. Error bars indicate s.d.