Three-dimensional models of the dual glycan-binding AAV2G9 chimera and its parental strains AAV2 and AAV9. A, three-dimensional structural model of an intact AAV2G9 capsid with existing HS and “grafted” Gal binding residues shown in purple and orange, respectively. B–D, illustrations of the three-dimensional surface models of VP3 trimers at the 3-fold symmetry axes of AAV2 (B), AAV9 (C), and AAV2G9 (D) capsids. ▴ indicate the 3-fold axes of symmetry. Residues involved in HS binding (AAV2 VP1 numbering Arg-487, Lys-527, Lys-532, Arg-585, and Arg-588) and Gal binding (AAV9 VP1 numbering Asp-271, Asn-272, Tyr-446, Asn-470, Ala-472, Val-473, and Trp-503) are highlighted as in A.

Heparin binding characteristics of AAV2, AAV2G9, and AAV9. A, heparin affinity column chromatogram of parental AAV2 vectors. A total of 1e11 vg containing particles of AAV2-CBA-Luc in 1 ml of 1× PBS was loaded onto a heparin affinity column. Bound virions were eluted from the heparin affinity column by elution with increasing NaCl concentration (right y axes, gray lines). The numbers of vg in the loaded sample and each eluted fraction were quantified using qPCR as described under “Experimental Procedures.” The numbers of vg in each fraction were normalized to the total loaded vg to obtain the percentage of recovered virions (left y axes, black lines). Heparin affinity column chromatograms of AAV2G9 (B) and AAV9 (C) were obtained through a similar approach.

In vitro characterization of the dual glycan-binding AAV2G9 chimera. A–C, inhibition of AAV2 (A), AAV2G9 (B), and AAV9 (C) transduction on CHO Lec2 cells with FITC-ECL and soluble heparin. CHO Lec2 cells were prechilled at 4 °C and incubated with FITC-ECL, soluble heparin, or both prior to infection with AAV2, AAV2G9, or AAV9 packaging a CBA-luciferase reporter transgene cassette. Transduction efficiency was measured 24 h post-infection as luciferase activity in relative light units. The percentage of transgene expression was calculated by normalizing transduction efficiency to relative light units from controls. Results are presented as mean ± S.E. (n = 4). D–F, inhibition of cell surface binding of AAV2 (D), AAV2G9 (E), and AAV9 (F) on CHO Lec2 cells with FITC-ECL and soluble heparin. Different AAV particles were bound to cells prechilled at 4 °C, and unbound virions were removed by washing with cold PBS. Bound virions were quantified using qPCR after viral genome extraction. The percentage of bound virions was determined by normalizing number of bound virions to that of corresponding controls. Results are presented as mean ± S.E. (n = 5). Statistical significance was analyzed using one-tailed Student's t test. *, p < 0.05; **, p < 0.01.

AAV2G9 mediates rapid onset and enhanced transgene expression in vivo. A, in vivo transgene expression kinetics of AAV2, AAV2G9, and AAV9 vectors packaging a CBA-luciferase transgene cassette. BALB/c mice (n = 4) were administered AAV vectors at a dose of 1 × 10¹¹ vg/animal through the tail vein, and bioluminescent images were collected at 3, 7, and 18 days post-injection using an Xenogen® Lumina imaging system. Representative live animal images are shown with bioluminescence on a rainbow-colored scale (1 × 10⁵-1 × 10⁶ photons/second/cm²/steradian). AAV2G9 maintains the hepatic tropism of AAV2 but demonstrates a more rapid and robust luciferase signal than both parental AAV strains. B and C, quantitation of the kinetics of light signal output (expressed as photons/second/cm²/steradian) was performed by marking regions of interest around images of the liver regions (B) and entire animals (C) obtained at different time intervals (n = 4). RLU, relative light unit. D, quantitation of luciferase transgene expression in major tissues from AAV2- (black), AAV2G9- (gray), or AAV9-treated (white) animals at 18 days post-administration (n = 4). BALB/c mice (n = 4) were administered AAV2, AAV2G9, or AAV9 packaging a CBA-Luciferase cassette at a dose of 1 × 10¹¹ vg/animal through the tail vein. At 18 days post-vector administration, animals were sacrificed to harvest brain, heart, liver, lung, spleen, skeletal muscle, and kidney tissues. Luciferase transgene expression in these tissue lysates was measured using a luminometer and normalized by the total amount of protein in each sample, determined using a Bradford assay. Statistical significance was assessed using one-tailed Student's t test. *, p < 0.05; **, p < 0.01.

Structural attributes of the AAV2i8G9 chimera. A, three-dimensional illustration of the intact AAV2i8G9 capsid. The surface-exposed motif derived from AAV8 (AAV8 VP1 numbering 588-QQNTAP-593) and the Gal binding site from AAV9 (AAV9 VP1 numbering Asp-271, Asn-272, Tyr-446, Asn-470, Ala-472, Val-473, and Trp-503) are shown in deep blue and orange, respectively. B, close-up view of the 3-fold axes of symmetry (▴) on the exterior of the AAV2i8G9 capsid. Three individual VP3 monomers are shown in pale green, light blue, and pale pink. The structural motifs derived from the AAV8 and AAV9 motifs are highlighted as described in A. C and D, heparin affinity column chromatograms of the parental AAV2i8 strain (C) and the AAV2i8G9 chimera (D). A total of 1e11 vg of AAV2i8 or AAV2i8G9 vectors in 1 ml of 1× PBS were loaded onto heparin affinity columns. Virions were eluted from the column with elution buffers containing increasing concentrations of NaCl (right y axes, gray lines). The percentage of recovered virions in each elution fraction (left y axes, black lines) was obtained by normalizing the total number of vg in each fraction to that in the loaded sample.

Competitive inhibition of AAV2G9 and AAV2i8G9 by parental AAV serotypes. A, CHO Lec2 cells were preincubated with AAV2 or AAV9-CBA-tdTomato at MOI ranging from 500–100,000 vg/cell for 2 h. Pretreated cells were then infected with AAV2G9-CBA-Luc particles (MOI = 1000 vg/cell). At 24 h post-transduction, cells were lysed to assess luciferase activity. Percentage inhibition of AAV2G9 transduction was calculated by normalizing luciferase transgene expression levels to that of control with no AAV competitors. B, competitive inhibition of AAV2i8G9-CBA-Luc transduction by AAV2i8-tdTomato and AAV9-tdTomato was carried out in a similar fashion. The percentage of luciferase expression was obtained by normalizing the luciferase transgene expression in each condition by that of the control condition without any competitor AAV. Results are presented as mean ± S.E. (n = 4). Statistical significance was analyzed using one-tailed Student's t test. *, p < 0.05; **, p < 0.01.

AAV2i8G9 demonstrates an enhanced and selective transduction profile in skeletal muscle. A, in vivo transgene expression kinetics of AAV2i8, AAV2i8G9, and AAV9 vectors packaging the CBA-luciferase transgene cassette. BALB/c mice (n = 4) were administered AAV2i8, AAV2i8G9, and AAV9 vectors at a dose of 1 × 10¹¹ vg/animal through the tail vein. Live animal bioluminescent images were collected at 3, 7, and 18 days post-injection using an Xenogen® Lumina imaging system. Representative live animal images are shown on a rainbow-colored scale (1 × 10⁵-1 × 10⁶ photons/second/cm²/steradian). B, in vitro transduction efficiency of AAV2i8, AAV2i8G9, and AAV9 on CHO-Pro5 and Lec2. Vectors packaging CBA-luciferase were bound to prechilled Pro5 or Lec2 at an MOI of 1000 vg/cell at 4 °C for 1 h. Unbound virions were removed by three washes with ice-cold 1× PBS, and luciferase transgene expression was measured at 18–24 h post-infection. Results are shown in relative light units (RLU) (n = 5). Statistical significance was analyzed using one-tailed Student's t test. *, p < 0.05; **, p < 0.01. C, quantitation of the kinetics of transgene expression by AAV2i8, AAV2i8G9, and AAV9 in BALB/c mice. Regions of interest were marked around the entire animals in live-animal images (A) to measure the light signal output (expressed as photons/second/cm²/steradian) from the mice at different time intervals (n = 4). Statistical significance was assessed to compare AAV2i8 and AAV2i8G9 as well as AAV9 and AAV2i8G9. D, in vivo transduction efficiency of AAV2i8, AAV2i8G9, and AAV9 in BALB/c mice. At 18 days post-administration, animals were sacrificed, and luciferase transgene expression in tissue lysates from brain, heart, liver, lung, spleen, muscle, and kidney were determined using a luminometer. Results are normalized to the amount of protein in each sample measured by Bradford Assay and presented as mean ± S.E. (n = 4). Statistical significance was assessed using one-tailed Student's t test. n.s., not significant; *, p < 0.05.