An engineered AAV targeting integrin alpha V beta 6 presents improved myotropism across species

Abstract

Current adeno-associated virus (AAV) gene therapy using nature-derived AAVs is limited by non-optimal tissue targeting. In the treatment of muscular diseases (MD), high doses are often required but can lead to severe adverse effects. Here, we rationally design an AAV capsid that specifically targets skeletal muscle to lower treatment doses. We computationally integrate binding motifs of human integrin alphaV beta6, a skeletal muscle receptor, into a liver-detargeting capsid. Designed AAVs show higher productivity and superior muscle transduction compared to their parent. One variant, LICA1, demonstrates comparable muscle transduction to other myotropic AAVs with reduced liver targeting. LICA1's myotropic properties are observed across species, including non-human primate. Consequently, LICA1, but not AAV9, effectively delivers therapeutic transgenes and improved muscle functionality in two mouse MD models (male mice) at a low dose (5E12 vg/kg). These results underline the potential of our design method for AAV engineering and LICA1 variant for MD gene therapy.

Fig. 1. Computational rational AAV capsid design to bind to αVβ6 integrin.

A Overview of the design pipeline, including three steps: 1. Capsid 3D structures were obtained either from the PDB database or predicted by AlphaFold2. 2. The capsid VR4 loop was completely replaced by integrating the binding motif, which was extracted from receptor’s natural binder, using RosettaRemodel protocol. 3. Top scored designs from the previous grafting step were docked onto the intended receptor in silico to verify the binding potential of the designed capsid. B An illustration of the sampling for low-energy sequence-structure pairs during motif-grafting process. Capsid VR4 after removing the loop was colored in blue, extracted binding motif was colored in red. The sampled linkers and sequences (Fig. S1F) were labeled in green. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en). C, D The three lowest energy designs after grafting TGFβ3 (C) and TGFβ1 (D) into the capsid VR4. All top designs showed convergence in structures and sequences, suggesting sampling approached the global optimum. E, F Retrospective docking of motif-grafted capsids (E Cap9rh74_4um9) and (F Cap9rh74_5ffo) onto the αVβ6 structure. The left panels are illustrations of the structures with the lowest energy at the interface of capsid and integrin proteins (dG_separated: difference in free energy of two proteins). Both two newly designed VR4s (colored in green) were predicted to bind to the αVβ6 complex at very similar positions to natural binding motifs (colored in red). The right panels are scatter plots of dG_separated energy versus root-mean-square deviation (RMSD) from the lowest energy structure of all sampled docking positions.

Fig. 2. Designed AAV_ITGs were well-produced and improved transduction via αVβ6 binding.

A AAV titers of different AAV variants in bulked small-scale production in suspension 3-day post-triple-transfection (2 ml production, 6 biological replicates, one-way ANOVA followed by FDR correction). B Western blot of VP proteins from purified AAVs showed similar VP ratios for designed AAV_ITGs capsids compared to AAV9 and AAV9rh74, suggesting successful capsid assembly. C, D VCN (C) and luciferase activity (D) of 293_αVβ6 after AAV infection (3 and 4 biological replicates, one-way ANOVA followed by FDR correction). Both the two designed AAV_ITGs showed enhanced VCN and luciferase activities compared to AAV9rh74 and AAV9. E Inhibition of cell entry of designed AAV_ITGs, but not for AAV9 or AAV9rh74, in 293_αVβ6 cells by αVβ6 recombinant protein. AAVs were preincubated with human αVβ6 recombinant protein (r.ITGAV-B6) for 30 min at 37 °C before infection (4 biological replicates, two-way ANOVA followed by FDR correction, 1 µg protein per 5E9vg AAV, 2E5 vg per cell). The same condition treated with recombinant SGCA protein (r.SGCA) was used as the control. F–J Enhanced transduction of AAV_ITGs in in vitro human differentiated myotubes, but not in myoblasts. F Representative images of the GFP signal of myotubes 48 h post-infection (scale bar: 400 µm). G–J VCN and luciferase activities of AAV_ITGs in comparison with AAV9 and AAV9rh74 in myoblasts (G, I, 3 and 3/4 biological replicates, respectively) and myotubes (H, J, 3 and 3/4/6 biological replicates, respectively) (one-way ANOVA followed by FDR correction). Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant. Source data are provided as a Source Data file.

Fig. 3. Designed AAV_ITGs showed enhanced transduction in skeletal and cardiac muscles while strongly liver-detargeted in vivo.

A Scheme of in vivo experiment. AAVs (CMV_GFP-Luciferase) were injected intravenously into 6wo C57BL6 mice at the dose of 1E13 vg/kg. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en). B Representative images of the bioluminescence signal 20 days post-infection. C, D VCN (C) and gene expression (D) (GFP mRNA level in the liver and luciferase activity in other organs) for different AAVs in liver, skeletal muscles, heart, lung, and kidney (4 biological replicates, one-way ANOVA followed by FDR correction). Both designed AAV_ITGs strongly detargeted from the liver compared to AAV9, while they significantly improved VCN and luciferase activities over AAV9rh74 (and AAV9 with AAV9rh74_4um9 variant) in skeletal and cardiac muscles, and were detected and expressed at low levels in lung and kidney. Data in (C, D) are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant. Source data are provided as a Source Data file.

Fig. 4. AAV9rh74_4um9 exhibits highest tropism towards the skeletal muscle among tested myotropic AAVs in mice.

Comparison of the AAV9rh74_4um9 variant with other public myotropic AAVs (mAAVs)^,. A Illustration of the differences between mAAVs and AAV9rh74_4um9 at modification sites in capsid protein and modification methods. B The VR8 loop sequences of mAAVs compared to VR8 of their backbone AAV9, and VR4 of AAV9rh74_4um9 compared to VR4 of AAV9rh74. C, D VCN (C) and gene expression (D) (GFP mRNA level in liver and luciferase activity in other organs) of different AAVs in liver, skeletal muscles, heart, lung, kidney, and brain (4 biological replicates, one-way ANOVA followed by FDR correction). AAV9rh74_4um9 showed similar VCN and gene expression in skeletal muscle to other mAAVs, while being significantly more strongly detargeted from the liver. Data in (C, D) are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant. Source data are provided as a Source Data file.

Fig. 5. Low-dose gene transfer by LICA1 was more effective and better at restoring dystrophic phenotypes than AAV9 in the DMD mouse model.

A, B Comparison of transduction efficacy between AAV9 and LICA1 in three skeletal muscles and heart, in terms of VCN (A), and µDys RNA level (B) (n = 4/5). C Comparison of percentage of successfully transduced (dystrophin-positive) fibers in the skeletal muscles (n = 3/5). D, E Comparison of restoration levels in dystrophic histological features between AAV9 and LICA1 in terms of percentage of centro-nucleated fibers (D) and fibrosis level (E) (n = 3/5). Illustrated images in (C–E) are of quadriceps muscles (scale bar: 100 µm). F Serum MYOM3 level at 4 weeks post-injection (n = 5). G–I Comparison of functional restoration between AAV9 and LICA1 by Escape test—global force measurement (G, n = 6), tetanus force of TA muscle (H, n = 10/12), and twitch force of TA muscle (I, n = 9/11/12). J–M Comparison of restoration in global transcriptomic changes in quadriceps muscle between AAV9 and LICA1 (n = 4, adjusted p-values < 0.05). J The heatmap shows the log2 fold-change (log2FC) in comparison to WT muscle for all 8717 DEGs in mdx muscle, displayed as row Z-scores from blue (lowest) to red (highest). K–M Volcano plots of multiple comparisons illustrate transcriptomic changes before and after AAV treatment. As a reference, 4216 downregulated and 4501 upregulated DEGs found in mdx were colored blue and red, respectively, in all volcano plots. Among these DEGs, the number of genes found to be significantly different in each pair-wise comparison were labeled in the upper corners. K mdx versus WT. L mdx versus AAV treatments (significant DEGs are the genes correctly restored). M AAV treatments versus WT (significant DEGs are the genes that are not or incompletely restored). Data in (A–I) are presented as mean ± SEM. n represents the number of biological replicates. Statistics in (A, B) and (F–I) were performed using one-way ANOVA followed by FDR correction (A, B) or post-hoc test (F–I). Statistics in (C–E) were performed using two-way ANOVA (~serotypes * IHF slides) followed by FDR correction. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant. Source data are provided as a Source Data file.

Fig. 6. Low-dose gene transfer by LICA1 was better at restoring dystrophic phenotypes and functionality than AAV9 in the LGMDR3 mouse model.

A Scheme of in vivo experiment. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en). B–D Comparison of transduction efficacy between AAV9 and LICA1 in terms of VCN (B, n = 4/5), hSGCA mRNA level (C, n = 4/5), and percentage of succesfully transduced (SGCA-positive) fibers (D, n = 3/4/5). E–G Comparison of restoration levels in dystrophic histological features between AAV9 and LICA1 in all three muscles that were tested in terms of percentage of centro-nucleated fibers (E, n = 3/4/5), fibrosis level (F, n = 3/4/5), and fiber size distribution (G, n = 3/4/5). Illustrated images in (D–F) are of quadriceps muscles (scale bar: 100 µm). H–J Comparison of functional restoration between AAV9 and LICA1 using the escape test—global force measurement (H, n = 3/5), tetanus force of TA muscle (I, n = 3/4/5), and serum MYOM3 level—indicator of muscle damage (J, n = 3/4/5). K–N Comparison of restoration in global transcriptomic changes in quadriceps muscle between AAV9 and LICA1 (n = 4, adjusted p-values < 0.05). K. The heatmap presents the log2FC in comparison to WT muscle for all 8591 DEGs found in KO muscle (compared to WT), displayed as row Z-scores from blue (lowest) to red (highest). L–N Volcano plots of multiple comparisons illustrate transcriptomic changes before and after AAV treatment. As a reference, 4035 downregulated and 4556 upregulated DEGs found in KO were colored blue and red, respectively, in all volcano plots. Among these DEGs, the number of genes found to be significantly different in each pair-wise comparison were labeled in the upper corners. L KO versus WT. M KO versus AAV treatments (significant DEGs are the genes correctly restored). N. AAV treatments versus WT (significant DEGs are the genes that are not or incompletely restored). Data in (B–F) and (H–J) are presented as mean ± SEM. n represents the number of biological replicates. Statistics in (B, C) and (H–J) were performed using one-way ANOVA followed by FDR correction. Statistics in (D–F) were performed using two-way ANOVA (~serotypes * IHF slides) followed by FDR correction. *p < 0.05; ** p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant. Source data are provided as a Source Data file.

Fig. 7. LICA1 showed conserved interaction with αVβ6 from multiple species.

A Structure of human αVβ6 binding to human TGF-β3-derived motif (pdb code: 4um9). The binding interface, defined as all amino acid in αVβ6 with distance to the binding motif of < 8 Å, is highlighted. B Aligment of ITGAV (upper panel) and ITGB6 (lower panel) protein sequence around the binding interface from multiple species. The binding interface defined in A is highlighted, and is identical across species being examined. Amino acids with distance to the binding motif of < 6 Å, are in bold and boxed. The sequence mismatches, only found outside the binding interface, are colored in red. C Transduction efficiency, measured by luciferase activity, of AAV_ITGs but not AAV9 was inhibited by pre-incubating AAVs before infection with recombinant αVβ6 protein from both human, rat, and mouse. 2E8vg AAVs were incubated with different αVβ6 concentrations (0–120 nM) for 1 h at 37 °C before added directly into cell medium (dose: 1E4 vg/cell, 96-well plate, duration: 24 h, cell line: 293_WT, n = 3 biological replicates). Same incubation conditions using 120 nM of recombinant SGCA protein were used as the control, which showed no significant difference with 0 nM αVβ6 condition. Data are presented as mean ± SEM. The statistics were performed to compare with the condition of no αVβ6 protein during incubation (0 nM αVβ6) by using two-way ANOVA (~AAV serotypes * αVβ6 treatments) followed by FDR correction. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant. Source data are provided as a Source Data file.

Fig. 8. A LICA1 showed cross-species enhanced transduction in the skeletal muscle.

Scheme of comparative study between multiple AAV capsids in multiple species. Transgene expression cassette is ITR_tMCK_hCAPN3-inactive_BC_SV40pA_ITR. Barcodes (BCs) allow quantitative measurements of transgene expression mRNA level. 2/3 barcodes were used for each capsid to minimize the sequence bias. AAV production of each capsid variant was done separately, before pooled together at the equimolar amount before AAV transduction in vitro (C, human myotube, n = 2 biological replicates, dose: 2E10/2E11 vg per 12wp well, duration: 48 h), or in vivo injection in C57Bl6 mice (B, n = 3 mice, dose: 5E12 vg/kg per capsid variant) and Macaca fascicularis NHP (D, n = 3 NHPs, dose: 3.2E12 vg/kg per capsid variant). Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en). B The mRNA enrichment (BC_mRNA/Rplp0/ BC_AAV) of different capsid variants in the liver and three skeletal muscles in C57Bl6 mice measured by RT-qPCR. Each dot correponds to the individual barcode. The color of each barcode corresponds to the individual mouse in Fig. S6C. The statistics were performed on the average on different barcodes (using for the same capsid variant in each mouse) by using two-way ANOVA ( ~ mice * capsids) followed by FDR correction. C The mRNA enrichment, measured by NGS, of different capsid variants in in vitro human myotubes at two AAV concentration. The log2FC compared to AAV9 of all variants is presented. Each dot correponds to the individual barcode. The colors correpsond to different biological replicates. D The mRNA enrichment (BC_mRNA/Rplp0/BC_AAV) of different capsid variants in the liver and skeletal muscles in NHP measured by RT-qPCR. Each dot correponds to the individual barcode. The data was presented for individual primates (P1-3). The statistics were to compare the mRNA level of LICA1 with natural capsids, AAV8/AAV9, performed on the fold change of LICA1 to AAV8/AAV9 (averages of barcodes using for the same capsid variant in each primate were used to calculate the fold-change) by using two-tailed one-sample t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant. Data in (B) and (D) are presented as mean ± SEM. Source data are provided as a Source Data file.