Engineering novel AAV capsids by broadly attenuated and subsequent muscle-specific tropism in mice and NHPs

Figure 1. Chimeric capsid variants exhibit dramatic reductions in liver transduction (A) Capsid spike formed by VRs IV and VIII of one AAV2 monomer interacting with the VR V of a second monomer. (B) Sequence alignments of the variable region (VR)-IV, VR-V, and VR-VIII of AAV.Zero1, AAV.Zero2, and AAV.Zero3 compared with AAV2 and AAV9. The blue dashed line represents the AAV2 backbone, while the red dashed line indicates the AAV9 backbone. (C) Different dilutions of AAV2 (left) or AAV9 (right) antibody-containing serum were incubated with constant amounts of AAV serotypes AAV9-, AAV2-, AAV.Zero1-, AAV.Zero2-, or AAV.Zero3-CAG-Fluc-P2A-EGFP at MOI = 1E6 and tested for neutralization of transduction in HEK293T. Data are presented as mean ± SEM (n = 4 wells for left image, and n = 6 wells for right image). (D) Schematic of virus administration and biodistribution assessment process. (1) CAG-luciferase-P2A-eGFP was packaged as reporter transgene. (2) Experimental workflow in mice. (E–I) 8-week-old C57BL/6 J mice systemically injected with 2 × 1011 vg per mouse (∼8 × 1012 vg/kg) of AAV9-, AAV2-, AAV.Zero1-, AAV.Zero2-, or AAV.Zero3-CAG-Fluc-P2A-EGFP, and data were collected 21 days…

Figure 2. RGD-containing peptides enable chimeric capsid selectively target to muscle with high efficiency in C57BL/6J mice (A) Sequence alignments of VR-IV, VR-V and VR-VIII of the capsid variants containing RGD peptides 2A, 4E, and 4A inserted into AAV.Zero3 at R585. The blue dashed line represents the AAV2 backbone, while the red dashed line represents the AAV9 backbone. (B) Representative images of in vitro transduction in HEK293T and C2C12 cell lines were transduced at MOI = 1E5 with AAV9-, AAV2-, MyoAAV 2A-, AAV.Zero3 2A-, MyoAAV 4E-, AAV.Zero3 4E-, MyoAAV 4A-, or AAV.eM-CAG-Fluc-P2A-EGFP 72 h after transduction. Scale bars, 100 μm. (C) Heparin competition assay for AAV-mediated LacZ reporter transduction. Data are presented as luminescence normalized to vehicle-treated controls (n = 3 technical replicates). Statistical significance was determined by two-way ANOVA followed by comparisons of each heparin dilution group against the corresponding vehicle-treated control for each AAV capsid. All values represent means ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. (D–F) Eight-week-old C57BL/6J mice were systemically injected with 2 × 1011 vg per mouse AAV9-, AAV2-, MyoAAV 2A-, AAV.Zero3…

Figure 3. AAV.eM is both efficient and specific in muscle-targeting across rodent species Eight-week-old BALB/c mice were systemically injected with 2 × 1011 vg per mouse of AAV9-, AAV2-, MyoAAV 4A-, or AAV.eM-CAG-Fluc-P2A-EGFP and data were collected 21 days post-injection. (A) Representative whole body in vivo bioluminescence images. (B) Quantification of firefly luciferase luminescence from hindlimbs. The Kruskal-Wallis test was used for comparisons between AAV.eM and each other group. Sample sizes were n = 10 for each group, except for AAV2 (n = 9) and the control group (n = 7), ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (C) Viral genome copy number per diploid from mouse tissues detected by qPCR using a standard curve. Comparisons between AAV9 and each other capsid across tissues were performed using ordinary one-way ANOVA, the Brown-Forsythe test and Welch’s ANOVA, or the Kruskal-Wallis test, as appropriate for the data distribution and variance homogeneity (n = 5), ∗p < 0.05, ∗∗p < 0.01. (D) Quantification of the Fluc mRNA in biceps, triceps, quadriceps, gastrocnemius, abdominal muscle, and heart muscles as well as the liver, brain, and kidney. For comparisons between AAV9 and each other group…

Figure 4. AAV.eM causes less liver toxicity without sacrificing the transduction efficiency in non-human primates NHPs were given intravenous administration of 3 × 1013 vg/kg of AAV.eM- or MyoAAV 4A-CAG-Fluc-P2A-EGFP (A) Hepatic enzyme (AST and ALT) and creatine kinase (CK) levels were monitored prior to and after dosing. The p values were calculated by Kruskal-Wallis test with Dunn’s multiple comparisons test for each capsid. The number of monkeys were 5 for AST and ALT, and 3 for CK, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. AST, aspartate aminotransferase; ALT, alanine aminotransferase. (B) Quantification of relative vector genome per diploid genome and Fluc mRNA relative fold-change in the liver of NHPs at 28 days post-injection. The p value was calculated by unpaired Student’s t test, the number of monkeys were 4 for mRNA and 2 for gDNA, ∗p < 0.05. (C) Representative western blot image detecting EGFP and GAPDH of biceps, triceps, quadriceps, gastrocnemius, and liver sampled at 14 days post-injection. (D) Representative cross-section fluorescent images of biceps, triceps, quadriceps, and gastrocnemius sampled 28 days post-injection. Green: EGFP, blue: DAPI, Scale bars, 100 μm. 1# and 2#…

Figure 5. Systemic injection of AAV.eM-MHCK7-microdystrophin results in widespread micro-dystrophin expression and effective restoration of muscle function in adult DMD mice 8-week-old wild-type B10 mice and B10 DMD-KO mice were systemically injected with vehicle or 2 × 1013 vg/kg of either AAV9 or AAV.eM-MHCK7-microdystrophin (μDys) (A and B) The grip strength (A) and serum creatine kinase (CK) levels (B) were assessed 14 weeks post-injection. The p values were calculated by one-way ANOVA with Tukey-Kramer multiple comparisons test, the number of mice were 14 of each group for grip strength, and 4–5 for CK concentration). ∗p < 0.05, ∗∗∗p < 0.001, ns, not significant. (C–F) Quantification of relative vector genome copy number (C), mRNA expression (D), representative fluorescence images of cross-section whole mount (E) and zoom in (F) for μDys 20 weeks post-injection. The AAV.eM-μDys expression data are normalized to the relative expression levels of AAV9-μDys. Comparisons between the two groups for each tissue were performed using the Mann-Whitney U test, Student’s t test, or Welch’s t test, as appropriate; the number of mice were 5 for AAV9-μDys, and 4 for AAV.eM-μDys except for kidney…