Immune transgene-dependent myocarditis in macaques after systemic administration of adeno-associated virus expressing human acid alpha-glucosidase

Figures

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  Figure 1. Pharmacology of AAVhu68.hGAA and immune responses. (A) GAA enzyme activity in plasma, (B) anti-hGAA total antibody titers, and (C) anti-AAVhu68 NAb titers. Each data point represents an individual animal and timepoint. Nine animals had a negative AAVhu68 NAb baseline while two had pre-existing NAb titers of 1:10 to 1:80 at screening and 1:20 to 1:160 at day 0 pre-dosing. Peripheral blood mononuclear cell (PBMC) ELISPOT responses to (D) hGAA peptide pools and (E) AAVhu68 peptide pools from eleven rhesus macaques that received IV AAVhu68.CAG.vIGF2.hGAA vectors at 1x1013 GC/kg or 5x1013 GC/kg or combined IV and ICM at 5x1013 GC/kg and 3x1013 GC, or at 1x1014 GC/kg and 1x1013 GC, respectively. Each data point represents a separate peptide pool from an individual animal.

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  Figure 2. Safety, bloodwork. (A) Alanine transaminase (ALT) levels from eleven rhesus macaques that received IV AAVhu68.CAG.vIGF2.hGAA vectors at 1x1013 GC/kg (IV low dose) or 5x1013 GC/kg (IV high dose) or combined IV and ICM at 5x1013 GC/kg and 3x1013 GC or at 1x1014 GC/kg and 1x1013 GC. Data are separated by dose level and the presence/absence of pre-existing NAbs to AAVhu68. (B) Complement factors sC5b-9 (terminal complex), Bb (alternative-pathway-specific), and C4a (classical-pathway-specific) from three animals presenting no, low, or moderate NAb titers at baseline. Each data point represents a separate peptide pool from an individual animal.

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  Figure 3. Safety, cardiac toxicity. (A) Cardiac troponin I levels and cardiac histopathology from the eleven rhesus macaques presented in the previous figures. Cardiac findings (infiltrate and/or necrosis) were graded by a board-certified veterinary pathologist (ELB) on the following severity scale: 0 (within normal limits), 1 (minimal mononuclear infiltrates, similar to what is commonly observed as background in macaques), 2 (mild), 3 (moderate), 4 (marked), and 5 (severe). When different scores were given to different cardiac regions (ventricle, atrium, septum), the worst score is presented here. For full scoring result see Table S4 . (B) Immunophenotyping of the mononuclear cell infiltrate by immunofluorescence on heart tissue sections via T-cell-specific markers.

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  Figure 4. Representative images showing the severity range of microscopic findings in heart. NHPs administered AAVhu68.vIGF2.hGAA at various routes of administration (ROA), including intravenous (IV 5x1013 GC/kg) and combination IV (5x1013 GC/kg) and ICM (3x1013 GC). (A, B) animal RA3679, minimal (grade 1) myocardial mononuclear cell infiltrates, left ventricle. (C, D) Animal RA3607, moderate (grade 3) myocardial mononuclear cell infiltrates, right atrium. (E, F) Animal 180717, severe (grade 5) myocardial mononuclear cell infiltrates with associated fibrosis and moderate (grade 3) cardiomyocyte degeneration and necrosis, septum. The extent of cardiomyocyte involvement in this animal warranted a separate diagnosis. See also Table S4 . Hematoxylin and Eosin; Scale bar: 4x (A, C, E)=500µm, 20x (B, D, F)=100µm.