High-dose systemic adeno-associated virus vector administration causes liver and sinusoidal endothelial cell injury

Abstract

We analyzed retrospective data from toxicology studies involving administration of high doses of adeno-associated virus expressing different therapeutic transgenes to 21 cynomolgus and 15 rhesus macaques. We also conducted prospective studies to investigate acute toxicity following high-dose systemic administration of enhanced green fluorescent protein-expressing adeno-associated virus to 10 rhesus macaques. Toxicity was characterized by transaminitis, thrombocytopenia, and alternative complement pathway activation that peaked on post-administration day 3. Although most animals recovered, some developed ascites, generalized edema, hyperbilirubinemia, and/or coagulopathy that prompted unscheduled euthanasia. Study endpoint livers from animals that recovered and from unscheduled necropsies of those that succumbed to toxicity were analyzed via hypothesis-driven histopathology and unbiased single-nucleus RNA sequencing. All liver cell types expressed high transgene transcript levels at early unscheduled timepoints that subsequently decreased. Thrombocytopenia coincided with sinusoidal platelet microthrombi and sinusoidal endothelial injury identified via immunohistology and single-nucleus RNA sequencing. Acute toxicity, sinusoidal injury, and liver platelet sequestration were similarly observed with therapeutic transgenes and enhanced green fluorescent protein at doses ≥1 × 10¹⁴ GC/kg, suggesting it was the consequence of high-dose systemic adeno-associated virus administration, not green fluorescent protein toxicity. These findings highlight a potential toxic effect of high-dose intravenous adeno-associated virus on nonhuman primate liver microvasculature.

Keywords: AAV; NHP; TMA; adeno-associated virus; endothelium; liver; nonhuman primate; thrombotic microangiopathy; toxicity.

Graphical abstract

Figure 1

Acute and subacute liver injury following high-dose systemic AAV.eGFP (A) Alanine transaminase (ALT), aspartate transaminase (AST), and total bilirubin levels in rhesus macaques that received intravenous (i.v.) AAV-PHP.eB or AAV9 gradient-purified vectors encoding enhanced green fluorescent protein (eGFP) at 1 × 10¹⁴ GC/kg. (B) ALT, AST, and total bilirubin levels in rhesus macaques that received column- or gradient-purified AAV9 vectors encoding eGFP at 2 × 10¹⁴ GC/kg i.v. (C) Representative images of liver stained with Masson trichrome (arrows: fibrosis) and immunostained with a GFP-specific antibody. Upper panel scale bar, 50 μm; lower panel scale bar, 20 μm.

Figure 2

Acute thrombocytopenia and LSEC injury following high-dose systemic AAV.eGFP (A) Platelet levels in rhesus macaques that received intravenous (i.v.) AAV-PHP.eB or AAV9 gradient-purified vectors encoding enhanced green fluorescent protein (eGFP) at 1 × 10¹⁴ GC/kg. (B) Platelet levels in rhesus macaques that received column- or gradient-purified AAV9 vectors encoding eGFP at 2 × 10¹⁴ GC/kg i.v. (C) Representative images and semi-quantitative scoring of liver section immunostaining of fibrinogen (fibrin thrombi marker), CD41 (platelet marker), LSECtin (liver sinusoidal endothelial cell lectin, a sinusoidal endothelium marker), VEGFR2 (sinusoidal endothelium marker), PECAM1 (CD31, non-sinusoidal endothelial marker), and FCGR2b (sinusoidal-specific FC γ receptor 2b). Scale bar, 50 μm. (D) Serum hyaluronic acid (HA) levels (biomarker of LSEC integrity) in rhesus macaques that received AAV-PHP.eB or AAV9 vectors encoding eGFP at 1 × 10¹⁴ GC/kg or 2 × 10¹⁴ GC/kg i.v. (E) Correlation between serum HA level and platelet reduction on day 3 post high-dose AAV. Pearson correlation R² 0.6, two-tailed p value 0.0075.

Figure 3

Acute liver and LSEC injury following high-dose systemic AAV expressing human therapeutic transgenes (A) Alanine transaminase (ALT), aspartate transaminase (AST), and platelet levels in 36 macaques that received AAVhu68 expressing one of five different human therapeutic transgenes at doses ranging from 5 × 10¹³ GC/kg to 2 × 10¹⁴ GC/kg. Line graph showing mean and standard deviation at each time point. (B) Liver enzyme and platelet levels at day 3 in animals that did or did not receive prophylactic steroids. Mean bar charts with standard deviation error bars and individual data points (each data point = one animal). (C) Representative images of liver LSECtin (sinusoidal endothelium marker) and CD41 (platelet marker). Scale bar, 50 μm. ∗p < 0.05; ns: not significant.

Figure 4

Acute complement and antibody responses following high-dose systemic AAV (A) Membrane attack complex (MAC, SC5b-9), complement Bb, C4a, and total C3 levels in rhesus macaques that received column- or gradient-purified AAV9 vectors encoding enhanced green fluorescent protein (eGFP) at 2 × 10¹⁴ GC/kg. (B) Pro-inflammatory cytokine levels in the same animals as in (A). See also Table S11 for additional cytokines. (C) Anti-AAV9 IgM and IgG titers in the same animals as in (A). (D) Examples of IgG and IgM deposit immunostaining in kidney glomeruli from rhesus macaques necropsied 3, 6, or 14 days post AAV-PHP.eB dosing. Scale bar, 20 μm.

Figure 5

snRNA-seq, cell clustering, and eGFP expression in the liver following high-dose systemic AAV (A) snRNA-seq study design and workflow. (B) UMAP plot showing nuclei clustering for complete dataset with cell-type annotation. (C) Cell-type proportions for NHPs that received high-dose AAV and were euthanized early (3–6 days post dosing due to SAEs) or at day 14 (no SAEs, scheduled time point), compared with AAV-naïve animals. (D) UMAP plots showing administered transgene (eGFP or therapeutic human transgene) expression intensity at the single nuclei level within the complete dataset, broken down by group: AAV-naïve, high dose eGFP early necropsy, high dose non-eGFP early necropsy, and high dose day 14 necropsy. (E) GFP expression presented as percentage of positive nuclei per cell type and per necropsy time point. (F) Confirmation of snRNA-seq transgene expression data using dual ISH probes specific for RNA (transcripts, red) or DNA (vector genome, green). Scale bar, 100 μm (naive, high-dose early necropsy) or 200 μm (high-dose day 14 necropsy). HD, high dose.