Impact of liver fibrosis on AAV-mediated gene transfer to mouse hepatocytes

Abstract

Liver fibrosis, characterized by scar tissue accumulation due to liver injury, poses significant barriers to liver-targeted gene therapy. Current clinical trials exclude patients with fibrosis, as intact liver architecture is considered essential for efficient and safe adeno-associated viral vector (AAV)-mediated gene delivery. Here, we show that liver fibrosis reduces the efficiency of hepatocyte transduction by AAV8 vectors across three mouse models with diverse fibrotic patterns. This inefficiency stems primarily from decreased vector uptake by the liver rather than loss of vector genomes due to hepatocyte turnover. Additionally, fibrosis alters blood vector clearance and redistributes AAV particles to extra-hepatic organs, such as spleen, lung, and kidney. At the cellular level, fibrosis decreases AAV genome content in hepatocytes while increasing it in non-parenchymal liver cells and splenic immune cells. Importantly, the capsid variant AAV-KP1 retains transduction efficiency in fibrotic livers, highlighting its potential for expanding gene therapy applications to fibrotic diseases.

Fig. 1. Reduced AAV8 transduction in liver fibrosis mouse models.

The AAV2/8-TBG-GFP vector was injected into male mice treated with thioacetamide (TAA, n = 8) or vehicle (PBS, n = 6) at a dose of 1 × 10¹³ gc/kg, and into 18-week-old male Atp7b^−/− (n = 6) and Atp7b^+/− (control, n = 5) mice, and 14-week-old male Abcb4^−/− (n = 5) and Abcb4^+/− (control, n = 7) mice at a dose of 5 × 10¹² gc/kg. Mice were sacrificed 42 days (TAA) or 14 days (Atp7b^−/− and Abcb4^−/−) post-injection. A Representative images of Sirius Red staining of liver sections. Scale bar: 100 µm. B Representative images of immunofluorescence performed on liver sections using an anti-GFP antibody. Nuclei are counterstained with DAPI. Scale bar: 100 µm. C Representative image from western blot analysis of whole liver lysates using an anti-GFP antibody. β-actin (ACTB) and p115 were used as loading controls. D Vector genome copy (GC) analysis performed by qPCR. (two-tailed t-test) Data are reported as mean ± standard error.

Fig. 2. Delayed clearance and altered biodistribution of AAV8 in TAA-treated and Atp7b^−/− mice.

A–D The AAV2/8-TBG.GFP vector was administered to 18-week-old male Atp7b^−/− (n = 11) and Atp7b^+/− (control, n = 13) mice. Mice were sacrificed 2 days post-injection. A Vector genome copy (GC) analysis in the liver. B Vector GC analysis in the plasma and C quantification of the area under the curve (AUC). D Vector GC analysis in extra-hepatic organs. E–G The AAV2/8-TBG-GFP vector was injected in male wild-type mice treated with thioacetamide (TAA, n = 8) or vehicle (PBS, n = 6) as control. Mice were sacrificed 42 days post-vector administration. E Vector GC analysis in the plasma and F quantification of the area under the curve (AUC). G Vector GC analysis in extra-hepatic organs. Data are reported as mean ± standard error. (two-tailed t-test).

Fig. 3. Delayed clearance and altered biodistribution of AAV8 in Abcb4^−/− mice.

14-week-old male Abcb4^−/− (n = 7) and Abcb4^+/− (control, n = 6) mice were administered with 5 × 10¹² gc/kg of AAV2/8-TBG-GFP vector and sacrificed 2 days post-injection. A Vector genome copy (GC) analysis in livers (P = 0.00002). B Vector GC analysis in the plasma and C quantification of the area under the curve (AUC) in Abcb4^−/− (n = 14) and Abcb4^+/− (control, n = 11) mice administered with AAV2/8-TBG-GFP. D Vector GC analysis in extra-hepatic organs from male Abcb4^−/− (n = 8) and Abcb4^+/− (control, n = 6) mice (two-tailed t-test or, B only, repeated measure two-way ANOVA plus Sidak post-hoc). Data are reported as mean ± standard error.

Fig. 4. Increased AAV8 distribution to liver non-parenchymal cells in Atp7b^−/− mice and to myeloid splenocytes in Abcb4^−/− mice.

A–C AAV2/8-TBG-GFP vector was administered to 18-week-old male Atp7b^−/− (n = 5) and Atp7b^+/− (control, n = 5) mice. Mice were sacrificed 4 h post-injection. A Vector genome copy (GC) analysis by digital PCR in hepatocytes (Hep) and non-parenchymal liver cells: dendritic cell (DC), endothelial cell (EC), hepatic stellate cell (HSC), and Kupffer cell (KC) fractions. B Representative images from electron microscopy analysis on liver tissues. Arrows indicate endothelial fenestrations. Scale bar: 500 nm. C Quantification of fenestrations. The number of endothelial fenestrae per 1 µm of capillary wall length was counted in three animals per group. D The AAV2/8-TBG-GFP vector was administered to 10-week-old female Abcb4^−/− (n = 11) and Abcb4^+/− (n = 12) mice. Mice were sacrificed 4 h post-injection. Vector genome copy (GC) analysis by digital PCR in hepatocyte (Hep) and hepatic dendritic cell (DC) fractions. E–G Gene expression analysis by qPCR of AAV8 receptor E AAVR and F LamR in hepatocyte (Hep), dendritic cell (DC), and hepatic stellate cell (HSC) fractions from Atp7b^−/− (n = 5) and Atp7b^+/− (control, n = 7) mice and G LamR in dendritic cell (DC) fractions from Abcb4^−/− (n = 5) and Abcb4^+/− (control, n = 7) mice. H Vector genome copy (GC) analysis by digital PCR in splenic macrophages (MΦ) and dendritic cell (DC) fractions from Abcb4^−/− (n = 5) and Abcb4^+/− (n = 8) mice (two-tailed t-test). Data are reported as mean ± standard error.

Fig. 5. Liver fibrosis impacts gene transfer in a capsid-dependent fashion and does not affect AAV-KP1.

A, B The AAV testing kit was administered to 18-week-old male Atp7b^−/− (n = 5), Atp7b^+/− (control, n = 5) mice, and to 14-week-old male Abcb4^−/− (n = 4) and Abcb4^+/− (control, n = 6) mice at the dose of 1 × 10¹² gc/kg. Mice were sacrificed at 5 days post-injection. Quantification of RNA expression for the twelve most represented AAV variants in A Atp7b^−/− and Atp7b^+/− control mice and B Abcb4^−/− and Abcb4^+/− control mice. The AAV2/KP1.TBG.GFP vector was administered to 18-week-old male Atp7b^−/− (n = 7), Atp7b^+/− (control, n = 7), and to 14-week-old male Abcb4^−/− (n = 7) and Abcb4^+/− (control, n = 2) mice at the dose of 5 × 10¹² gc/kg. Mice were sacrificed at 14 days post-injection. C Representative images of immunofluorescence performed on liver sections using an anti-GFP antibody. Nuclei are counterstained with DAPI. Scale bar: 100 µm. D Representative image from western blot analysis of whole liver lysates using an anti-GFP antibody. p115 was used as a loading control. E Vector genome copy (GC) analysis performed by qPCR (two-tailed t-test). Data are reported as mean ± standard error.