Liver-directed AAV gene therapy normalizes disease symptoms and provides cross-correction in a model of lysosomal acid lipase deficiency

Abstract

Lysosomal acid lipase deficiency (LAL-D) is caused by mutations in the LIPA gene, which encodes the lysosomal enzyme that hydrolyzes triglycerides and cholesteryl esters to free fatty acids and free cholesterol. The objective of this study was to develop a curative single-treatment therapy for LAL-D using adeno-associated virus (AAV). Treatment at both early (1-2 days) and late (8-week) timepoints with rscAAVrh74.LP1.LIPA, a liver-directed AAV gene therapy, normalized many disease measures in Lipa^-/- mice when measured at 24 weeks of age, including hepatosplenomegaly, serum transaminase activity, organ triglyceride and cholesterol levels, and biomarkers of liver inflammation and fibrosis. For most measures, liver-directed therapy was superior to therapy utilizing a constitutive tissue expression approach. rscAAVrh74.LP1.LIPA treatment elevated LAL enzyme activity above wild-type levels in all tissues tested, including liver, spleen, intestine, muscle, and brain, and treatment elicited minimal serum antibody responses to transgenic protein. AAV treatment at 8 weeks of age with 1 × 10¹³ vg/kg extended survival significantly, with all AAV-treated mice surviving beyond the maximal lifespan of untreated Lipa^-/- mice. These results show that this liver-directed LIPA gene therapy has the potential to be a transformative treatment for LAL-D.

Keywords: adeno-associated virus; enzyme; fibrosis; gene therapy; hepatitis; lipid metabolism; liver; lysosomal acid lipase deficiency; lysosome; muscle.

Graphical abstract

Figure 1

Treatment with rscAAVrh74.LP1.LIPA reduces hepatosplenomegaly (A) Schematic of treatment plan. Mice were intravenously dosed with different gene therapy constructs. Early treatment (1–2 days) or late treatment (8 weeks) were the treatment timepoints used. Study endpoint was at 24 weeks of age. (B) Gross pathology of liver and spleen at 24 weeks of age after treatment with different constructs and doses. Scale bar, 1 cm. (C) Relative weight of the liver (weight of liver normalized to body weight) at 24 weeks of age. (D) Relative weight of the spleen (weight of spleen normalized to body weight) at 24 weeks of age. All data presented as mean (SD) (n = 2–7). Statistical significance between groups is denoted with different letters at p ≤ 0.05 using one-way ANOVA with Tukey’s post hoc test. Schematic created with BioRender.com.

Figure 2

Biodistribution and gene expression of gene therapy constructs (A–D) Detected AAV vector genomes (vg) per diploid nucleus in various organs with all tested constructs and doses as quantified by qPCR. (E–H) Gene expression of human LIPA normalized to 18S ribosomal RNA and compared with the expression of endogenous mouse Lipa in wild-type mice in various organs. All data presented as mean (SD) (n = 2–6). Statistical significance between groups is denoted with different letters at p ≤ 0.05 using one-way ANOVA with Tukey’s post hoc test.

Figure 3

Lysosomal acid lipase enzyme activity levels (A–I) Lysosomal acid lipase enzyme (LAL) enzyme activity was measured in various tissue homogenates or serum. All data presented as mean (SD) (n = 2–7). Statistical significance between groups is denoted with different letters at p ≤ 0.05 using one-way ANOVA with Tukey’s post hoc test.

Figure 4

Lipid accumulation is reduced and dyslipidemia is improved by rscAAVrh74.LP1.LIPA therapy (A) Oil red O staining (red) of neutral lipids in liver, spleen, and intestines at 24 weeks of age after treatment with various doses of rscAAVrh74.LP1.LIPA. Tissues are counter-stained with hematoxylin (purple). Scale bar, 50 μm. (B–G) Measure of cholesterol or triglyceride levels per mg of tissue for liver, spleen, and intestines. (H–L) Serum panel of various lipid and lipoprotein species. QNS: quantity not sufficient to perform the assay. All data presented as mean (SD) (n = 2–7). Statistical significance between groups is denoted with different letters at p ≤ 0.05 using one-way ANOVA with Tukey’s post hoc test.

Figure 5

Inflammation is reduced after treatment with rscAAVrh74.LP1.LIPA (A) Immunohistochemical staining of livers. Tissues were immunostained for CD68 (brown) and counter-stained with hematoxylin (purple). Scale bar, 50 μm. (B–E) RT-qPCR expression of various genes involved in inflammation and immune response in liver. All data presented as mean (SD) (n = 2–7). Statistical significance between groups is denoted with different letters at p ≤ 0.05 using one-way ANOVA with Tukey’s post hoc test.

Figure 6

Prevalence of anti-LIPA antibodies in treated mice Serum samples were assayed by ELISA for total immunoglobulin G binding against LIPA protein at a dilution of 1:50 (n = 2–6).

Figure 7

Long-term survival and sustained activity after rscAAVrh74.LP1.LIPA treatment (A) Kaplan-Meier survival curve comparing untreated Lipa^−/− mice (n = 44) vs. Lipa^−/− mice treated at 8 weeks with 1.0 × 10¹³ vg/kg of rscAAVrh74.LP1.LIPA (n = 20). (B) LAL enzyme activity in serum at 40 weeks of age, 32 weeks post-injection. All data presented as mean (SD) (n = 2–20). Statistical significance between groups was defined as p ≤ 0.05 (∗∗∗∗p ≤ 0.0001), using one-way ANOVA with Tukey’s post hoc test.