Exosome-based Ldlr gene therapy for familial hypercholesterolemia in a mouse model

Abstract

Familial hypercholesterolemia (FH), with high LDL (low-density lipoprotein) cholesterol levels, is due to inherited mutations in genes, such as low-density lipoprotein receptor (LDLR). Development of therapeutic strategies for FH, which causes atherosclerosis and cardiovascular disease, is urgently needed. Methods: Mice with low-density lipoprotein receptor (Ldlr) deletion (Ldlr^-/- mice) were used as an FH model. Ldlr mRNA was encapsulated into exosomes by forced expression of Ldlr in the donor AML12 (alpha mouse liver) cells, and the resultant exosomes were denoted as Exo^Ldlr. In vivo distribution of exosomes was analyzed by fluorescence labeling and imaging. The delivery efficiency of Ldlr mRNA was analyzed by qPCR and Western blotting. Therapeutic effects of Exo^Ldlr were examined in Ldlr^-/- mice by blood lipids and Oil Red O staining. Results: The encapsulated mRNA was stable and could be translated into functional protein in the recipient cells. Following tail vein injection, exosomes were mainly delivered into the liver, producing abundant LDLR protein, resembling the endogenous expression profile in the wild-type mouse. Compared with control exosomes, Exo^Ldlr treatment significantly decreased lipid deposition in the liver and lowered the serum LDL-cholesterol level. Significantly, the number and size of atherosclerotic plaques and inflammation were reduced in the Exo^Ldlr-treated mice. Conclusions: We have shown that exosome-mediated Ldlr mRNA delivery effectively restored receptor expression, treating the disorders in the Ldlr^-/- mouse. Our study provided a new therapeutic approach for the treatment of FH patients and managing atherosclerosis.

Keywords: atherosclerosis; exosomes; familial hypercholesterolemia; gene therapy; low-density lipoprotein receptor.

Figure 1

Construction and characterization of Exo^Ldlr. A. Cloning of Ldlr-expressing plasmid. The CDS of Ldlr was cloned into the plasmid backbone with restriction endonuclease sites as indicated. B. Schematic illustration of Ldlr mRNA encapsulation procedure into the exosomes. The donor cells were forced to express Ldlr upon transfection of the plasmid or infection with the Ldlr-expressing virus. The high level of Ldlr was thus passively enriched in the exosomes. C. Western blot analysis of the exclusive and inclusive exosome markers in the isolated exosomes and parental cells. Cells were transfected/infected with control or Ldlr vector. D. Size distribution of the indicated exosomes analyzed by ZetaView Particle Metrix. E. Representative transmission electron microscope images of the indicated exosomes. Scale bar=100 nm. F. Expression of Ldlr mRNA in AML12 cells treated as indicated. GAPDH served as an internal control. Data are expressed as mean ±SEM of three independent experiments. *, p< 0.05 by t-test. G. Ldlr mRNA abundance in exosomes derived from AML12 cells treated as indicated. H. Western blot analysis of LDLR at the protein level in parental cells and derived exosomes. Cells were transfected with control or Ldlr vector. GAPDH served as the loading control. Representative data of 3 independent experiments.

Figure 2

Exo^Ldlr efficiently delivers functional Ldlr mRNA in vitro. A. Schematic illustration of the exosome-mediated Ldlr mRNA delivery into the recipient cells, where the mRNA is translated into the functional protein. B. Fluorescence microscopy images showing the endocytosis of exosomes in the recipient cells. The intracellular distribution of DiI-labeled exosomes was analyzed by fluorescence microscopy. Nuclei were counterstained with Hoechst. PBS served as the negative control. Scale bar=5 μm. C. qPCR analysis of Ldlr mRNA expression in HEK 293T cells treated as indicated. Data are expressed as mean ± SEM of three independent experiments. *, p< 0.05 by one-way ANOVA. D. Western blot analysis of LDLR protein expression in HEK 293T cells treated as indicated. GAPDH served as the loading control. Representative data from three independent experiments.

Figure 3

In vivo distribution of Exo^Ldlr after tail vein injection. A. Representative IVIS images of mice injected with 100 μL PBS, 100 μg (in 100 μL) DiR labeled Exo^empty or Exo^Ldlr via the tail vein. IVIS imaging was performed 4 h after injection. B. Ex vivo fluorescence imaging analysis of the distribution of the DiR-labeled exosomes in different organs, including the liver, spleen, heart, kidney, and lung. C. Quantification of the fluorescence signal intensity in Fig 3B. n=4, ns, no significance. D. Representative fluorescence microscopic images of the localization of DiI-labeled exosomes. Mice were injected with 100 μL DiI-labeled Exo^empty or Exo^Ldlr via tail vein and sacrificed 4 h after injection. Scale bar = 20 μm. E. Representative images of localization of the exosomes, immune cells, and the epithelial cells in the liver or lung sections. Scale bar = 10 μm.

Figure 4

Exo^Ldlr effectively delivers Ldlr mRNA into the liver in vivo. A. Schematic illustration of the experimental procedure. Ldlr^-/- mice were fed with a high-fat diet for 8 weeks, followed by the injection of indicated exosomes. The expression of Ldlr at both mRNA and protein levels in the liver was examined 3 days after injection. B. Semi-quantitative PCR analysis of Ldlr mRNA expression in livers from mice treated as indicated. Lane 1 is DNA ladder. The lower 289 bp band represents the endogenous truncated Ldlr from knockout mice and the 371 bp band represents the exogenous wild-type Ldlr. Data shown are representative of 4 independent experiments. C. Quantitative data of Figure 4B. Data are expressed as mean ± SEM. *, p < 0.05 by one-way ANOVA. D. qPCR analysis of the wild-type Ldlr mRNA in livers from mice treated as indicated. n=4. Data are expressed as mean ± SEM. *, p < 0.05 by one-way ANOVA. E. Western blot analysis of the LDLR expression at the protein level in livers from mice treated as indicated. Notably, there was no endogenous LDLR protein expression in the Ldlr^-/- mice with PBS treatment. F. Quantification of Western blot bands by densitometry. Data are expressed as mean ± SEM. *, p < 0.05 by one-way ANOVA.

Figure 5

Exo^Ldlr effectively reduces liver steatosis in Ldlr^-/- mice. A. Schematic illustration of the experimental procedure. Ldlr^-/- mice were fed with a high-fat diet for 8 weeks, followed by the injection of indicated exosomes once a week for 8 weeks. At the end of the experiment, lipid deposition in the liver and liver function were examined. B. Representative images of Oil Red O staining of the liver samples from indicated groups. C. Percentage of Oil Red O positive area in livers from indicated groups. (D-E) Plasma ALT (D) and AST (E) levels in Ldlr^-/- mice treated as indicated. (F-H) qPCR analysis of the expression of Tnfα (F), Mcp1 (G), and Col1a1 (H) in Ldlr^-/- mice treated as indicated. Data are expressed as mean ± SEM. *, p < 0.05 by one-way ANOVA. ALT, Alanine aminotransferase; AST, Aspartate aminotransferase.

Figure 6

Exo^Ldlr reduces the LDL level in Ldlr^-/- mice. A. Schematic illustration of the experimental procedure. Ldlr^-/- mice were fed with high fat-diet for 8 weeks, followed by injection of indicated exosomes once a week for 8 weeks. B. Representative images of the appearance of serum samples from Ldlr^-/- mice treated as indicated. (C-F) Examination of the total cholesterol (C), total triglyceride (D), LDL-C (E), and HDL-C (F) in Ldlr^-/- mice treated as indicated. Data are expressed as mean ± SEM. *, p < 0.05 by one-way ANOVA. ns, no significant difference.

Figure 7

Exo^Ldlr alleviates atherosclerotic lesions in Ldlr^-/- mice. A. Representative aortic arch view of the atherosclerotic lesions in Ldlr^-/- mice treated as indicated. AA, ascending aorta; BA, brachiocephalic artery; LCCA, left common carotid artery; LSA, left subclavian artery; DA, descending aorta. B. Percentage of the atherosclerotic area in the aortic arch. Data are expressed as mean ± SEM. *, p < 0.05 by one-way ANOVA. C. Representative images of Oil Red O staining of the atherogenic lesion areas in mice treated as above. D. Representative images of the cross-sectional view of the aortic roots stained with Oil red O from Ldlr^-/- mice treated as indicated. E. Percentage analysis of the atherosclerotic region from C. F. Statistical data of the oil O red positive plaque area from D. Data are expressed as mean ± SEM. *, p < 0.05 by one-way ANOVA.

Figure 8

Schematic illustration of the study. In Ldlr^-/- mice, deficiency of Ldlr results in abnormal lipid metabolism and atherosclerosis. However, exosome-mediated Ldlr mRNA delivery could robustly restore Ldlr expression and thus reverse the phenotype.