Gene Therapy in a Humanized Mouse Model of Familial Hypercholesterolemia Leads to Marked Regression of Atherosclerosis

Familial hypercholesterolemia (FH) is an autosomal codominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Homozygous FH patients (hoFH) have severe hypercholesterolemia leading to life threatening atherosclerosis in childhood and adolescence. Mice with germ line interruptions in the Ldlr and Apobec1 genes (Ldlr(-/-)Apobec1(-/-)) simulate metabolic and clinical aspects of hoFH, including atherogenesis on a chow diet.In this study, vectors based on adeno-associated virus 8 (AAV8) were used to deliver the gene for mouse Ldlr (mLDLR) to the livers of Ldlr(-/-)Apobec1(-/-) mice. A single intravenous injection of AAV8.mLDLR was found to significantly reduce plasma cholesterol and non-HDL cholesterol levels in chow-fed animals at doses as low as 3×10(9) genome copies/mouse. Whereas Ldlr(-/-)Apobec1(-/-) mice fed a western-type diet and injected with a control AAV8.null vector experienced a further 65% progression in atherosclerosis over 2 months compared with baseline mice, Ldlr(-/-)Apobec1(-/-) mice treated with AAV8.mLDLR realized an 87% regression of atherosclerotic lesions after 3 months compared to baseline mice. Immunohistochemical analyses revealed a substantial remodeling of atherosclerotic lesions.Collectively, the results presented herein suggest that AAV8-based gene therapy for FH may be feasible and support further development of this approach. The pre-clinical data from these studies will enable for the effective translation of gene therapy into the clinic for treatment of FH

Identification of the Active Form of Endothelial Lipase, a Homodimer in a Head-to-Tail Conformation*

Endothelial lipase (EL) is a member of a subfamily of lipases that act on triglycerides and phospholipids in plasma lipoproteins, which also includes lipoprotein lipase and hepatic lipase. EL has a tropism for high density lipoprotein, and its level of phospholipase activity is similar to its level of triglyceride lipase activity. Inhibition or loss-of-function of EL in mice results in an increase in high density lipoprotein cholesterol, making it a potential therapeutic target. Although hepatic lipase and lipoprotein lipase have been shown to function as homodimers, the active form of EL is not known. In these studies, the size and conformation of the active form of EL were determined. Immunoprecipitation experiments suggested oligomerization. Ultracentrifugation experiments showed that the active form of EL had a molecular weight higher than the molecular weight of a simple monomer but less than a dimer. A construct encoding a covalent head-to-tail homodimer of EL (EL-EL) was expressed and had similar lipolytic activity to EL. The functional molecular weights determined by radiation inactivation were similar for EL and the covalent homodimer EL-EL. We previously showed that EL could be cleaved by proprotein convertases, such as PC5, resulting in loss of activity. In cells overexpressing PC5, the covalent homodimeric EL-EL appeared to be more stable, with reduced cleavage and conserved lipolytic activity. A comparative model obtained using other lipase structures suggests a structure for the head-to-tail EL homodimer that is consistent with the experimental findings. These data confirm the hypothesis that EL is active as a homodimer in head-to-tail conformation

Evaluation of AAV8 encoding mouse <i>Vldlr</i> or mouse <i>Ldlr</i> in Ldlr-/-Apobec1-/- Mice.

<p>(A) Plasma cholesterol levels in <i>Ldlr-/-Apobec1-/-</i> mice after treatment with AAV8.TBG.<i>mVLDLR</i> or AAV8.TBG.<i>mLDLR</i> (n = 5 animals per dose group). Each point represents mean ± s.d. *P<0.05, **P<0.01, ***P<0.001 (B and C) Pooled mouse plasma from AAV-injected <i>Ldlr-/-Apobec1-/-</i> (n = 5) were analyzed by FPLC fractionation and the cholesterol content of each fraction was determined. (B) Lipoprotein profile of animals injected with 1×10∧12 GC of vector 28 days after treatment. (C) Lipoprotein profile of animals injected with 3×10∧11 GC of vector 28 days after treatment. (D) Plasma ALT levels in <i>Ldlr-/-Apobec1-/-</i> mice after treatment with AAV8.TBG.<i>mVLDLR</i> or AAV.TBG.<i>mLDLR</i> (n = 5 animals per dose group). Each point represents mean ± s.d. At all time points and doses examined, no significant differences in ALT were detected between AAV8.TBG.<i>mLDLR</i> and AAV8.TBG.<i>mVLDLR</i>.</p

Evaluation of AAV8.TBG.<i>mLDLR</i> vector in high fat fed <i>Ldlr-/-Apobec1-/-</i> mice.

<p>Amounts of (A) Plasma cholesterol (B) non-HDL cholesterol, and (C) Alanine transaminase were evaluated in <i>Ldlr-/-Apobec1-/-</i> mice up to day 60 after treatment with 1x10∧11 GC of AAV8.TBG.<i>mLDLR</i> (n = 10) or 1×1011 GC of AAV8.TBG.<i>nLacZ</i> (n = 9). Each point represents mean ± s.d. *P<0.05, ‡ P<0.001.</p

Immunohistochemical analysis of mouse atherosclerotic lesions.

<p>Representative aortic root sections immunostained for the foam cell marker CD68 (A), VCAM-1 (B), or Masson trichrome blue stain for collagen content (C). Original magnification, 40×. Note abundant immunostaining for foam cell marker, CD68 (brown), VCAM-1 adhesion molecules (also brown), and presence of collagen αblue) within lesion in baseline and AAV.TBG.<i>nLacZ</i> injected <i>Ldlr-/-Apobec1-/-</i>animals.</p

AAV8.TBG.<i>mLDLR</i> mediated regression of atherosclerotic lesions in high-fat fed <i>Ldlr-/-Apobec1-/-</i>mice.

<p>(A) En face Sudan IV staining. Mouse aortas were pinned and stained with Sudan IV, which stains neutral lipids. Representative aortas from animals treated with 1×10∧11 of AAV8.TBG.<i>nLacZ,</i> 1×10∧11 of AAV8.TBG.<i>mLDLR</i> at day 60 after vector administration (day 120 on high-fat diet), or at baseline (day 60 on high-fat diet) are shown. (B) The percent Sudan IV staining of the total aortic surface in baseline (n = 10), AAV.TBG.<i>nLacZ</i> (n = 9) and AAV8.TBG.<i>mLDLR</i> (n = 10) was determined as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013424#s2" target="_blank">Materials and Methods</a>. Aortic roots from these mice were stained with oil red o (C) or hematoxylin and eosin (H&E) (D) 10× magnification. Quantification was conducted on oil red o lesions (E) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013424#s2" target="_blank">materials and methods</a>. Each column represents mean ± s.d. *P<0.05, **P<0.01, ***P<0.001, ‡ P<0.001. (F) H&E stained aortic roots at 40× magnification show a thin fibrous cap and expanded necrotic core in lesions of baseline and AAV8.nLacZ treated mice compared to AAV8.mLDLR injected animals.</p

Evaluation of the minimum effective dose of AAV.TBG.<i>mLDLR</i> vector in <i>Ldlr-/-Apobec1-/-</i> Mice.

<p>Amounts of (A) Plasma cholesterol and (B) non-HDL cholesterol were evaluated in <i>Ldlr-/-Apobec1-/-</i> mice up to day 35 after treatment with different doses of AAV8.mLDLR (n = 9 animals per dose group). Each point represents mean ± s.d. *P<0.05, **P<0.01, ***P<0.001. (C) Pooled mouse plasma from AAV-injected <i>Ldlr-/-Apobec1-/-</i> (n = 5, per dose group) were analyzed by FPLC fractionation and the cholesterol content of each fraction was determined. (D) Dose response analysis of Day 60 samples examining cholesterol levels as a function of vector dose. (E) Plasma cholesterol and (F) Alanine transaminase were evaluated in <i>Ldlr-/-Apobec1-/-</i> mice up to day 180 days after treatment with 1×10∧11 GC of AAV8.TBG.<i>mLDLR</i> (n = 10) or 1×10∧11 GC of AAV8.TBG.<i>nLacZ</i> (n = 9). Each point represents mean ± s.d.</p