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

Association of Low-Frequency and Rare Coding-Sequence Variants with Blood Lipids and Coronary Heart Disease in 56,000 Whites and Blacks

Low-frequency coding DNA sequence variants in the proprotein convertase subtilisin/kexin type 9 gene (PCSK9) lower plasma low-density lipoprotein cholesterol (LDL-C), protect against risk of coronary heart disease (CHD), and have prompted the development of a new class of therapeutics. It is uncertain whether the PCSK9 example represents a paradigm or an isolated exception. We used the “Exome Array” to genotype >200,000 low-frequency and rare coding sequence variants across the genome in 56,538 individuals (42,208 European ancestry [EA] and 14,330 African ancestry [AA]) and tested these variants for association with LDL-C, high-density lipoprotein cholesterol (HDL-C), and triglycerides. Although we did not identify new genes associated with LDL-C, we did identify four low-frequency (frequencies between 0.1% and 2%) variants (ANGPTL8 rs145464906 [c.361C>T; p.Gln121∗], PAFAH1B2 rs186808413 [c.482C>T; p.Ser161Leu], COL18A1 rs114139997 [c.331G>A; p.Gly111Arg], and PCSK7 rs142953140 [c.1511G>A; p.Arg504His]) with large effects on HDL-C and/or triglycerides. None of these four variants was associated with risk for CHD, suggesting that examples of low-frequency coding variants with robust effects on both lipids and CHD will be limited

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 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

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

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

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

Association of low-frequency and rare coding-sequence variants with blood lipids and coronary heart disease in 56,000 whites and blacks

Low-frequency coding DNA sequence variants in the proprotein convertase subtilisin/kexin type 9 gene (PCSK9) lower plasma low-density lipoprotein cholesterol (LDL-C), protect against risk of coronary heart disease (CHD), and have prompted the development of a new class of therapeutics. It is uncertain whether the PCSK9 example represents a paradigm or an isolated exception. We used the "Exome Array" to genotype >200,000 low-frequency and rare coding sequence variants across the genome in 56,538 individuals (42,208 European ancestry [EA] and 14,330 African ancestry [AA]) and tested these variants for association with LDL-C, high-density lipoprotein cholesterol (HDL-C), and triglycerides. Although we did not identify new genes associated with LDL-C, we did identify four low-frequency (frequencies between 0.1% and 2%) variants (ANGPTL8 rs145464906 [c.361C>T; p.Gln121*], PAFAH1B2 rs186808413 [c.482C>T; p.Ser161Leu], COL18A1 rs114139997 [c.331G>A; p.Gly111Arg], and PCSK7 rs142953140 [c.1511G>A; p.Arg504His]) with large effects on HDL-C and/or triglycerides. None of these four variants was associated with risk for CHD, suggesting that examples of low-frequency coding variants with robust effects on both lipids and CHD will be limited

Association of low-frequency and rare coding-sequence variants with blood lipids and coronary heart disease in 56,000 whites and blacks.

Low-frequency coding DNA sequence variants in the proprotein convertase subtilisin/kexin type 9 gene (PCSK9) lower plasma low-density lipoprotein cholesterol (LDL-C), protect against risk of coronary heart disease (CHD), and have prompted the development of a new class of therapeutics. It is uncertain whether the PCSK9 example represents a paradigm or an isolated exception. We used the "Exome Array" to genotype >200,000 low-frequency and rare coding sequence variants across the genome in 56,538 individuals (42,208 European ancestry [EA] and 14,330 African ancestry [AA]) and tested these variants for association with LDL-C, high-density lipoprotein cholesterol (HDL-C), and triglycerides. Although we did not identify new genes associated with LDL-C, we did identify four low-frequency (frequencies between 0.1% and 2%) variants (ANGPTL8 rs145464906 [c.361C>T; p.Gln121(∗)], PAFAH1B2 rs186808413 [c.482C>T; p.Ser161Leu], COL18A1 rs114139997 [c.331G>A; p.Gly111Arg], and PCSK7 rs142953140 [c.1511G>A; p.Arg504His]) with large effects on HDL-C and/or triglycerides. None of these four variants was associated with risk for CHD, suggesting that examples of low-frequency coding variants with robust effects on both lipids and CHD will be limited

Association of low-frequency and rare coding-sequence variants with blood lipids and coronary heart disease in 56,000 whites and blacks

Low-frequency coding DNA sequence variants in the proprotein convertase subtilisin/kexin type 9 gene (PCSK9) lower plasma low-density lipoprotein cholesterol (LDL-C), protect against risk of coronary heart disease (CHD), and have prompted the development of a new class of therapeutics. It is uncertain whether the PCSK9 example represents a paradigm or an isolated exception. We used the "Exome Array" to genotype >200,000 low-frequency and rare coding sequence variants across the genome in 56,538 individuals (42,208 European ancestry [EA] and 14,330 African ancestry [AA]) and tested these variants for association with LDL-C, high-density lipoprotein cholesterol (HDL-C), and triglycerides. Although we did not identify new genes associated with LDL-C, we did identify four low-frequency (frequencies between 0.1% and 2%) variants (ANGPTL8 rs145464906 [c.361C>T; p.Gln121-], PAFAH1B2 rs186808413 [c.482C>T; p.Ser161Leu], COL18A1 rs114139997 [c.331G>A; p.Gly111Arg], and PCSK7 rs142953140 [c.1511G>A; p.Arg504His]) with large effects on HDL-C and/or triglycerides. None of these four variants was associated with risk for CHD, suggesting that examples of low-frequency coding variants with robust effects on both lipids and CHD will be limited