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Gene therapy in a humanized mouse model of familial hypercholesterolemia leads to marked regression of atherosclerosis.

Kassim SH, Li H, Vandenberghe LH, Hinderer C, Bell P, Marchadier D, Wilson A, Cromley D, Redon V, Yu H, Wilson JM, Rader DJ - PLoS ONE (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT

Background: 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.

Methods/principal findings: 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. 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.

Conclusions/significance: 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.

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Evaluation of the minimum effective dose of AAV.TBG.mLDLR vector in Ldlr-/-Apobec1-/- Mice.Amounts of (A) Plasma cholesterol and (B) non-HDL cholesterol were evaluated in Ldlr-/-Apobec1-/- 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 Ldlr-/-Apobec1-/- (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 Ldlr-/-Apobec1-/- mice up to day 180 days after treatment with 1×10∧11 GC of AAV8.TBG.mLDLR (n = 10) or 1×10∧11 GC of AAV8.TBG.nLacZ (n = 9). Each point represents mean ± s.d.
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pone-0013424-g002: Evaluation of the minimum effective dose of AAV.TBG.mLDLR vector in Ldlr-/-Apobec1-/- Mice.Amounts of (A) Plasma cholesterol and (B) non-HDL cholesterol were evaluated in Ldlr-/-Apobec1-/- 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 Ldlr-/-Apobec1-/- (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 Ldlr-/-Apobec1-/- mice up to day 180 days after treatment with 1×10∧11 GC of AAV8.TBG.mLDLR (n = 10) or 1×10∧11 GC of AAV8.TBG.nLacZ (n = 9). Each point represents mean ± s.d.

Mentions: Correction of hypercholesterolemia was found to be dose dependent. With respect to total cholesterol (Fig. 2A) and non-HDL cholesterol (Fig. 2B), reduction was realized within seven days of vector treatment. The most significant decreases were observed at the highest treatment doses. Surprisingly, reduction was observed with doses as low as 3×109 GC/mouse with total cholesterol levels dropping 35% from 397±26 mg/dl at baseline to 256±29 mg/dl on day 35 and non-HDL cholesterol levels decreasing by 45% from 284±19 mg/dl at baseline to 154±17 mg/dl (Fig. 2B) on day 35. This magnitude of correction is comparable to that seen in Ldlr−/−Apobec1−/− mice treated with 1×1011 GC/mouse of AAV8.TBG.mVLDLR (Fig. 1A), suggesting that AAV8.TBG.mLDLR is at least 30 fold more effective than AAV8.TBG.mVLDLR. With respect to FPLC fraction cholesterol levels, all doses of AAV8.TBG.mLDLR, except 3×108 GC, led to substantial reductions in the IDL/LDL peak compared to AAV8.TBG.nLacZ treated Ldlr−/−Apobec1−/− mice. Only the higher doses of 1×1011 GC/mouse and 1×1010 GC/mouse also reduced the HDL-C levels compared to control Ldlr−/−Apobec1−/− mice (Fig. 2C). Regression analysis of the day 35 post-injection data revealed a clear dose-dependent response with regard to reduction of total cholesterol levels (Fig. 2D). Long-term studies revealed that treatment of Ldlr−/−Apobec1−/− mice with 1×1011 GC AAV8.TBG.mLDLR resulted in significant correction of cholesterol (Fig. 2E) in the absence of ALT elevation for up to 6 months after injection of vector (Fig. 2F). Collectively, these data reveal that 1×1010 GC/mouse is the minimum effective dose required for complete correction; furthermore, partial correction can be attained with as little as 3×109 GC/mouse. Importantly, this correction was maintained for up to 6 months after treatment with no detectable inflammation.


Gene therapy in a humanized mouse model of familial hypercholesterolemia leads to marked regression of atherosclerosis.

Kassim SH, Li H, Vandenberghe LH, Hinderer C, Bell P, Marchadier D, Wilson A, Cromley D, Redon V, Yu H, Wilson JM, Rader DJ - PLoS ONE (2010)

Evaluation of the minimum effective dose of AAV.TBG.mLDLR vector in Ldlr-/-Apobec1-/- Mice.Amounts of (A) Plasma cholesterol and (B) non-HDL cholesterol were evaluated in Ldlr-/-Apobec1-/- 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 Ldlr-/-Apobec1-/- (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 Ldlr-/-Apobec1-/- mice up to day 180 days after treatment with 1×10∧11 GC of AAV8.TBG.mLDLR (n = 10) or 1×10∧11 GC of AAV8.TBG.nLacZ (n = 9). Each point represents mean ± s.d.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2957433&req=5

pone-0013424-g002: Evaluation of the minimum effective dose of AAV.TBG.mLDLR vector in Ldlr-/-Apobec1-/- Mice.Amounts of (A) Plasma cholesterol and (B) non-HDL cholesterol were evaluated in Ldlr-/-Apobec1-/- 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 Ldlr-/-Apobec1-/- (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 Ldlr-/-Apobec1-/- mice up to day 180 days after treatment with 1×10∧11 GC of AAV8.TBG.mLDLR (n = 10) or 1×10∧11 GC of AAV8.TBG.nLacZ (n = 9). Each point represents mean ± s.d.
Mentions: Correction of hypercholesterolemia was found to be dose dependent. With respect to total cholesterol (Fig. 2A) and non-HDL cholesterol (Fig. 2B), reduction was realized within seven days of vector treatment. The most significant decreases were observed at the highest treatment doses. Surprisingly, reduction was observed with doses as low as 3×109 GC/mouse with total cholesterol levels dropping 35% from 397±26 mg/dl at baseline to 256±29 mg/dl on day 35 and non-HDL cholesterol levels decreasing by 45% from 284±19 mg/dl at baseline to 154±17 mg/dl (Fig. 2B) on day 35. This magnitude of correction is comparable to that seen in Ldlr−/−Apobec1−/− mice treated with 1×1011 GC/mouse of AAV8.TBG.mVLDLR (Fig. 1A), suggesting that AAV8.TBG.mLDLR is at least 30 fold more effective than AAV8.TBG.mVLDLR. With respect to FPLC fraction cholesterol levels, all doses of AAV8.TBG.mLDLR, except 3×108 GC, led to substantial reductions in the IDL/LDL peak compared to AAV8.TBG.nLacZ treated Ldlr−/−Apobec1−/− mice. Only the higher doses of 1×1011 GC/mouse and 1×1010 GC/mouse also reduced the HDL-C levels compared to control Ldlr−/−Apobec1−/− mice (Fig. 2C). Regression analysis of the day 35 post-injection data revealed a clear dose-dependent response with regard to reduction of total cholesterol levels (Fig. 2D). Long-term studies revealed that treatment of Ldlr−/−Apobec1−/− mice with 1×1011 GC AAV8.TBG.mLDLR resulted in significant correction of cholesterol (Fig. 2E) in the absence of ALT elevation for up to 6 months after injection of vector (Fig. 2F). Collectively, these data reveal that 1×1010 GC/mouse is the minimum effective dose required for complete correction; furthermore, partial correction can be attained with as little as 3×109 GC/mouse. Importantly, this correction was maintained for up to 6 months after treatment with no detectable inflammation.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT

Background: 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.

Methods/principal findings: 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. 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.

Conclusions/significance: 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.

Show MeSH
Related in: MedlinePlus