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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 AAV8 encoding mouse Vldlr or mouse Ldlr in Ldlr-/-Apobec1-/- Mice.(A) Plasma cholesterol levels in Ldlr-/-Apobec1-/- mice after treatment with AAV8.TBG.mVLDLR or AAV8.TBG.mLDLR (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 Ldlr-/-Apobec1-/- (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 Ldlr-/-Apobec1-/- mice after treatment with AAV8.TBG.mVLDLR or AAV.TBG.mLDLR (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.mLDLR and AAV8.TBG.mVLDLR.
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pone-0013424-g001: Evaluation of AAV8 encoding mouse Vldlr or mouse Ldlr in Ldlr-/-Apobec1-/- Mice.(A) Plasma cholesterol levels in Ldlr-/-Apobec1-/- mice after treatment with AAV8.TBG.mVLDLR or AAV8.TBG.mLDLR (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 Ldlr-/-Apobec1-/- (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 Ldlr-/-Apobec1-/- mice after treatment with AAV8.TBG.mVLDLR or AAV.TBG.mLDLR (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.mLDLR and AAV8.TBG.mVLDLR.

Mentions: A proximal step in the development of a clinical trial of gene therapy for hoFH is the selection of the transgene which, in the case of hoFH, could be LDLR or VLDLR. We performed a direct comparison of AAV8 mediated gene transfer of the murine versions of both genes at three doses (1×1012, 3×1011 and 1×1011 GC/mouse) in chow-fed Ldlr−/−Apobec1−/− mice using a liver specific promoter, thyroxine binding globulin (TBG). Within seven days of vector administration, a marked reduction in plasma cholesterol was realized in Ldlr−/−Apobec1−/− mice injected with AAV8.TBG.mLDLR at all doses compared to control AAV8.TBG.nLacZ treated animals, in which serum lipids were maintained at the high baseline levels throughout the study (Fig. 1A). In the low-dose (1×1011 GC/mouse) group, for example, plasma cholesterol levels significantly fell from a baseline level of 505±59 mg/dl to 112±34 mg/dl. AAV8.TBG.mVLDLR treated animals realized much less significant decreases in levels of total plasma cholesterol (Fig. 1A). We also examined FPLC fraction cholesterol levels 28 days after treatment with the high and (Fig. 1B) and intermediate-dose of vector (Fig. 1C). AAV8.TBG.mLDLR at both doses was considerably more effective than AAV8.TBG.mVLDLR in reducing levels of VLDL and IDL/LDL in Ldlr−/−Apobec1−/− mice; in contrast, a prominent IDL/LDL peak was detected at both doses of AAV8.TBG.mVLDLR treatment. No elevation of transaminases was detected in any group (Fig. 1D). Given these results, the remaining experiments focused exclusively on characterizing AAV8.TBG.mLDLR function in vivo.


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 AAV8 encoding mouse Vldlr or mouse Ldlr in Ldlr-/-Apobec1-/- Mice.(A) Plasma cholesterol levels in Ldlr-/-Apobec1-/- mice after treatment with AAV8.TBG.mVLDLR or AAV8.TBG.mLDLR (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 Ldlr-/-Apobec1-/- (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 Ldlr-/-Apobec1-/- mice after treatment with AAV8.TBG.mVLDLR or AAV.TBG.mLDLR (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.mLDLR and AAV8.TBG.mVLDLR.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2957433&req=5

pone-0013424-g001: Evaluation of AAV8 encoding mouse Vldlr or mouse Ldlr in Ldlr-/-Apobec1-/- Mice.(A) Plasma cholesterol levels in Ldlr-/-Apobec1-/- mice after treatment with AAV8.TBG.mVLDLR or AAV8.TBG.mLDLR (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 Ldlr-/-Apobec1-/- (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 Ldlr-/-Apobec1-/- mice after treatment with AAV8.TBG.mVLDLR or AAV.TBG.mLDLR (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.mLDLR and AAV8.TBG.mVLDLR.
Mentions: A proximal step in the development of a clinical trial of gene therapy for hoFH is the selection of the transgene which, in the case of hoFH, could be LDLR or VLDLR. We performed a direct comparison of AAV8 mediated gene transfer of the murine versions of both genes at three doses (1×1012, 3×1011 and 1×1011 GC/mouse) in chow-fed Ldlr−/−Apobec1−/− mice using a liver specific promoter, thyroxine binding globulin (TBG). Within seven days of vector administration, a marked reduction in plasma cholesterol was realized in Ldlr−/−Apobec1−/− mice injected with AAV8.TBG.mLDLR at all doses compared to control AAV8.TBG.nLacZ treated animals, in which serum lipids were maintained at the high baseline levels throughout the study (Fig. 1A). In the low-dose (1×1011 GC/mouse) group, for example, plasma cholesterol levels significantly fell from a baseline level of 505±59 mg/dl to 112±34 mg/dl. AAV8.TBG.mVLDLR treated animals realized much less significant decreases in levels of total plasma cholesterol (Fig. 1A). We also examined FPLC fraction cholesterol levels 28 days after treatment with the high and (Fig. 1B) and intermediate-dose of vector (Fig. 1C). AAV8.TBG.mLDLR at both doses was considerably more effective than AAV8.TBG.mVLDLR in reducing levels of VLDL and IDL/LDL in Ldlr−/−Apobec1−/− mice; in contrast, a prominent IDL/LDL peak was detected at both doses of AAV8.TBG.mVLDLR treatment. No elevation of transaminases was detected in any group (Fig. 1D). Given these results, the remaining experiments focused exclusively on characterizing AAV8.TBG.mLDLR function in vivo.

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