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Efficient Gene Knockdown in the Liver via Intrasplenic Injection of Adeno-Associated Virus Serotype 8 (AAV8)-Delivered Small Hairpin RNA
Published on: November 1, 2024
Apolipoprotein B knockdown by AAV-delivered shRNA lowers plasma cholesterol in mice
Annemart Koornneef1, Piotr Maczuga, Richard van Logtenstein
1Department of Research and Development, Amsterdam Molecular Therapeutics, Amsterdam, The Netherlands. p.konstantinova@amtbiopharma.com
Abstract:
Serum low-density lipoprotein cholesterol (LDL-C) levels are proportionate to the risk of atherosclerotic cardiovascular disease. In order to reduce serum total cholesterol and LDL-C levels in mice, RNA interference (RNAi) was used to inhibit expression of the structural protein of LDL-C, apolipoprotein B100 (ApoB). We developed and screened 19 short hairpin RNAs (shRNAs) targeting conserved sequences in human, mouse, and macaque ApoB mRNAs (shApoB) and subsequently narrowed our focus to one candidate for in vivo testing. Self-complementary adeno-associated virus serotype 8 (scAAV8) was used for long-term transduction of murine liver with shApoB. A strong dose-dependent knockdown of ApoB mRNA and protein was observed, which correlated with a reduction in total cholesterol levels, without obvious signs of toxicity. Furthermore, shApoB was found to specifically reduce LDL-C in diet-induced dyslipidemic mice, whereas high-density lipoprotein cholesterol (HDL-C) remained unaffected. Finally, elevated lipid accumulation was shown in murine liver transduced with shApoB, a known phenotypic side effect of lowering ApoB levels. These results demonstrate a robust dose-dependent knockdown of ApoB by AAV-delivered shRNA in murine liver, thus providing an excellent candidate for development of RNAi-based gene therapy for the treatment of hypercholesterolemia.
Insights
RNA interference targeting apolipoprotein B100 (ApoB) effectively reduced LDL-C in mice. This study demonstrates a promising RNAi-based gene therapy candidate for treating high cholesterol by lowering ApoB levels.
Area of Science:
- Biochemistry
- Molecular Biology
- Gene Therapy
Background:
- Elevated low-density lipoprotein cholesterol (LDL-C) is a primary risk factor for atherosclerotic cardiovascular disease.
- Apolipoprotein B100 (ApoB) is the main structural protein of LDL-C, making it a key therapeutic target.
Purpose of the Study:
- To develop and evaluate RNA interference (RNAi) using short hairpin RNAs (shRNAs) delivered via adeno-associated virus (AAV) to inhibit ApoB expression.
- To assess the efficacy and safety of ApoB knockdown in reducing LDL-C levels in a mouse model.
Main Methods:
- Screened 19 shRNAs targeting conserved ApoB mRNA sequences.
- Utilized self-complementary AAV serotype 8 (scAAV8) for liver transduction in mice.
- Administered varying doses of shApoB via AAV to assess dose-dependent effects.
Main Results:
- Achieved robust, dose-dependent knockdown of ApoB mRNA and protein in the liver.
- Observed significant reduction in serum total cholesterol and LDL-C levels.
- Demonstrated specific reduction of LDL-C without affecting high-density lipoprotein cholesterol (HDL-C) levels in dyslipidemic mice.
Conclusions:
- AAV-delivered shRNA targeting ApoB is a potent strategy for lowering LDL-C.
- This approach shows promise as a gene therapy for hypercholesterolemia with manageable side effects like hepatic lipid accumulation.
- Further development is warranted for clinical translation of this RNAi-based therapy.
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