CRISPR/dCas9-induced upregulation of endogenous Apoa1/Pon1 in apoE-/- mice halved the area of aortic lipid deposits and revealed new mechanisms for APOA1/PON1 anti-atherosclerotic action.
Key Findings
Results
CRISPR/dCas9 technology successfully upregulated endogenous APOA1 and PON1 gene expression in human Huh7 hepatocytes, with proteins secreted into culture medium both in the presence and absence of TNFα.
The CRISPR/dCas9 (dCas9) activation system was used to target transcription of endogenous APOA1/PON1 genes in the human hepatocyte cell line Huh7.
Upregulation was confirmed at both the mRNA and protein level, with secreted proteins detected in conditioned medium.
The system functioned under both basal conditions and inflammatory conditions induced by tumor necrosis factor-α (TNFα).
Results
APOA1-rich conditioned medium from Huh7 hepatocytes exerted antioxidant and anti-inflammatory effects in TNFα-activated EA.hy926 endothelial cells.
Conditioned medium collected from APOA1-upregulated Huh7 cells was applied to EA.hy926 endothelial cells stimulated with TNFα.
The conditioned medium reduced markers of oxidative stress and inflammation in the activated endothelial cells.
This in vitro experiment modeled the paracrine protective effects of hepatocyte-secreted APOA1 on the vascular endothelium.
Results
A single intravenous dose of CRISPR/dCas9 plasmids increased hepatic Apoa1 and Pon1 expression and elevated their serum levels for up to four weeks in apoE-/- mice.
A single i.v. injection of CRISPR/dCas9 plasmids targeting Apoa1 and/or Pon1 was administered to apoE-/- mice.
Hepatic mRNA and serum protein levels of both APOA1 and PON1 were elevated for up to four weeks post-injection.
The duration of effect demonstrates sustained transcriptional activation from a single-dose delivery.
Results
FPLC analysis revealed that increased serum APOA1 in treated apoE-/- mice was distributed among HDL, LDL, and lipid-free fractions.
Fast protein liquid chromatography (FPLC) was used to fractionate serum lipoproteins from treated apoE-/- mice.
Elevated APOA1 was not confined exclusively to HDL particles but was also found associated with LDL fractions and in a lipid-free form.
This distribution pattern indicates that upregulated APOA1 participates in multiple lipoprotein pools, not solely HDL.
Results
Treated apoE-/- mice showed high levels of hepatic, gallbladder, and fecal cholesterol associated with upregulation of hepatic SR-B1, CYP7A1, and ABCG8.
Hepatic scavenger receptor class-B1 (SR-B1), cholesterol 7-alpha-hydroxylase (CYP7A1), and ATP-binding cassette sub-family-G-member-8 (ABCG8) transporter were all upregulated in treated mice.
Elevated expression of these transporters and enzymes corresponded with increased cholesterol content in the liver, gallbladder, and feces.
These findings suggest enhanced reverse cholesterol transport and biliary cholesterol excretion as mechanisms contributing to athero-protection.
Results
No increased inflammatory stress or innate immune activation was detected in apoE-/- mice with upregulated Apoa1/Pon1, while lipid peroxides were decreased in PON1-treated mice.
Safety assessment showed absence of elevated inflammatory or innate immune markers in treated mice, suggesting the CRISPR/dCas9 plasmid delivery did not induce overt immune responses.
Lipid peroxide levels were specifically reduced in PON1-upregulated mice, consistent with the known antioxidant enzymatic activity of PON1.
These findings support the tolerability and antioxidant efficacy of the treatment approach in vivo.
Results
The area of aortic lipid deposits was halved in apoE-/- mice treated with CRISPR/dCas9-mediated upregulation of Apoa1 and/or Pon1.
Quantification of aortic lipid deposits (atherosclerotic lesion area) showed approximately 50% reduction in treated versus untreated apoE-/- mice.
This reduction was described as 'of major interest' by the authors and represents the primary in vivo efficacy endpoint.
The apoE-/- mouse model is a well-established model of atherosclerosis used to study lipid accumulation in the aorta.
What This Means
This research suggests that a gene-activation technology called CRISPR/dCas9 can be used to turn up the production of two naturally protective proteins — apolipoprotein A1 (APOA1) and paraoxonase 1 (PON1) — in the liver, which in turn reduces the buildup of fatty deposits in arteries. The study first showed this worked in human liver cells grown in a lab, where boosted APOA1 production led to protective effects on blood vessel cells under inflammatory conditions. When the same approach was tested in mice prone to developing atherosclerosis (clogged arteries), a single injection caused increased production of both proteins for up to four weeks, and the amount of fatty deposits in the aorta was cut in half.
The study also uncovered several mechanisms that may explain how this works: the extra APOA1 appeared not just in HDL ('good cholesterol') particles but also in other lipid fractions, and the treated mice showed signs of enhanced cholesterol removal via the liver and bile — essentially speeding up the body's natural process for clearing cholesterol. Importantly, the treatment did not trigger unwanted inflammation or immune reactions in the mice, and PON1-treated animals specifically showed lower levels of lipid peroxides, which are harmful byproducts of oxidative stress linked to artery damage.
This research is significant because it demonstrates that CRISPR-based gene activation — rather than gene editing or replacement — can safely amplify the body's own protective proteins to combat cardiovascular disease. Rather than introducing foreign genes, this approach simply increases the activity of genes that already exist, which could represent a safer and potentially longer-lasting strategy for treating atherosclerosis compared to conventional drug therapies. Further development and safety testing would be needed before such an approach could be considered for human use.
Toma L, Barbălată T, Hărătău J, Sanda G, Fuior E, Fenyo I, et al.. (2026). CRISPR/dCas9-induced upregulation of endogenous apolipoprotein A1 and paraoxonase 1 genes reduces the aortic lipid deposits in apoE-/- mice.. Molecular biomedicine. https://doi.org/10.1186/s43556-026-00580-8