Researchers from the University of Barcelona and the University of Oregon say they have found a way to control levels of low-density lipoprotein cholesterol (LDL-C). This may prevent it from accumulating in the arteries without the side effects seen from statin-based medications. The new method, detailed in a study published in Biochemical Pharmacology, inhibits PCSK9 gene expression using polypurine hairpins (PPRHs) to increase LDL receptor levels (LDLRs) on cells surfaces which promotes cholesterol uptake from the bloodstream.
PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) is a protease that binds to LDL receptors and marks them for lysosomal degradation. This reduces the number of LDLRs available to clear LDL-cholesterol from circulation. Existing therapies, such as monoclonal antibodies like evolocumab and the siRNA-based drug inclisiran, block PCSK9 function or reduce its production. But these medications are costly and can raise circulating PCSK9 levels or require repeated administration.
To develop this new method, the team designed two PPRHs, HpE9 and HpE12, that bind specifically to exons 9 and 12 of the PCSK9 gene. “Specifically, one of the arms of each chain of the HpE9 and HpE12 polypurines binds specifically to polypyrimidine sequences of exons 9 and 12 of PCSK9, respectively, via Watson-Crick bonds,” said corresponding author Carles J. Ciudad, PhD, a professor of biochemistry and molecular biology, University of Barcelona. This binding halts transcription, thereby reducing PCSK9 expression.
To validated their approach, the team tested the new method in both cultured human liver cells (HepG2) and in transgenic mice expressing human PCSK9. Their testing showed that PPRHs effectively reduced PCSK9 mRNA levels (63% and 74% for HpE9 and HpE12, respectively) and protein (by 76% and 87%) at 24h. In vivo, a single injection of HpE12 in mice led to a 50% drop in plasma PCSK9 and a 47% reduction in total cholesterol within three days. These levels returned to baseline by day 15.
This study builds on prior work using PPRHs in cancer research, where these molecules have been shown to affect the DHFR gene, as wells as the proteins survivin and telomerase. “This is the first report describing the use of these therapeutic oligonucleotides as a strategy in the treatment of metabolic diseases such as hypercholesterolemia,” the researchers wrote.
Compared to statins, which reduce cholesterol by inhibiting HMG-CoA reductase, the PPRH method works upstream by increasing LDL receptor availability. Unlike statins, the study showed that PPRHs do not induce myopathies or cause liver enzyme abnormalities. “PPRHs, especially HpE12, are therapeutic oligonucleotides with many advantages, including low cost of synthesis, stability and lack of immunogenicity,” the researchers noted.
The researchers noted that their experiment showed variability in PCSK9 expression among transgenic mice indicating a the need for improved delivery of this potential therapy. “We are conscious that this can be a limitation since its delivery is not specific,” the authors noted, proposing future use of liver-targeting agents such as N-acetylgalactosamine (GalNac), already employed in approved RNA-based therapies.
Clinical implications are substantial, given the global prevalence of cardiovascular disease and the limitations of current lipid-lowering therapies. Next steps include co-testing HpE9 and HpE12 with statins and other lipid-lowering agents evaluating their comparative performance against current PCSK9 inhibitors, larger studies to explore sex-dependent effects, and finding ways to optimize delivery strategies.
