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Overview of causes of in-stent thrombosis after balloon angioplasty.
Cell-selective inhibition of restenosis
The development of percutaneous coronary intervention (e.g., balloon angioplasty) revolutionized the treatment of cardiovascular disease caused by atherosclerosis. These interventions have evolved from simple balloons that squeeze the plaque flat to bare metal stents that hold the expanded blood vessel open and drug-eluting stents that inhibit the overgrowth of smooth muscle cells into the artery (restenosis). However, the presence of the stent and the non-specific activity of the cell cycle inhibitors employed on drug-eluting stents have led to an increased incidence of late-onset adverse events (i.e., in-stent thrombus formation). An ideal post-angioplasty therapy would spare the endothelial cells that line blood vessels to promote healing and minimize thrombosis risk.
Immunoflourescence imaging of rat carotid arteries after balloon injury and local treatment with adenovirus with the indicated constructs. (Adapted from Santulli et al. 2014 JCI)
Hana Totary-Jain (my PhD advisor) developed a viral therapy for restenosis capable of selectively sparing endothelial cells. The inclusion of target sites for microRNA 126 in the 3' UTR of p27 (an inhibitory regulator of the cell cycle) only prevented the overgrowth of smooth muscle cells after angioplasty while allowing the regrowth of the damaged endothelium. The treated vessels showed an accelerated healing rate compared to untreated vessels or vessels treated with a non-selective version of the treatment (Santulli et al. 2014 JCI). However, this approach required two crucial advances to make it clinically viable: firstly, a non-viral vector was needed, and secondly, the vector should be delivered systemically and still reach the injured vessel.
Overview of innate immune response to exogenous RNAs.
Building an effective, regulatable synthetic mRNA
A, G, C, and U: The four nucleotides that make up RNA... if you don't count the numerous modified versions of these nucleotides that are also found in RNA. These modifications play a key role in distinguishing a cell's own RNA from that of an invading virus. By incorporating these modified nucleotides during in vitro transcription, synthetic mRNAs can avoid the innate immune response triggered by Toll-like receptors. This was first shown to increase the efficacy of modified mRNAs by Katalin Kariko and paved the way for the development of the Pfizer/BioNTech SARS-CoV-2 vaccine in 2020.
Western blots for GFP in 293T cells 24 hours after transfection of the indicated microRNA mimics and modified mRNAs (24 hours after mimics). (Taken from Lockhart et al. 2019 MTNA)
microRNAs post-transcriptionally regulate mRNAs by binding (typically) to the 3' UTR to inhibit translation and promote mRNA degradation. One common modified nucleotide, 5-methylcytidine (m5C), is enriched near microRNA binding sites. We found that incorporating modified nucleotides into synthetic mRNA can alter its regulation by microRNA whose target sites were included in the 3' UTR (Panels A - D). However, moving that microRNA target site to the 5' UTR significantly improved the silencing capacity of the cognate microRNAs, particularly for one of the most potent nucleotide modifications, N-1-methyl-pseudouridine (Panels E-F).
This work was published in "Nucleotide modification alters microRNA-dependent silencing of microRNA switches" (Lockhart et al. 2019 MTNA)
Diagram of the RNA delivery mechanism of p5RHH. (Taken from Hou et al. 2013 ACS Nano).
Delivering mRNA payloads with a peptide nanoparticle
mRNA therapeutics hold a lot of promise for the treatment of human diseases. The rapid development of vaccines against SARS-CoV-2 (Covid-19) highlighted the potential of this approach. However, delivering the mRNA to the target tissue is the (current) primary limitation for the widespread application of mRNA therapeutics. Lipid nanoparticles are the most common method of mRNA delivery currently employed in both the lab and the clinic. However, Samuel Wickline's group developed a modified form of melittin (the primary component of honey bee venom) that was capable of forming nanoparticles with small RNA molecules (Hou et al. 2013 ACS Nano). In addition, Wickline's group had shown that siRNA-p5RHH nanoparticles exhibited a predilection to accumulate in regions of disrupted endothelium (Pan et al. 2018, Int J Nano), which suggested a similar accumulation might occur after endothelial damage during balloon angioplasty.
Demonstration that mRNA-p5RHH nanoparticles are RNAse resistant and effective at transfecting cells in vitro. (Taken from Lockhart et al. 2021 Mol Ther)
We performed several optimization experiments to achieve robust transfection of mRNA that orders-of-magnitude larger than the small interfering RNA previously used by the Wickline lab. The mRNA-p5RHH nanoparticles possessed several qualities that made them ideal for in vitro use: 1) the nanoparticles formed spontaneously in normal transfection conditions, 2) the nanoparticles were extremely resistant to RNase degradation (Panels A-B), and 3) transfection with mRNA-p5RHH nanoparticles was well tolerated even by primary cells that are sensitive to commercial lipid-based transfection reagents.
In addition, we observe that endothelial cells were less likely to uptake the mRNA-p5RHH nanoparticles compared to smooth muscle cells. The synergy of the microRNA-126 detargeting strategy and the selective uptake of the mRNA-p5RHH nanoparticles allowed us specifically inhibit the growth and migration of smooth muscle cells while simultaneously allowing endothelial cells to continue the wound healing process.
This work was published in "Self-assembled miRNA-switch nanoparticles target denuded regions and prevent restenosis" (Lockhart et al. 2021 Mol Ther).
Fluorescence microscopy images of injured/uninjured mouse femoral arteries after systemic administration of niRFP mRNA-p5RHH nanoparticles. (Taken from Lockhart et al. 2021 Mol Ther)
Putting it all together
Now that we had an optimized mRNA vector to replace the adenoviral vector used in the original study by Hana Totary-Jain and an effective delivery platform, all that remained was to test the approach in an animal model. Our preliminary studies using the mRNA-p5RHH nanoparticles showed that the treatments were well tolerated by the mice and specific overexpression of the cargo mRNA in the denuded blood vessels. Importantly, this overexpression was seen after systemic administration of the nanoparticles, allowing us to circumvent the localized treatment employed in Dr. Totary-Jain's original work (Santulli et al. 2014 JCI).
Analysis of restenosis after wire injury in mice treated with our therapeutic nanoparticles or control nanoparticles. (Taken from Lockhart et al. 2021 Mol Ther)
After treating the mice for two weeks with mRNA-p5RHH nanoparticles loaded with a nucleotide-modified mRNA encoding p27 with one target site in the 5' UTR, we saw a complete inhibition of restenosis. In contrast, mice treated with nanoparticles containing a control near-infrared fluorescent protein (niRFP) encoding mRNA had significant restenosis (Panels A-C). Notably, the endothelial cells of the animals that received the therapeutic nanoparticles regrew into the damaged area. The regrowth of the endothelial layer is a critical shortcoming of the drugs currently used to prevent restenosis and highlights the power of this approach.
This work was published in "Self-assembled miRNA-switch nanoparticles target denuded regions and prevent restenosis" (Lockhart et al. 2021 Mol Ther).
Dr. Totary-Jain's lab is continuing to develop this approach for clinical applications.
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