The Totary-Jain Lab

Exploring the role of non-coding RNAs in immunity and the progression of atherosclerosis.

    

Atherosclerotic Cardiovascular Disease

Endothelial cell coverage of balloon
injured arteries treated with cell selective
Ad-p27-126Ts miRNA switch
En-Face Endothelial cell staining of artery
exhibiting restenosis after balloon injury

Our lab aimed to develop cell-selective nanotherapy to combat atherosclerotic cardiovascular disease. This therapy specifically targets inflammatory and vascular smooth muscle cells, which are key players in plaque buildup, while carefully preserving healthy endothelial cells.

Initially, we employed an adenoviral vector, but our current focus is on a more advanced synthetic mRNA-based miRNA switch technology encapsulated in nanoparticles.

This innovative approach effectively delivers therapeutic payloads directly to damaged vessel areas, significantly reducing plaque formation and promoting vessel repair. We are actively enhancing this technology by combining it with siRNA to further suppress inflammation, in an effort to restore the endothelial barrier while inhibiting the progression of atherosclerotic plaque.

Transposable Elements and Antiviral Defense

Our laboratory was the first to use CRISPR technology to transcriptionally activate the C19MC, a primate-specific microRNA cluster located on chromosome 19. This breakthrough enabled us to uncover a compelling example of convergent evolution involving the C19MC and a rodent-specific microRNA cluster on chromosome 2, C2MC. These clusters are primarily expressed in trophoblasts and are enriched with short interspersed nuclear elements (SINEs) known as Alu (primate) or B1 (rodent) repeats.

Our research has revealed that the SINEs of these clusters produce double-stranded RNA (dsRNA), which triggers a potent immune response akin to viral mimicry. This immune activation involves type III interferon signaling, providing crucial antiviral protection to the fetus during development. Furthermore, we also demonstrated that C19MC plays a pivotal role in regulating epithelial-to-mesenchymal transition and accelerating cell reprogramming processes.

Relevant Publications

  • Wickramage, Ishani, Jeffrey VanWye, Klaas Max, John H Lockhart, Ismet Hortu, Ezinne F Mong, John Canfield, et al. (2023) 2023. “SINE RNA of the Imprinted MiRNA Clusters Mediates Constitutive Type III Interferon Expression and Antiviral Protection in Hemochorial Placentas.”. Cell Host & Microbe 31 (7): 1185-1199.e10. https://doi.org/10.1016/j.chom.2023.05.018.
    Publisher's Version
    Publisher's Version

    Hemochorial placentas have evolved defense mechanisms to prevent the vertical transmission of viruses to the immunologically underdeveloped fetus. Unlike somatic cells that require pathogen-associated molecular patterns to stimulate interferon production, placental trophoblasts constitutively produce type III interferons (IFNL) through an unknown mechanism. We demonstrate that transcripts of short interspersed nuclear elements (SINEs) embedded in miRNA clusters within the placenta trigger a viral mimicry response that induces IFNL and confers antiviral protection. Alu SINEs within primate-specific chromosome 19 (C19MC) and B1 SINEs within rodent-specific microRNA cluster on chromosome 2 (C2MC) produce dsRNAs that activate RIG-I-like receptors (RLRs) and downstream IFNL production. Homozygous C2MC knockout mouse trophoblast stem (mTS) cells and placentas lose intrinsic IFN expression and antiviral protection, whereas B1 RNA overexpression restores C2MCΔ/Δ mTS cell viral resistance. Our results uncover a convergently evolved mechanism whereby SINE RNAs drive antiviral resistance in hemochorial placentas, placing SINEs as integral players in innate immunity.

  • Lockhart, John H, Jeffrey VanWye, Richa Banerjee, Samuel A Wickline, Hua Pan, and Hana Totary-Jain. (2021) 2021. “Self-Assembled MiRNA-Switch Nanoparticles Target Denuded Regions and Prevent Restenosis.”. Molecular Therapy : The Journal of the American Society of Gene Therapy 29 (5): 1744-57. https://doi.org/10.1016/j.ymthe.2021.01.032.
    Publisher's Version
    Publisher's Version

    Cardiovascular disease is the leading cause of death and disability worldwide. Effective delivery of cell-selective therapies that target atherosclerotic plaques and neointimal growth while sparing the endothelium remains the Achilles heel of percutaneous interventions. The current study utilizes synthetic microRNA switch therapy that self-assembles to form a compacted, nuclease-resistant nanoparticle <200 nM in size when mixed with cationic amphipathic cell-penetrating peptide (p5RHH). These nanoparticles possess intrinsic endosomolytic activity that requires endosomal acidification. When administered in a femoral artery wire injury mouse model in vivo, the mRNA-p5RHH nanoparticles deliver their payload specifically to the regions of endothelial denudation and not to the lungs, liver, kidney, or spleen. Moreover, repeated administration of nanoparticles containing a microRNA switch, consisting of synthetically modified mRNA encoding for the cyclin-dependent kinase inhibitor p27Kip1 that contains one complementary target sequence of the endothelial cell-specific miR-126 at its 5' UTR, drastically reduced neointima formation after wire injury and allowed for vessel reendothelialization. This cell-selective nanotherapy is a valuable tool that has the potential to advance the fight against neointimal hyperplasia and atherosclerosis.

  • Santulli, Gaetano, Anetta Wronska, Kunihiro Uryu, Thomas G Diacovo, Melanie Gao, Steven O Marx, Jan Kitajewski, et al. (2014) 2014. “A Selective MicroRNA-Based Strategy Inhibits Restenosis While Preserving Endothelial Function.”. The Journal of Clinical Investigation 124 (9): 4102-14. https://doi.org/10.1172/JCI76069.
    Publisher's Version
    Publisher's Version

    Drugs currently approved to coat stents used in percutaneous coronary interventions do not discriminate between proliferating vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). This lack of discrimination delays reendothelialization and vascular healing, increasing the risk of late thrombosis following angioplasty. We developed a microRNA-based (miRNA-based) approach to inhibit proliferative VSMCs, thus preventing restenosis, while selectively promoting reendothelialization and preserving EC function. We used an adenoviral (Ad) vector that encodes cyclin-dependent kinase inhibitor p27(Kip1) (p27) with target sequences for EC-specific miR-126-3p at the 3' end (Ad-p27-126TS). Exogenous p27 overexpression was evaluated in vitro and in a rat arterial balloon injury model following transduction with Ad-p27-126TS, Ad-p27 (without miR-126 target sequences), or Ad-GFP (control). In vitro, Ad-p27-126TS protected the ability of ECs to proliferate, migrate, and form networks. At 2 and 4 weeks after injury, Ad-p27-126TS-treated animals exhibited reduced restenosis, complete reendothelialization, reduced hypercoagulability, and restoration of the vasodilatory response to acetylcholine to levels comparable to those in uninjured vessels. By incorporating miR-126-3p target sequences to leverage endogenous EC-specific miR-126, we overexpressed exogenous p27 in VSMCs, while selectively inhibiting p27 overexpression in ECs. Our proof-of-principle study demonstrates the potential of using a miRNA-based strategy as a therapeutic approach to specifically inhibit vascular restenosis while preserving EC function.