ABSTRACT:Cell-free RNA (cf-RNA) has emerged as a critical mediator of intercellular communication and a potential regulator of hemostasis. In this study, plasminogen (Plg), the zymogen precursor of plasmin, was demonstrated to function as a secreted ribonucleoprotein that carries regulatory, extracellular small noncoding RNAs (sRNAs). Purified human and bovine Plg, isolated via lysine-affinity chromatography, were found to transport 25- to 60-nucleotide-long sRNAs derived from both host and microbial sources. In vitro studies revealed that Plg accepted sRNA cargo from primary macrophages and bound candidate sRNAs with moderate (micromolar) affinity. Notably, Plg-sRNA complexes exhibited a distinct RNA profile compared to high-density lipoproteins, and their compositions were sensitive to hypercholesterolemic conditions. Functionally, removal of sRNA cargo from Plg via ribonuclease digestion significantly increased plasmin enzymatic activity and accelerated clot lysis, while also attenuating Plg-induced proinflammatory cytokine expression in both mouse and human macrophages. These findings reveal a dual regulatory role for sRNAs in modulating both the fibrinolytic and immunogenic properties of Plg, offering novel insights into the cross talk between cf-RNA biology and coagulation pathways. This work positions Plg-sRNA interactions as promising targets for therapeutic intervention in thrombotic and inflammatory diseases.

Plasminogen (Plg), the pro-enzyme of the serine-protease plasmin, is known for its role in cleaving blood clots within the fibrinolytic pathway. More recently, Plg has been reported to associate with oxidized phospholipids and accept cholesterol from macrophages, two processes normally observed with lipoproteins. Other non-enzymatic functions of Plg include aiding monocyte migration, activation of immune cells, and angiogenesis. We have previously reported the biological functions of cell-free RNA circulating on lipoproteins. Based on these newly described functions of Plg as a lipoprotein-like particle, we hypothesized that Plg transports functional cell-free RNA. Moreover, we predicted that small RNA (sRNA) cargo regulates Plg’s classic enzymatic and alternative inflammatory capacities. Plg were isolated from plasma by lysine-slurry chromatography and checked for purity and quality by electrophoresis and endotoxin tests, respectively. Highly-pure human and bovine Plg samples were observed to dose-dependently transport RNAs in plasma, namely sRNAs (25-60nt in length). High-throughput small RNA sequencing analyses found that Plg carries both host and non-host (microbial) sRNA. Plg samples were observed to accept sRNAs from donor macrophages in efflux studies, and Plg were observed to have moderate affinity (Kd=nM-µM range) towards candidate host and microbial sRNAs, as quantified by microscale thermophoresis. We also observed that Plg samples were markedly pro-inflammatory towards primary macrophages; however, we demonstrated through PCR and bulk sequencing that sRNA cargo on Plg confers, in large part, Plg’s pro-inflammatory functions towards macrophages, as RNase treated Plg failed to activate cytokine expression in treated human and mouse primary macrophages. Most importantly, RNase treatments of both human and bovine PLG were found to significantly increase Plg’s enzymatic activity, as observed through activity assay and clot lysis assays. Overall, these results support a negative regulatory role for sRNA cargo on Plg’s enzymatic function and a positive role for sRNA cargo driving Plg’s inflammatory properties. Collectively, results from this study suggest that Plg is a novel circulating ribonucleoprotein and that sRNA cargo likely governs the enzymatic capacity and inflammation within the clot or wound microenvironment. Therefore, Plg-sRNA cargo and its receptors provide new therapeutic targets to control fibrinolysis and systemic inflammation.

Background and aims Epitranscriptomic RNA modifications play a crucial role in RNA processing, stability, and function. Small non-coding RNAs (sRNAs), such as tRNA-derived fragments (tDRs), likely inherit modifications from parent transcripts. Evidence supports that cell-free sRNA modifications regulate gene expression, modulate immune responses, and are closely linked to the pathogenesis of immune related diseases. Given that high-density lipoproteins (HDL) transport sRNAs from parent transcripts with extensive modifications, we predicted that many HDL-sRNAs harbor immune regulatory modifications that contribute to HDL functionality. Methods To assess HDL-sRNA modifications in atherosclerotic cardiovascular disease (ASCVD), total RNAs were isolated from HDL of healthy subjects and those with advanced ASCVD. HDL-sRNA modifications were quantified using liquid chromatography-tandem mass-spectrometry (LC-MS/MS), AlkB-facilitated RNA (de)Methylation Sequencing (ARM-seq), and qPCR. Results LC-MS/MS revealed a significant increase in the relative levels of modified nucleosides on HDL in ASCVD and healthy individuals. ARM-seq identified candidate tDR-ArgACG fragments harboring 1-methyladenosine (m1A) modifications that were enriched in ASCVD subjects compared to controls. Bulk mRNA sequencing showed significant macrophage gene expression changes in response to ASCVD-HDL-sRNA uptake, including TMEM123, a transmembrane protein linked to immune cell migration and adhesion. Reconstituted HDL loaded with m1A-tDR-ArgACG were also found to significantly increase TMEM123 expression in primary macrophages, and blocking m1A using an anti-m1A neutralizing antibody attenuated this effect. Conclusions Results support a model in which HDL-delivered m1A-HDL-tDRs regulate immune signaling, macrophage activation, and pro-inflammatory sub-phenotypes within the atherosclerotic lesion.

BACKGROUND:Chronic inflammation is a major driver of atherosclerotic cardiovascular disease, and therapeutics that target inflammation reduce cardiac events beyond levels seen with strategies targeting cholesterol alone. RNA sequencing revealed increased expression of CaMK4 (calcium/calmodulin-dependent protein kinase IV) in advanced/unstable human carotid artery plaque. We validated this finding in mouse and human atherosclerotic lesions, demonstrating increased CaMK4 in plaque macrophages. Therefore, we hypothesized that CaMK4 would promote inflammation and impair resolution in atherosclerosis. METHODS:We obtained mice in which exon 3 within the kinase domain of CaMK4 is deleted, leading to degradation and deletion of the gene (Camk4-/-). Control and Camk4-/- mice were injected with a gain-of-function AAV (adeno-associated virus) 8-PCSK9 (proprotein convertase subtilisin/kexin type 9) virus, rendering them hypercholesterolemic, and fed a high-fat/high-cholesterol diet for 12 weeks. RESULTS:Hypercholesterolemic Camk4-/- mice developed smaller and more stable lesions compared with control mice. Surprisingly, Camk4-/- mice had a peripheral monocytosis with skewing of monocyte populations toward the nonclassical Ly6clow subset, suggesting a less inflammatory monocyte population. Silencing or inhibition of CaMK4 in human monocytes recapitulated this phenotype. In response to hypercholesterolemia, which promotes myelopoiesis, Camk4-/- mice had markedly more myeloid progenitors. Camk4-/- monocytes expressed higher levels of genes associated with myeloid differentiation and recruitment of ATF6 (activating transcription factor 6) to conserved binding sites. In addition, Camk4-/- monocytes expressed higher levels of Nr4a1, which promotes conversion of Ly6chigh to Ly6clow monocytes. Camk4-/- monocytes failed to efficiently traffic in vitro and in vivo. Bone marrow-derived macrophages generated from Camk4-/- marrow had a more proreparative phenotype than control macrophages, consistent with our in vivo observations in the plaque. CONCLUSIONS:These findings suggest that CaMK4 is an important regulator of the myelopoietic response to hypercholesterolemia through ATF6-mediated transcriptional regulation and that loss of functional CaMK4 promotes a proreparative phenotype in myeloid cells. Therefore, targeting CaMK4 may offer a unique way to target the progression of atherosclerosis.

The clinical neurosciences are in the midst of a renaissance spurred by the development of new therapeutic modalities. Short interfering RNAs (siRNAs), in particular, are gaining interest for treating neurological diseases owing to their capacity to sustain inhibition of nearly any gene target. However, to be effective, siRNA therapies must achieve delivery and on-target gene silencing activity in specific sites and cells in the brain. To this end, we developed a lipid-siRNA conjugate (L2-siRNA) that transports effectively throughout the brain when injected into cerebrospinal fluid (CSF). We provide a detailed examination of regional bulk tissue gene silencing in mice, highlighting potent knockdown 5 months after a single injection without detectable toxicity. Intrathecal delivery of L2-siRNA in rats further illustrates effective transport and knockdown using a clinically relevant route of administration. Single-cell RNA sequencing was additionally performed in mice to generate an atlas of cell type-specific knockdown. Lastly, we benchmarked L2-siRNA gene silencing activity in different brain regions against antisense oligonucleotides, a related but different gene silencing modality. Collectively, this work examines properties of lipid-siRNA conjugates that facilitate CSF to brain delivery and supports L2-siRNA as a promising platform for silencing genes implicated in central nervous system disorders.

Biofluids contain a heterogeneous mixture of extracellular vesicles and non-vesicular nanoparticles (including exomeres and supermeres) that transport a diverse array of proteins, RNA, and lipids. Our previous efforts to characterize the contents of these carriers in colorectal cancer relied on 2D culture systems requiring large-scale setups and time-consuming ultracentrifugation-based isolation. To streamline this process, we have combined 3D hollow-fiber bioreactor production and fast-protein liquid chromatography-based size-exclusion chromatography. Here, we compare the impact of culture methods and purification strategies on small extracellular vesicle, exomere, and supermere cargo. Proteomic analyses show consistently distinct profiles for extracellular vesicles, exomeres, and supermeres regardless of culture conditions or isolation method. In contrast, these two variables influence small RNAs, their base modifications, and lipidomic profiles. We present an online tool to query these and future secretome datasets (https://superomics.shinyapps.io/browse).

Osteoarthritis and rheumatoid arthritis are debilitating joint diseases marked by pain, inflammation and cartilage destruction. Current osteoarthritis treatments only relieve symptoms, while rheumatoid arthritis therapies can cause immune suppression and provide variable efficacy. Here we developed an optimized small interfering RNA targeting matrix metalloproteinase 13 for preferential delivery to arthritic joints. Chemical modifications in a stabilizing 'zipper' pattern improved RNA resistance to degradation, and two independent linkers with 18 ethylene glycol repeats connecting to tandem C18 lipids enhanced albumin binding and targeted delivery to inflamed joints following intravenous administration. In preclinical models of post-traumatic osteoarthritis and rheumatoid arthritis, a single intravenous injection of the albumin-binding small interfering RNA achieved long-term joint retention, sustained gene silencing and reduced matrix metalloproteinase 13 activity over 30 days, resulting in decreased cartilage erosion and improved clinical outcomes, including reduced joint swelling and pressure sensitivity. This approach demonstrated superior efficacy over corticosteroids and small-molecule MMP inhibitors, highlighting the therapeutic promise of albumin 'hitchhiking' for targeted, systemic delivery of gene-silencing therapeutics to treat osteoarthritis and rheumatoid arthritis.

Atherosclerotic cardiovascular disease is the leading global cause of death. Atheromas linked to clinical events are characterized by large necrotic cores, resulting from defective efferocytosis, the clearance of apoptotic cells (ACs). Recently, a phenomenon that facilitates long-lasting innate immune memory called ‘trained immunity’ has been identified. Trained immunity is driven by epigenetic and metabolic rewiring of innate immune cells in response to specific stimuli, such as oxidized low-density lipoproteins (oxLDL). This ‘priming’ results in macrophages (Mφs) that are rewired to mount an augmented inflammatory response. We hypothesize that trained immunity in Mφs promotes inflammation by impairing efferocytosis. Murine bone marrow progenitors treated with oxLDL for 24 hours and differentiated into Mφs (BMDMφs) were able to ingest a first round of ACs better than untrained BMDMφs yet had an impaired ability to take up additional ACs, reflecting a defect in continual efferocytosis. We then transplanted bone marrow from Ldlr-/- mice on chow or Western diets into naïve C57BL/6 recipients. After recovery, elicited peritoneal Mφs from recipients receiving marrow from Western diet fed Ldlr-/- mice not only displayed impaired efferocytosis, but also significantly upregulated PGE2 production. These findings suggest that oxLDL/Western diet training impairs efferocytosis and resolution, with heritable effects. Future work will explore the implications for atheroprogression 5R01HL159487-04 (NIH/NHLBI) 1F31HL172670-01 (NIH/NHLBI) 5T32AI138932-05 (NIH/NIAID) Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

The high potential of siRNAs to silence oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, divalent lipid-conjugated siRNAs are optimized for in situ binding to albumin to improve pharmacokinetics and tumor delivery. Systematic variation of the siRNA conjugate structure reveals that the location of the linker branching site dictates tendency toward albumin association versus self-assembly, while the lipid hydrophobicity and reversibility of albumin binding also contribute to siRNA intracellular delivery. The lead structure increases tumor siRNA accumulation 12-fold in orthotopic triple negative breast cancer (TNBC) tumors over the parent siRNA. This structure achieves approximately 80% silencing of the anti-apoptotic oncogene MCL1 and yields better survival outcomes in three TNBC models than an MCL-1 small molecule inhibitor. These studies provide new structure-function insights on siRNA-lipid conjugate structures that are intravenously injected, associate in situ with serum albumin, and improve pharmacokinetics and tumor treatment efficacy.

Objective High‐density lipoprotein (HDL) has well‐characterized anti‐atherogenic cholesterol efflux and antioxidant functions. Another function of HDL uncharacterized in rheumatoid arthritis (RA) is its ability to transport microRNAs (miRNAs) between cells and thus alter cellular function. The study's purpose was to determine if HDL‐miRNA cargo is altered and affects inflammation in RA. Methods HDL‐microRNAs were characterized in 30 RA and 30 control participants by next generation sequencing and quantitative polymerase chain reaction. The most abundant differentially expressed miRNA was evaluated further. The function of miR‐1246 was assessed by miRNA mimics, antagomiRs, small interfering RNA knockdown, and luciferase assays. Monocyte‐derived macrophages were treated with miR‐1246‐loaded HDL and unmodified HDL from RA and control participants to measure delivery of miR‐1246 and its effect on interleukin‐6 (IL‐6). Results The most abundant miRNA on HDL was miR‐1246; it was significantly enriched two‐fold on HDL from RA versus control participants. HDL‐mediated miR‐1246 delivery to macrophages significantly increased IL6 expression 43‐fold. miR‐1246 delivery significantly decreased DUSP3 1.5‐fold and DUSP3 small interfering RNA knockdown increased macrophage IL6 expression. Luciferase assay indicated DUSP3 is a direct target of miR‐1246. Unmodified HDL from RA delivered 1.6‐fold more miR‐1246 versus control participant HDL. Unmodified HDL from both RA and control participants attenuated activated macrophage IL6 expression, but this effect was significantly blunted in RA so that IL6 expression was 3.4‐fold higher after RA versus control HDL treatment. Conclusion HDL‐miR‐1246 was increased in RA versus control participants and delivery of miR‐1246 to macrophages increased IL‐6 expression by targeting DUSP3 . The altered HDL‐miRNA cargo in RA blunted HDL's anti‐inflammatory effect. image

Short-interfering RNA (siRNA) has gained significant interest for treatment of neurological diseases by providing the capacity to achieve sustained inhibition of nearly any gene target. Yet, efficacious drug delivery throughout deep brain structures of the CNS remains a considerable hurdle for intrathecally administered therapeutics. We herein describe an albumin-binding lipid-siRNA conjugate that transports along meningeal and perivascular CSF pathways, leading to broad dispersion throughout the CNS parenchyma. We provide a detailed examination of the temporal kinetics of gene silencing, highlighting potent knockdown for up to five months from a single injection without detectable toxicity. Single-cell RNA sequencing further demonstrates gene silencing activity across diverse cell populations in the parenchyma and at brain borders, which may provide new avenues for neurological disease-modifying therapies.

Aim: Extracellular communication via the transfer of vesicles and nanoparticles is now recognized to play an important role in tumor microenvironment interactions. Cancer cells upregulate and secrete abundant levels of miR-100 and miR-125b that can alter gene expression in donor and recipient cells. In this study, we sought to identify targets of miR-100 and miR-125b and conclusively demonstrate that microRNAs (miRNAs) can be functionally transferred from donor to recipient cells. Methods: To identify targets of miR-100 and miR-125b , we used bioinformatic approaches comparing multiple colorectal cancer (CRC) cell lines, including knockout lines lacking one or both of these miRNAs. We also used spheroid and 3D growth conditions in collagen to test colony growth and invasiveness. We also used Transwell co-culture systems to demonstrate functional miRNA transfer. Results: From an initial list of 96 potential mRNA targets, we identified and tested 15 targets, with 8 showing significant downregulation in the presence of miR-100 and miR-125b . Among these, cingulin (CGN) and protein tyrosine phosphatase receptor type-R (PTPRR) are downregulated in multiple cancers, consistent with regulation by increased levels of miR-100 and miR-125b. We also show that increased cellular levels of miR-100 and miR-125b enhance 3D growth and invasiveness in CRC and glioblastoma cell lines. Lastly, we demonstrate that extracellular transfer of miR-100 and miR-125b can silence both reporter and endogenous mRNA targets in recipient cells and also increase the invasiveness of recipient spheroid colonies when grown under 3D conditions in type I collagen. Conclusion: miR-100 and miR-125b target multiple mRNAs that can regulate 3D cell-autonomous growth and invasiveness. By extracellular transfer, miR-100 and miR-125b can also increase colony growth and invasiveness in recipient cells through non-cell-autonomous mechanisms.

Chronic inflammation is a major driver of atherosclerotic cardiovascular disease, and therapeutics that target inflammation reduce clinical cardiac events beyond levels seen with conventional strategies targeting cholesterol alone. Recent findings suggest innate immune cells maintain ‘memory’ of prior exposure to inflammatory stimuli, a phenomenon known as ‘trained immunity’. In response to inflammatory stimuli, macrophages undergo metabolic and epigenetic rewiring that primes them to mount an augmented response upon a second exposure. Oxidized low-density lipoproteins (oxLDL) have recently been shown to be potent triggers of trained immunity. While trained immunity has been shown to promote inflammation, little is known about how immune training impacts efferocytosis. Therefore, we hypothesize that trained immunity in macrophages promotes inflammation by impairing efferocytosis. We treated murine bone marrow progenitors with oxLDL for 24 hours, then washed and differentiated them into macrophages (BMDMs). Upon assessing efferocytosis, trained BMDMs were able to ingest a first apoptotic cell (AC) better than untrained BMDMs yet had an impaired ability to take up additional ACs, reflecting a defect in continual efferocytosis. Using an in vivo approach, we transplanted donor bone marrow from Ldlr -/- mice fed a chow or Western diet into naïve C57BL/6 recipients. After recovery, we elicited peritoneal macrophages to assess efferocytosis and found that recipients receiving marrow from Western diet fed Ldlr -/- mice not only displayed impaired efferocytosis, but also significantly upregulated PGE 2 production, suggesting impaired resolution. To determine whether PGE 2 is a mediator of trained immunity, we primed bone marrow progenitors with PGE 2 and differentiated them into BMDMs. We found that macrophages primed with PGE 2 elaborated higher levels of inflammatory cytokines in response to LPS stimulation than controls. Overall, these findings demonstrate that oxLDL/Western diet-training impinges on the resolution program by impairing efferocytosis and are durable effects that demonstrate heritability. Future directions include determining the impact of these findings on the development of atherosclerosis.

Rationale: Multiciliated cell (MCC) loss/dysfunction is common in the small airways of patients with COPD but it is unclear if this contributes to COPD lung pathology. Objectives: To determine if loss of p73 causes a COPD-like phenotype in mice and explore whether smoking or COPD impact p73 expression. Methods: p73floxE7-E9 mice were crossed with Shh-Cre mice to generate mice lacking MCCs in the airway epithelium. The resulting p73Δairway mice were analyzed using electron microscopy, flow cytometry, morphometry, forced oscillation technique, and single-cell RNA sequencing. Further, the effects of cigarette smoke on p73 transcript and protein expression were examined using in vitro and in vivo models and in studies including airway epithelium from smokers and COPD patients. Measurements and Main Results: Loss of functional p73 in the respiratory epithelium resulted in a near-complete absence of MCCs in p73Δairway mice. In adulthood, these mice spontaneously developed neutrophilic inflammation and emphysema-like lung remodeling and had progressive loss of secretory cells. Exposure of normal airway epithelium cells to cigarette smoke rapidly and durably suppressed p73 expression in vitro and in vivo. Further, TP73 mRNA expression was reduced in the airways of current smokers (n=82) compared to former smokers (n=69) and p73-expressing MCCs were reduced in the small airways of COPD patients (n=11) compared to non-COPD controls (n=12). Conclusions: Loss of functional p73 in murine airway epithelium results in the absence of MCCs and promotes COPD-like lung pathology. In smokers and patients with COPD, loss of p73 may contribute to MCC loss or dysfunction.

Abstract Introduction: Extracellular vesicles have been found to be important regulators of intercellular communication between cancer cells. EV cargo varies greatly between different cell types, the composition of which provides important insights into the role of donor and recipient cells. Cetuximab Resistant Colorectal Cancer (CCCR) cells upregulate miR-100 and miR-125b and select them as cargo for their respective secreted EVs. These miRNAs are responsible for altered gene expression in the CCCR cells and can also be functionally transferred as part of EVs between donor and recipient cells. Bioinformatic analysis identified Cingulin (CGN) mRNA as one of the possible targets of miR-100 and miR-125b. The action of miRNAs leads to degradation of CGN mRNA ultimately decreasing cellular concentration of the functional protein. Absence of CGN has been associated with increased invasiveness and metastatic potential in the cancer cells. We carried out immunofluorescent imaging of CCCR cells which revealed downregulation of CGN in CCCR cells compared to the parental Cetuximab sensitive Colorectal Cancer (CC) cells. Similar effects were observed in recipient cells that obtained the miRNAs as part of EVs originating from CCCR donor cells. This project helps elucidate the roles of miR-100 and miR-125b in CCCR cells and how EVs facilitate their functional transfer between nearby cancer cells. Methods: CCCR cell lines were derived from parental CC cells in the lab of Dr. Robert Coffey by iterative selection of cetuximab resistance after growth in 3D. CRISPR/Cas9 technology was used to knockout miR-100 and miR-125b in CCCR cells. Transwell co-culture experiments were carried out to analyze EV transfer of miRNAs. Transfer was monitored by immunofluorescence using antibodies against CGN. Cells were stained and imaged as Z stacks under the 63x Immersion setting. ImageJ was used to analyze the stacks and CGN concentrations between the cells. For quantification of immunofluorescence, additional Z stack images were obtained from an inverted microscope and fluorescence digital camera. Images were analyzed using Fiji software to obtain specific CGN concentrations between the cells. Results and Discussion: Immunofluorescent imaging revealed statistically significant downregulation of CGN in CCCR cells. The protein was also downregulated in the recipient knockout cells as part of the Transwell co-culture experiment with CCCR donor cells. CGN is a tight junction protein and its absence confers increased invasiveness in 3D growth with enhanced metastasis. Decreased CGN concentration in the miR-100 and miR-125b enriched EV recipient knockout cells is indicative of functional transfer of the two miRNAs between cancer cells. These distinctive cellular dynamics could be harnessed to develop potential cancer therapies that provide a better prognosis and limit the metastatic potential of targeted cancer cells. Citation Format: Muhammad Shameer, Hannah M Nelson, Shimian Qu, Liyu Huang, Kevin C Corn, Sydney N Chapman, Nicole L Luthcke, Sara A Schuster, Tellie D Stamaris, Lauren A Turnbull, Lucas L Guy, Xiao Liu, Danielle L Michell, Elizabeth M Semler, Kasey C Vickers, Qi Liu, Jeffrey L Franklin, Alissa M Weaver, Marjan Rafat, Robert J Coffey, James G Patton. Characterizing invasiveness in CCCR cells: Role of miR-100, miR-125b and EVs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B022.

5-Fluorouracil (5-FU) has been used for chemotherapy for colorectal and other cancers for over 50 years. The prevailing view of its mechanism of action is inhibition of thymidine synthase leading to defects in DNA replication and repair. However, 5-FU is also incorporated into RNA causing defects in RNA metabolism, inhibition of pseudouridine modification, and altered ribosome function. We examined the impact of 5-FU on post-transcriptional small RNA modifications (PTxMs) and the expression and export of RNA into small extracellular vesicles (sEVs). EVs are secreted by all cells and contain a variety of proteins and RNAs that can function in cell-cell communication. We found that treatment of colorectal cancer (CRC) cells with 5-FU represses sEV export of miRNA and snRNA-derived RNAs, but promotes export of snoRNA-derived RNAs. Strikingly, 5-FU treatment significantly decreased the levels of pseudouridine on both cellular and sEV small RNA profiles. In contrast, 5-FU exposure led to increased levels of cellular small RNAs containing a variety of methyl-modified bases. These unexpected findings show that 5-FU exposure leads to altered RNA expression, base modification, and aberrant trafficking and localization of small RNAs.

The high potential for therapeutic application of siRNAs to silence traditionally undruggable oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, siRNAs were optimized for in situ binding to albumin through C18 lipid modifications to improve pharmacokinetics and tumor delivery. Systematic variation of siRNA conjugates revealed a lead structure with divalent C18 lipids each linked through three repeats of hexaethylene glycol connected by phosphorothioate bonds. Importantly, we discovered that locating the branch site of the divalent lipid structure proximally (adjacent to the RNA) rather than at a more distal site (after the linker segment) promotes association with albumin, while minimizing self-assembly and lipoprotein association. Comparison to higher albumin affinity (diacid) lipid variants and siRNA directly conjugated to albumin underscored the importance of conjugate hydrophobicity and reversibility of albumin binding for siRNA delivery and bioactivity in tumors. The lead conjugate increased tumor siRNA accumulation 12-fold in orthotopic mouse models of triple negative breast cancer over the parent siRNA. When applied for silencing of the anti-apoptotic oncogene MCL-1, this structure achieved approximately 80% MCL1 silencing in orthotopic breast tumors. Furthermore, application of the lead conjugate structure to target MCL1 yielded better survival outcomes in three independent, orthotopic, triple negative breast cancer models than an MCL1 small molecule inhibitor. These studies provide new structure-function insights on optimally leveraging siRNA-lipid conjugate structures that associate in situ with plasma albumin for molecular-targeted cancer therapy.

HDL are dynamic transporters of diverse molecular cargo and play critical roles in lipid metabolism and inflammation. We have previously reported that HDL transport both host and nonhost small RNAs (sRNA) based on quantitative PCR and sRNA sequencing approaches; however, these methods require RNA isolation steps which have potential biases and may not isolate certain forms of RNA molecules from samples. HDL have also been reported to accept functional sRNAs from donor macrophages and deliver them to recipient endothelial cells; however, using PCR to trace HDL-sRNA intercellular communication has major limitations. The present study aims to overcome these technical barriers and further understand the pathways involved in HDL-mediated bidirectional flux of sRNAs between immune cells. To overcome these technical limitations, SYTO RNASelect, a lipid-penetrating RNA dye, was used to quantify a) overall HDL-sRNA content, b) bidirectional flux of sRNAs between HDL and immune cells, c) HDL-mediated intercellular communication between immune cells, and d) HDL-mediated RNA export changes in disease. Live cell imaging and loss-of-function assays indicate that the endo-lysosomal system plays a critical role in macrophage storage and export of HDL-sRNAs. These results identify HDL as a substantive mediator of intercellular communication between immune cells and demonstrate the importance of endocytosis for recipient cells of HDL-sRNAs. Utilizing a lipid-penetrating RNA-specific fluorescence dye, we were able to both quantify the absolute concentration of sRNAs transported by HDL and characterize HDL-mediated intercellular RNA transport between immune cells.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are joint diseases that are associated with pain and lost quality of life. No disease modifying OA drugs are currently available. RA treatments are better established but are not always effective and can cause immune suppression. Here, an MMP13-selective siRNA conjugate was developed that, when delivered intravenously, docks onto endogenous albumin and promotes preferential accumulation in articular cartilage and synovia of OA and RA joints. MMP13 expression was diminished upon intravenous delivery of MMP13 siRNA conjugates, consequently decreasing multiple histological and molecular markers of disease severity, while also reducing clinical manifestations such as swelling (RA) and joint pressure sensitivity (RA and OA). Importantly, MMP13 silencing provided more comprehensive OA treatment efficacy than standard of care (steroids) or experimental MMP inhibitors. These data demonstrate the utility of albumin ‘hitchhiking’ for drug delivery to arthritic joints, and establish the therapeutic utility of systemically delivered anti-MMP13 siRNA conjugates in OA and RA. Editorial summary Lipophilic siRNA conjugates optimized for albumin binding and “hitchhiking” can be leveraged to achieve preferential delivery to and gene silencing activity within arthritic joints. Chemical stabilization of the lipophilic siRNA enables intravenous siRNA delivery without lipid or polymer encapsulation. Using siRNA sequences targeting MMP13, a key driver of arthritis-related inflammation, albumin hitchhiking siRNA diminished MMP13, inflammation, and manifestations of osteoarthritis and rheumatoid arthritis at molecular, histological, and clinical levels, consistently outperforming clinical standards of care and small molecule MMP antagonists.