RNA Loading Research Articles

Molecular Mediators of RNA Loading into Extracellular Vesicles.

In the last decade, an increasing number of studies have demonstrated that non-coding RNA (ncRNAs) cooperate in the gene regulatory networks with other biomolecules, including coding RNAs, DNAs and proteins. Among them, microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) are involved in transcriptional and translation regulation at different levels. Intriguingly, ncRNAs can be packed in vesicles, released in the extracellular space, and finally internalized by receiving cells, thus affecting gene expression also at distance. This review focuses on the mechanisms through which the ncRNAs can be selectively packaged into extracellular vesicles (EVs).

Thinking Quantitatively of RNA-Based Information Transfer via Extracellular Vesicles: Lessons to Learn for the Design of RNA-Loaded EVs.

Extracellular vesicles (EVs) are 50–1000 nm vesicles secreted by virtually any cell type in the body. They are expected to transfer information from one cell or tissue to another in a short- or long-distance way. RNA-based transfer of information via EVs at long distances is an interesting well-worn hypothesis which is ~15 years old. We review from a quantitative point of view the different facets of this hypothesis, ranging from natural RNA loading in EVs, EV pharmacokinetic modeling, EV targeting, endosomal escape and RNA delivery efficiency. Despite the unique intracellular delivery properties endowed by EVs, we show that the transfer of RNA naturally present in EVs might be limited in a physiological context and discuss the lessons we can learn from this example to design efficient RNA-loaded engineered EVs for biotherapies. We also discuss other potential EV mediated information transfer mechanisms, among which are ligand–receptor mechanisms.

A Microfluidics-Based Scalable Approach to Generate Extracellular Vesicles with Enhanced Therapeutic MicroRNA Loading for Intranasal Delivery to Mouse Glioblastomas.

Extracellular vesicles (EVs), including exosomes and microvesicles derived from different cell sources, are used as promising nanovesicles for delivering therapeutic microRNAs (miRNAs) and drugs in cancer therapy. However, their clinical translation is limited by the quantity, size heterogeneity, and drug or small RNA loading efficiency. Herein, we developed a scalable microfluidic platform that can load therapeutic miRNAs (antimiRNA-21 and miRNA-100) and drugs while controlling the size of microfluidically processed EVs (mpEVs) using a pressure-based disruption and reconstitution process. We prepared mpEVs of optimal size using microvesicles isolated from neural stem cells engineered to overexpress CXCR4 receptor and characterized them for charge and miRNA loading efficiency. Since the delivery of therapeutic miRNAs to brain cancer is limited by the blood-brain barrier (BBB), we adopted intranasal administration of miRNA-loaded CXCR4-engineered mpEVs in orthotopic GBM mouse models and observed a consistent pattern of mpEVs trafficking across the nasal epithelia, bypassing the BBB into the intracranial compartment. In addition, the CXCR4-engineered mpEVs manifested selective tropism toward GBMs by stromal-derived factor-1 chemotaxis to deliver their miRNA cargo. The delivered miRNAs sensitized GBM cells to temozolomide, resulting in prominent tumor regression, and improved the overall survival of mice. A simple and efficient approach of packaging miRNAs in mpEVs using microfluidics, combined with a noninvasive nose-to-brain delivery route presents far-reaching potential opportunities to improve GBM therapy in clinical practice.

Performance of a scalable RNA extraction-free transcriptome profiling method for adherent cultured human cells

RNA sequencing enables high-content/high-complexity measurements in small molecule screens. Whereas the costs of DNA sequencing and RNA-seq library preparation have decreased consistently, RNA extraction remains a significant bottleneck to scalability. We evaluate the performance of a bulk RNA-seq library prep protocol optimized for analysis of many samples of adherent cultured cells in parallel. We combined a low-cost direct lysis buffer compatible with cDNA synthesis (in-lysate cDNA synthesis) with Smart-3SEQ and examine the effects of calmidazolium and fludrocortisone-induced perturbation of primary human dermal fibroblasts. We compared this method to normalized purified RNA inputs from matching samples followed by Smart-3SEQ or Illumina TruSeq library prep. Our results show the minimal effect of RNA loading normalization on data quality, measurement of gene expression patterns, and generation of differentially expressed gene lists. We found that in-lysate cDNA synthesis combined with Smart-3SEQ RNA-seq library prep generated high-quality data with similar ranked DEG lists when compared to library prep with extracted RNA or with Illumina TruSeq. Our data show that small molecule screens or experiments based on many perturbations quantified with RNA-seq are feasible at low reagent and time costs.

Role of RNA Motifs in RNA Interaction with Membrane Lipid Rafts: Implications for Therapeutic Applications of Exosomal RNAs.

RNA motifs may promote interactions with exosomes (EXO-motifs) and lipid rafts (RAFT-motifs) that are enriched in exosomal membranes. These interactions can promote selective RNA loading into exosomes. We quantified the affinity between RNA aptamers containing various EXO- and RAFT-motifs and membrane lipid rafts in a liposome model of exosomes by determining the dissociation constants. Analysis of the secondary structure of RNA molecules provided data about the possible location of EXO- and RAFT-motifs within the RNA structure. The affinity of RNAs containing RAFT-motifs (UUGU, UCCC, CUCC, CCCU) and some EXO-motifs (CCCU, UCCU) to rafted liposomes is higher in comparison to aptamers without these motifs, suggesting direct RNA-exosome interaction. We have confirmed these results through the determination of the dissociation constant values of exosome-RNA aptamer complexes. RNAs containing EXO-motifs GGAG or UGAG have substantially lower affinity to lipid rafts, suggesting indirect RNA-exosome interaction via RNA binding proteins. Bioinformatics analysis revealed RNA aptamers containing both raft- and miRNA-binding motifs and involvement of raft-binding motifs UCCCU and CUCCC. A strategy is proposed for using functional RNA aptamers (fRNAa) containing both RAFT-motif and a therapeutic motif (e.g., miRNA inhibitor) to selectively introduce RNAs into exosomes for fRNAa delivery to target cells for personalized therapy.

Structural Investigation of Self-Assembly and Target Binding of Anti-CRISPR AcrIIC2.

Anti-CRISPR (Acr) proteins are phage-borne inhibitors of the CRISPR-Cas immune system in archaea and bacteria. AcrIIC2 from prophages of Neisseria meningitidis disables the nuclease activity of type II-C Cas9, such that dimeric AcrIIC2 associates with the bridge helix (BH) region of Cas9 to compete with guide RNA loading. AcrIIC2 in solution readily assembles into oligomers of variable lengths, but the oligomeric states are not clearly understood. In this study, we investigated the dynamic assembly of AcrIIC2 oligomers, and identified key interactions underlying the self-association. We report that AcrIIC2 dimers associate into heterogeneous high-order oligomers with the equilibrium dissociation constant K D ∼8 μM. Oligomerization is driven by electrostatic interactions between charged residues, and rational mutagenesis produces a stable AcrIIC2 dimer with intact Cas9 binding. Remarkably, the BH peptide of Cas9 is unstructured in solution, and undergoes a coil-to-helix transition upon AcrIIC2 binding, revealing a unique folding-upon-binding mechanism for Acr recognition.

RNA-binding proteins contribute to small RNA loading in plant extracellular vesicles.

Plants utilize extracellular vesicles (EVs) to transport small RNAs (sRNAs) into their fungal pathogens and silence fungal virulence-related genes through a phenomenon called “cross-kingdom RNAi.” It remains unknown, however, how sRNAs are selectively loaded into EVs. Here, we identified several RNA-binding proteins (RBPs) in Arabidopsis, including Argonaute 1 (AGO1), RNA helicases (RHs) and Annexins (ANN), which are secreted by exosome-like EVs. AGO1, RH11 and RH37 selectively bind to EV-enriched sRNAs but not non-EV enriched sRNAs, suggesting that they contribute to the selective loading of sRNAs into EVs. Conversely, ANN1 and ANN2 bind to sRNAs non-specifically. The ago1, rh11rh37 and ann1ann2 mutants showed reduced secretion of sRNAs in EVs, demonstrating that these RBPs play an important role in sRNA loading and/or stabilization in EVs. Furthermore, rh11rh37 and ann1ann2 showed increased susceptibility to Botrytis cinerea, supporting that RH11, RH37, ANN1 and ANN2 positively regulate plant immunity against B. cinerea.

Tumor RNA-loaded nanoliposomes increases the anti-tumor immune response in colorectal cancer

Purpose Tumor RNA vaccines can activate dendritic cells to generate systemic anti-tumor immune response. However, due to easily degraded of RNA, direct RNA vaccine is less effective. In this study, we optimized the method for preparing PEGylated liposom-polycationic DNA complex (LPD) nanoliposomes, increased encapsulate amount of total RNA derived from CT-26 colorectal cancer cells. Tumor RNA LPD nanoliposomes vaccines improved anti-tumor immune response ability of tumor RNA and can effectively promote anti-tumor therapeutic effect of oxaliplatin. Methods Total tumor-derived RNA was extracted from colorectal cancer cells (CT-26 cells), and loaded to our optimized the LPD complex, resulting in the LPD nanoliposomes. We evaluated the characteristics (size, zeta potential, and stability), cytotoxicity, transfection ability, and tumor-growth inhibitory efficacy of LPD nanoliposomes. Results The improved LPD nanoliposomes exhibited a spherical shape, RNA loading efficiency of 9.07%, the average size of 120.37 ± 2.949 nm and zeta potential was 3.34 ± 0.056 mV. Also, the improved LPD nanoliposomes showed high stability at 4 °C, with a low toxicity and high cell transfection efficacy toward CT-26 colorectal cancer cells. Notably, the improved LPD nanoliposomes showed tumor growth inhibition by activating anti-tumor immune response in CT-26 colorectal cancer bearing mice, with mini side effects toward the normal organs of mice. Furthermore, the effect of the improved LPD nanoliposomes in combination with oxaliplatin can be better than that of oxaliplatin alone. Conclusion The improved LPD nanoliposomes may serve as an effective vaccine to induce antitumor immunity, presenting a new treatment option for colorectal cancer.

Binding of RNA Aptamers to Membrane Lipid Rafts: Implications for Exosomal miRNAs Transfer from Cancer to Immune Cells.

Intraluminal vesicles (ILVs) are released into the extracellular space as exosomes after the fusion of multivesicular bodies (MVBs) with the plasma membrane. miRNAs are delivered to the raft-like region of MVB by RNA-binding proteins (RBPs). RNA loading into exosomes can be either through direct interaction between RNA and the raft-like region of the MVB membrane, or through interaction between an RBP–RNA complex with this raft-like region. Selection of RNA aptamers that bind to lipid raft region of liposomal membranes was performed using the selection-amplification (SELEX) method. The pool of RNA aptamers was isolated, and the binding of this pool to lipid-raft regions was demonstrated. Sequencing of clones from rafted liposome-eluted RNAs showed sequences apparently of independent origin. Bioinformatics analysis revealed the most frequent raft-motifs present within these sequences. Four raft RNA motifs, one of them an EXO motif, have been identified. These motifs appear to be most frequent both in the case of raft RNA aptamers and in the case of exosomal pro-tumoral miRNAs transferred from cancer cells to macrophages, natural killer cells and dendritic cells, thus suggesting that the selection for incorporation of these miRNAs into ILVs is based on their affinity to the raft-like region of the MVB membrane.

In situ self imine-crosslinked nanocomplexes loaded with small noncoding RNA for efficient osteoarthritis attenuation

Small noncoding RNA molecules which can help to recover cartilage homeostasis exhibit great potential in OA alleviation. However, effective small RNA transfection to target cells dwelled in cartilage tissue is quite a challenging work because of the inhibition of dense and anion cartilage matrix as well as the limited RNA transmembrane ability. In this study, we developed an in-situ imine crosslinking nanocomplexes (PAR) formed by polyethyleneimine (PEI), aldehyde group modified hyaluronic acid (AHA), and small noncoding RNA (miR-140). Our in vitro study showed that covalently crosslinked and positively charged PAR nanocomplexes could effectively load and transfect miR-140 to chondrocytes and further exert chondrocyte protection under IL-1β induction even in the strong and persistent interference of HA and chondroitin sulfate (CS), which are widely distributing anion biomacromolecules in cartilage matrix or articular cavity. And in vivo evaluation showed that PAR could alleviate OA progression in mice effectively. However, non-crosslinked positively charged nanocomplexes failed in miR-140 transfection both in HA and CS interference condition and finally failed in in vivo OA attenuation in mice model. This study not only provides an effective, simple, and low-cost delivery vehicle for small noncoding RNA based therapy in OA but also demonstrates that covalent crosslinking in positively charged vehicles is quite necessary for the design of RNA transfection materials to cartilage because of the strong and persistent impact of anion biomacromolecules (HA and CS) that distribute widely in cartilage matrix on the stability of RNA loading carriers.

Structural Optimization of Polymeric Carriers to Enhance the Immunostimulatory Activity of Molecularly Defined RIG-I Agonists.

RNA ligands of retinoic acid-inducible gene I (RIG-I) hold significant promise as antiviral agents, vaccine adjuvants, and cancer immunotherapeutics, but their efficacy is hindered by inefficient intracellular delivery to the cytosol where RIG-I is localized. Here, we address this challenge through the synthesis and evaluation of a library of polymeric carriers rationally designed to promote the endosomal escape of 5′-triphosphate RNA (3pRNA) RIG-I agonists. We synthesized a series of PEG-block-(DMAEMA-co-AnMA) polymers, where AnMA is an alkyl methacrylate monomer ranging from n = 2–12 carbons, of variable composition, and examined effects of polymer structure on the intracellular delivery of 3pRNA. Through in vitro screening of 30 polymers, we identified four lead carriers (4–50, 6–40, 8–40, and 10–40, where the first number refers to the alkyl chain length and the second number refers to the percentage of hydrophobic monomer) that packaged 3pRNA into ∼100-nm-diameter particles and significantly enhanced its immunostimulatory activity in multiple cell types. In doing so, these studies also revealed an interplay between alkyl chain length and monomer composition in balancing RNA loading, pH-responsive properties, and endosomal escape, studies that establish new structure–activity relationships for polymeric delivery of 3pRNA and other nucleic acid therapeutics. Importantly, lead carriers enabled intravenous administration of 3pRNA in mice, resulting in increased RIG-I activation as measured by increased levels of IFN-α in serum and elevated expression of Ifnb1 and Cxcl10 in major clearance organs, effects that were dependent on polymer composition. Collectively, these studies have yielded novel polymeric carriers designed and optimized specifically to enhance the delivery and activity of 3pRNA with potential to advance the clinical development of RIG-I agonists.

Helminth genome analysis reveals conservation of extracellular vesicle biogenesis pathways but divergence of RNA loading machinery between phyla

A comparative genomics approach was used to determine whether established extracellular vesicle biogenesis pathways are conserved in helminths. This revealed conservation of membrane and cytoskeletal organising proteins as well as the endosomal sorting complex required for transport previously described in mammalian cells. Domain level analysis of this complex in helminths, however, indicated that some species may rely on atypical proteins to support subunit interactions and cargo recruitment. Interestingly, helminths displayed phylum level divergence of proteins associated with loading RNA into extracellular vesicles. These findings provide a framework for functional studies of helminth extracellular vesicle biogenesis and cargo sorting.

Gene Expression Profiling Using Nanostring™ Reveals Differences According to Location, Histology and MIPI Score in Mantle Cell Lymphoma

Background Mantle cell lymphoma (MCL) is an aggressive B cell neoplasm characterized the by t(11;14)(q13;q32)/CCND1-IGH resulting in overexpression of cyclin D1. Although indolent variants are recognized, MCL is generally aggressive and incurable. The MCL International Prognostic Index (MIPI), gene expression-based proliferation signatures, Ki-67 proliferation index, p53 expression, and aggressive histologic features are used for risk stratification, but the prognostic groups defined by each of these markers are clinically heterogeneous, demonstrating that each parameter alone does not fully account for the clinical behavior of these tumors. In this study, we investigated the gene expression profile of 60 cases of MCL and evaluated which genes had expression patterns that correlate with different clinical and histopathological parameters. Design A total of 60 excisional biopsy specimens of MCL were selected for gene expression profiling. Forty (66%) and 16 (26%) cases were untreated and relapse/persistent samples, respectively; in 4 this unknown was unknown. Biopsy sites included lymph nodes (n=38), spleen (n=10), tonsil (n=6) and various other extranodal tissue sites (n=6). The morphologic features were classic in 27 and aggressive (blastoid/pleomorphic) in 33 patients. MIPI was calculated in selected patients with nodal involvement and whose biopsy was an obtained prior to treatment. Each block contained tumor cells representing ≥ 80% of all cells in the biopsy specimen. RNA was extracted from formalin-fixed paraffin-embedded tissue using the Qiagen Allprep FFPE Kit after deparaffinization, according to the manufacturer's instructions. Gene expression was quantified in 200ng of RNA on the Nanostring™ platform. Normalization for RNA loading was performed using the geometric mean of 40 housekeeping genes with a cutoff value 20. Standard QC and data processing were performed using the nSolver™ Analysis Software. Adjusted p-values were used according to the Benjamini-Hochberg procedure. Results Gene expression profiling was different according to the site of involvement of MCL. Compared with nodal involvement, MCL involving spleen showed overexpression of VEGFA (p <0.01) and NOS (p <0.05). These genes are associated with angiogenesis via the PI3K signaling pathway. Similarly, tonsillar MCL showed overexpression of SFN and HSPB1 (p <0.01) compared with nodal MCL; these genes are related to the MAPK pathway. Different gene expression profiles were observed with respect to histology. Compared to classic variant, blastoid variant demonstrated overexpression of CDK4 (p <0.01) and underexpression of KAT2B and PIK3CG (p <0.01) in all cases. However, no significant differences were found in the gene expression profiles between the classic and pleomorphic variants. We also compared gene expression profiles based on the MIPI score. Compared to cases with a low MIPI score, those with a high MIPI score showed significant overexpression of several genes including SFN, COL11A2, HDAC6 and TP53 (p <0.01). Discussion MCL is a heterogeneous neoplasm, both clinically and genetically. In the present study, significant differences in gene expression profiles were observed based on site of involvement (spleen vs lymph node vs tonsil). However, we cannot exclude the possibility that the result reflects gene expression of non-lymphoma cells in the microenvironment since we did not extract RNA from lymphoma cells. We observed different gene expression profiles in the blastoid variant, but not in the pleomorphic variant compared to the classic variant, suggesting that the biology of pleomorphic variant is probably between the blastoid and the classic variants, possibly closer to the latter. Different gene expression profiles were found with respect to MIPI score, providing scientific support that the MIPI score, in addition to being a useful stratification factor in MCL patients, is reflecting differences in underlying biology of MCL. Disclosures No relevant conflicts of interest to declare.

Multidomain Convergence of Argonaute during RISC Assembly Correlates with the Formation of Internal Water Clusters

Despite the relevance of Argonaute proteins in RNA silencing, little is known about the structural steps of small RNA loading to form RNA-induced silencing complexes (RISCs). We report the 1.9Å crystal structure of human Argonaute4 with guide RNA. Comparison with the previously determined apo structure of Neurospora crassa QDE2 revealed that the PIWI domain has two subdomains. Binding of guide RNA fastens the subdomains, thereby rearranging the active-site residues and increasing the affinity for TNRC6 proteins. We also identified two water pockets beneath the nucleic acid-binding channel that appeared to stabilize the mature RISC. Indeed, mutating the water-pocket residues of Argonaute2 and Argonaute4 compromised RISC assembly. Simulations predict that internal water molecules are exchangeable with the bulk solvent but always occupy specific positions at the domain interfaces. These results suggest that after guide RNA-driven conformational changes, water-mediated hydrogen-bonding networks tie together the converged domains to complete the functional RISC structure.

Maximizing RNA Loading for Gene Silencing Using Porous Silicon Nanoparticles.

Gene silencing by RNA interference is a powerful technology with broad applications. However, this technology has been hampered by the instability of small interfering RNA (siRNA) molecules in physiological conditions and their inefficient delivery into the cytoplasm of target cells. Porous silicon nanoparticles have emerged as a potential delivery vehicle to overcome these limitations-being able to encapsulate RNA molecules within the porous matrix and protect them from degradation. Here, key variables were investigated that influence siRNA loading into porous silicon nanoparticles. The effect of modifying the surface of porous silicon nanoparticles with various amino-functional molecules as well as the effects of salt and chaotropic agents in facilitating siRNA loading was examined. Maximum siRNA loading of 413 μg/(mg of porous silicon nanoparticles) was found when the nanoparticles were modified by a fourth generation polyamidoamine dendrimer. Low concentrations of urea or salt increased loading capacity: an increase in RNA loading by 19% at a concentration of 0.05 M NaCl or 21% at a concentration of 0.25 M urea was observed when compared to loading in water. Lastly, it was demonstrated that dendrimer-functionalized nanocarriers are able to deliver siRNA against ELOVL5, a target for the treatment of advanced prostate cancer.

New copolymers as hosts of ribosomal RNA

Functionalized copolymers were synthesized and are proposed as hosts of RNA. The copolymers are based on carboxymethyl cellulose and poly-(ethylene glycol)-OH. These copolymers were functionalized with two amino acids, either lysine or histidine, through amide bond formation. The functionalized copolymer was then used to adsorb ribosomal RNA. The RNA loading was based on the nature of the amino acid functionalization of the copolymer. The array of RNA-copolymers was observed to be soft sphere-like, where the density of spheres was a function of the molecular weight of the carboxymethyl cellulose and the nature of the amino acid. Such RNA-copolymer systems are very sensitive to changes in pH.

Tuberculosis's cargoman: bacteria load RNA into host extracellular vesicles.

Tuberculosis remains one of the deadliest infectious diseases worldwide. Mycobacterium tuberculosis ( M.tb) has developed various mechanisms to manipulate the human host, in particular by disrupting the host phagosome and the immune response. It is becoming evident that secreted extracellular vesicles (EVs) are involved in the dynamic crosstalk between M.tb and the host cells. These vesicles shuttle different cargo components, such as RNA, lipids, and proteins, between cells. In this issue of EMBO Reports , Cheng and Schorey [1] describe a previously unknown EV‐mediated process, regulating M.tb RNA loading into EVs and their internalization by naive macrophages. They identify the mycobacterial Sec2 secretion system as involved in RNA loading into EVs and show that secreted vesicles contain bacterial RNA that not only promotes IFN‐β production upon entry into target cells, but also leads to M.tb clearance via the activation of the host9s RIG‐I/MAVS signaling pathway. Importantly, combined treatment with secreted EVs and antibiotics decreases bacterial load in a mouse model, improving lung pathology compared to treatment with antibiotics alone.

Bcd1p controls RNA loading of the core protein Nop58 during C/D box snoRNP biogenesis.

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins (C/D snoRNPs) is guided by conserved trans-acting factors that act collectively to assemble the core proteins SNU13/Snu13, NOP58/Nop58, NOP56/Nop56, FBL/Nop1, and box C/D small nucleolar RNAs (C/D snoRNAs), in human and in yeast, respectively. This finely elaborated process involves the sequential interplay of snoRNP-related proteins and RNA through the formation of transient pre-RNP complexes. BCD1/Bcd1 protein is essential for yeast cell growth and for the specific accumulation of box C/D snoRNAs. In this work, chromatin, RNA, and protein immunoprecipitation assays revealed the ordered loading of several snoRNP-related proteins on immature and mature snoRNA species. Our results identify Bcd1p as an assembly factor of C/D snoRNP biogenesis that is likely recruited cotranscriptionally and that directs the loading of the core protein Nop58 on RNA.

Light‐Inducible Exosome‐Based Vehicle for Endogenous RNA Loading and Delivery to Leukemia Cells

AbstractExosomes are a novel and promising drug delivery platform because of their endogenous origin, stability, biocompatibility, and other unique features. As the efficient loading and delivery of long RNA to target cells for therapeutic purposes remains challenging, a new exosome‐based RNA delivery system is proposed using a controllable RNA enrichment and releasing protocol. The system employs RNA aptamer–protein interactions and reversible light‐inducible protein–protein interaction modules by remolding exosome producer cells. Endogenous microRNA 21 (miR‐21) sponges, inhibitors of miR‐21, are successfully enriched on the plasma membrane and are sorted into exosomes by the biogenesis of the exosomes. The loading capacity of miR‐21 sponges is enhanced by 14‐fold in the light‐inducible loading system. In addition, targeted delivery of miR‐21 to leukemia cells is achieved by modifying exosomes with the cholesterol‐conjugated aptamer AS1411, resulting in significant cell apoptosis by blocking the function of miR‐21 in leukemia cells. This work provides an exosome‐based light‐inducible vehicle to efficiently load and deliver long endogenous RNA, which can enable more RNA‐based therapeutics for personalized cancer medicine.

In Vitro and in Vivo RNA Inhibition by CD9-HuR Functionalized Exosomes Encapsulated with miRNA or CRISPR/dCas9.

In vitro and in vivo delivery of RNAs of interest holds promise for gene therapy. Recently, exosomes are considered as a kind of rational vehicle for RNA delivery, especially miRNA and/or siRNA, while the loading efficiency is limited. In this study, we engineered the exosomes for RNA loading by constructing a fusion protein in which the exosomal membrane protein CD9 was fused with RNA binding protein, while the RNA of interest either natively harbors or is engineered to have the elements for the binding. By proof-of-principle experiments, we here fused CD9 with HuR, an RNA binding protein interacting with miR-155 with a relatively high affinity. In the exosome packaging cells, the fused CD9-HuR successfully enriched miR-155 into exosomes when miR-155 was excessively expressed. Moreover, miR-155 encapsulated in the exosomes in turn could be efficiently delivered into the recipient cells and recognized the endogenous targets. In addition, we also revealed that the CD9-HuR exosomes could enrich the functional miRNA inhibitor or CRISPR/dCas9 when the RNAs were engineered to have the AU rich elements. Taken together, we here have established a novel strategy for enhanced RNA cargo encapsulation into engineered exosomes, which in turn functions in the recipient cells.