mRNA initiation and termination are spatially coordinated.

The expression of a precise mRNA transcriptome is crucial for establishing cell identity and function, with dozens of alternative isoforms produced for a single gene sequence. The regulation of mRNA isoform usage occurs by the coordination of co-transcriptional mRNA processing mechanisms across a gene. Decisions involved in mRNA initiation and termination underlie the largest extent of mRNA isoform diversity, but little is known about any relationships between decisions at both ends of mRNA molecules. Here, we systematically profile the joint usage of mRNA transcription start sites (TSSs) and polyadenylation sites (PASs) across tissues and species. Using both short and long read RNA-seq data, we observe that mRNAs preferentially using upstream TSSs also tend to use upstream PASs, and congruently, the usage of downstream sites is similarly paired. This observation suggests that mRNA 5' end choice may directly influence mRNA 3' ends. Our results suggest a novel "Positional Initiation-Termination Axis" (PITA), in which the usage of alternative terminal sites are coupled based on the order in which they appear in the genome. PITA isoforms are more likely to encode alternative protein domains and use conserved sites. PITA is strongly associated with the length of genomic features, such that PITA is enriched in longer genes with more area devoted to regions that regulate alternative 5' or 3' ends. Strikingly, we found that PITA genes are more likely than non-PITA genes to have multiple, overlapping chromatin structural domains related to pairing of ordinally coupled start and end sites. In turn, PITA coupling is also associated with fast RNA Polymerase II (RNAPII) trafficking across these long gene regions. Our findings indicate that a combination of spatial and kinetic mechanisms couple transcription initiation and mRNA 3' end decisions based on ordinal position to define the expression mRNA isoforms.

Widespread occurrence of hybrid internal-terminal exons in human transcriptomes.

Messenger RNA isoform differences are predominantly driven by alternative first, internal, and last exons. Despite the importance of classifying exons to understand isoform structure, few tools examine isoform-specific exon usage. We recently observed that alternative transcription start sites often arise near internal exons, often creating "hybrid" first/internal exons. To systematically detect hybrid exons, we built the hybrid-internal-terminal (HIT) pipeline to classify exons depending on their isoform-specific usage. On the basis of splice junction reads in RNA sequencing data and probabilistic modeling, the HIT index identified thousands of previously misclassified hybrid first-internal and internal-last exons. Hybrid exons are enriched in long genes and genes involved in RNA splicing and have longer flanking introns and strong splice sites. Their usage varies considerably across human tissues. By developing the first method to classify exons according to isoform contexts, our findings document the occurrence of hybrid exons, a common quirk of the human transcriptome.

Engineering exosomes for pulmonary delivery of peptides and drugs to inflammatory lung cells by inhalation.

Exosomes have been investigated as delivery vesicles for various drugs. However, exosome-mediated peptide delivery into the lungs has not been studied. In this study, exosomes were engineered for the pulmonary delivery of RAGE-binding peptide (RBP), an anti-inflammatory peptide. To load the peptide into exosomes, RBP was linked to an exosome membrane integral protein, Lamp2b, to produce RBP-linked exosomes (RBP-exo). The anti-inflammatory effects of RBP-exo were confirmed by cytokine assays in lipopolysaccharide (LPS)-activated macrophage cells. To increase anti-inflammatory effects, curcumin was loaded into RBP-exo. Curcumin loaded RBP-exo (RBP-exo/Cur) had higher intracellular curcumin delivery efficiency than curcumin alone or curcumin loaded into unmodified exosomes (unmod-exo/Cur). This suggests that RBP on the surface of RBP-exo may interact with RAGE and increase the intracellular delivery efficiency of curcumin. In addition, RBP-exo/Cur had higher anti-inflammatory effects than curcumin alone, a mixture of RBP and curcumin, and unmod-exo/Cur in vitro. For in vivo evaluation, RBP-exo/Cur was administrated by intratracheal instillation into the lungs of an acute lung injury (ALI) model. The results showed that RBP-exo/Cur reduced pro-inflammatory cytokines more efficiently than curcumin alone, RBP-exo, and unmod-exo/Cur. Hematoxylin and eosin staining confirmed that the inflammation reaction was inhibited in the RBP-exo and RBP-exo/Cur groups. Immunostaining assays showed that RBP-exo was co-localized mostly with type I epithelial cells. In conclusion, RBP was successfully delivered with exosomes into the lungs by inhalation. A combination of RBP and curcumin using exosomes as carriers may be useful as ALI therapy.

Brain gene delivery using histidine and arginine-modified dendrimers for ischemic stroke therapy.

Polyamidoamine dendrimer has been studied as an efficient gene carrier. Due to its anti-inflammatory properties, polyamidoamine is a useful gene carrier, especially for inflammatory diseases. However, the commonly used polyamidoamine generation 6 dendrimer (PG6) has higher cytotoxicity than low-molecular weight polyamidoamines, which limits its applications. Therefore, early-generation polyamidoamine dendrimers, such as generation 2 (PG2), have been investigated as an alternative to PG6, although PG2 has a lower transfection efficiency. In this study, to improve gene delivery efficiency, histidine and arginine were conjugated on the primary amines of PG2, synthesizing PG2HR. The gene delivery efficiency of PG2HR was higher than that of PG2 or of PG2 conjugated with only arginine (PG2R), which may be due to higher cellular uptake and endosomal escape of the plasmid DNA (pDNA)/PG2HR complex. In addition, PG2HR had lower cytotoxicity than polyethylenimine (25 kDa, PEI25k), PG2, and PG2R. Mechanism studies showed that PG2HR delivered pDNA into the cells mainly by clathrin-independent endocytosis and partly by macropinocytosis. The therapeutic potential of PG2HR-mediated gene delivery was evaluated in middle cerebral artery occlusion (MCAO)-reperfusion stroke animal models. Heme oxygenase-1 (HO-1) plasmid was delivered into the brain by local injection. The results showed that PG2HR had higher gene delivery efficiency in the brain than did PEI25k, PG2, or PG2R. Furthermore, compared to the pHO-1/PEI25k, pHO-1/PG2, and pHO-1/PG2R complexes, the pHO-1/PG2HR complex had reduced apoptosis levels and infarct sizes in ischemic brains. Therefore, because of its low cytotoxicity and high gene delivery efficiency, PG2HR may be useful for gene therapy of inflammatory diseases including ischemic stroke.

Systemic delivery of microRNA-21 antisense oligonucleotides to the brain using T7-peptide decorated exosomes.

Antisense miRNA oligonucleotides against miR-21 (AMO-21) have a therapeutic potential for treatment of glioblastoma. However, glioblastoma-targeted delivery through systemic injection requires development of an efficient targeting carrier. For this purpose, a glioblastoma-targeting carrier was developed using the T7 peptide and exosomes. The transferrin receptor is overexpressed on the surface of glioblastoma cells, and T7 is a transferrin receptor-binding peptide. A T7 peptide-decorated exosome (T7-exo) was produced by incorporation of T7 into the exosome membrane as a fusion protein of T7 and Lamp2b. As a control, rabies virus glycoprotein (RVG) peptide targeting brain neuron cells was incorporated into the exosome membrane. AMO-21 was loaded into the exosomes by electroporation. In vitro studies of AMO-21 delivery showed that T7-exo had a higher delivery efficiency to C6 glioblastoma cells than unmodified exosome (Unmod-exo) and RVG-decorated exosome (RVG-exo). For in vivo delivery studies, T7-exo with AMO-21 was delivered into intracranial glioblastoma rat models by intravenous injection through the tail vein. The results showed that T7-exo delivered AMO-21 into the brain more efficiently than Unmod-exo and RVG-exo. In addition, delivery of AMO-21 using T7-exo reduced the miR-21 level in the glioblastoma efficiently. Reduction of miR-21 by AMO-21 induced the expression of PDCD4 and PTEN in tumors, resulting in reduction of tumor sizes. Taken together, these findings indicate that T7-exo is an efficient carrier of AMO-21 into the glioblastoma and may be useful in development of glioblastoma therapy.

Delivery of High Mobility Group Box-1 siRNA Using Brain-Targeting Exosomes for Ischemic Stroke Therapy.

Ischemic strokes are caused by decreased blood flow into the brain, due to narrowed cerebral arteries. In the ischemic brain, high-mobility group box 1 (HMGB1) is released into extracellular spaces and induces inflammatory reactions. In this study, HMGB1 small interfering RNA (siRNA) was delivered into ischemic brains by intravenous administration using rabies virus glycoprotein (RVG) peptide-decorated exosomes. A fusion protein of RVG and Lamp2b was expressed in 293T cells. Since Lamp2b is an exosome membrane-integral protein, RVG-Lamp2b is integrated into the exosomes, producing RVG-decorated exosomes (RVG-Exo). HMGB1-siRNA was loaded into RVG-Exo and unmodified exosomes (Unmod-Exo) by electroporation. The exosomes were homogenous with a size of less than 50 nm and a negative surface charge. In vitro delivery assays showed that RVG-Exo showed higher efficiency to Neuro2A cells than Unmod-Exo. Also, HMGB1 levels were reduced more effectively by RVG-Exo/HMGB1-siRNA. In vivo delivery efficiency and therapeutic effects of RVG-Exo/HMGB1-siRNA were evaluated in a middle cerebral artery occlusion (MCAO) model. RVG-Exo/HMGB1-siRNA, Unmod-Exo/HMGB1-siRNA, and PEI25k/HMGB1-siRNA were administrated into the MCAO model intravenously through the tail vein. The results showed that HMGB1, tumor necrosis factor-α (TNF-α), and apoptosis levels in the brain were reduced in the RVG-Exo/HMGB1-siRNA group more efficiently than the other groups. In addition, the infarct size was decreased in the RVG-Exo/HMGB1 group more effectively than the other groups. These results suggest that RVG-Exo with HMGB1-siRNA may have potential as a therapeutic system for the treatment of ischemic strokes.

Combined delivery of curcumin and the heme oxygenase-1 gene using cholesterol-conjugated polyamidoamine for anti-inflammatory therapy in acute lung injury.

BACKGROUND: Acute lung injury (ALI) is an inflammatory lung disease with a high mortality rate. In this study, combined delivery of the anti-inflammatory compound curcumin and the heme-oxygenase-1 (HO-1) gene using cholesterol-conjugated polyamidoamine was evaluated in a mouse model as a therapeutic option for ALI. METHODS: Curcumin was loaded into cholesterol-conjugated polyamidoamine (PamChol) micelles, and curcumin-loaded PamChol (PamChol-Cur) was then complexed with plasmid DNA (pDNA) through charge interactions. The pDNA/PamChol-Cur complex was physically characterized by dynamic light scattering, gel retardation, and heparin competition assay. Gene delivery efficiency was measured by luciferase assay. The HO-1 expression plasmid (pHO-1)/PamChol-Cur complex was administrated into the ALI model via intratracheal injection. The anti-inflammatory effect of the pDNA/PamChol-Cur complex was evaluated by ELISA, immunohistochemistry, and hematoxylin and eosin staining. RESULTS: The pDNA/PamChol-Cur complex had a size of approximately 120 nm with a positive surface charge. The in vitro plasmid DNA (pDNA) delivery efficiency of the pDNA/PamChol-Cur complex into L2 lung epithelial cells was higher than that of pDNA/PamChol. In addition, the curcumin in the pDNA/PamChol-Cur complex inhibited the nuclear translocation of NF-κB, suggesting an anti-inflammatory effect of curcumin. In the ALI animal model, the pHO-1/PamChol-Cur complex delivered the pHO-1 gene more efficiently than pHO-1/PamChol. In addition, the pHO-1/PamChol-Cur complex showed greater anti-inflammatory effects by reducing anti-inflammatory cytokine levels more than delivery of pHO-1/PamChol or PamChol-Cur only. CONCLUSION: The pHO-1/PamChol-Cur complex had a higher pHO-1 gene-delivery efficiency and greater anti-inflammatory effects than the pHO-1/PamChol complex or PamChol-Cur. Therefore, the combined delivery of curcumin and pHO-1 using PamChol-Cur may be useful for treatment of ALI.

Inhalable Gene Delivery System Using a Cationic RAGE-Antagonist Peptide for Gene Delivery to Inflammatory Lung Cells.

Acute lung injury (ALI) is a severe lung inflammatory disease. In ALI, the receptor for advanced glycation end-products (RAGE) is overexpressed in lung epithelial cells and involved in inflammatory reactions. A previous report showed that a RAGE-antagonist peptide (RAP), from high-mobility group box-1, bound to RAGE and reduced inflammatory reactions. RAP has high levels of positive amino acids, which suggests that RAP may form a complex with plasmid DNA (pDNA) by charge interactions. Because the charge density of RAP is lower than polyethylenimine (25 kDa, PEI25k), it may be able to avoid capture by the negatively charged mucus layer more easily and deliver pDNA into RAGE-positive lung cells of ALI animals by RAGE-mediated endocytosis. To prove this hypothesis, RAP was evaluated as a delivery carrier of adiponectin plasmid (pAPN) in lipopolysaccharide (LPS)-induced ALI animal models. In vitro transfection assays showed that RAP had lower transfection efficiency than PEI25k in L2 lung epithelial cells. However, in vivo administration to ALI animal models by inhalation showed that RAP had higher gene delivery efficiency than PEI25k. Particularly, due to a higher expression of RAGE in lung cells of ALI animals, the gene delivery efficiency of RAP was higher in ALI animals than that in normal animals. Delivery of the pAPN/RAP complex had anti-inflammatory effects, reducing pro-inflammatory cytokines. Hematoxylin and eosin staining confirmed that pAPN/RAP decreased inflammation in ALI models. Therefore, the results suggest that RAP may be useful as a carrier of pDNA into the lungs for ALI gene therapy.

Self-assembled polymeric micelles for combined delivery of anti-inflammatory gene and drug to the lungs by inhalation.

Acute lung injury (ALI) is a lung inflammatory disease for which pulmonary delivery of drug and gene could be a useful strategy. In this study, cholesterol-conjugated polyamidoamine (PAM-Chol) was synthesized and characterized as a carrier for combined delivery of anti-inflammatory gene and drug into the lungs by inhalation. The PAM-Chol formed self-assembled micelles in an aqueous solution with a critical micelle concentration of 0.22 mg ml-1. An in vitro transfection assay to L2 lung epithelial cells showed that the PAM-Chol micelle had higher transfection efficiency than lipofectamine and polyethylenimine (25 kDa, PEI25k). As the anti-inflammatory drug, resveratrol was loaded into the cores of the PAM-Chol micelles using the oil-in-water emulsion/solvent evaporation method. In lipopolysaccharide (LPS)-activated macrophage cells, resveratrol-loaded PAM-Chol (PAM-Chol/Res) reduced pro-inflammatory cytokines, confirming the anti-inflammatory effects of resveratrol. In in vitro transfection assays to L2 cells, the PAM-Chol/Res micelles had transfection efficiency similar to that of PAM-Chol. The delivery of resveratrol or the heme oxygenase-1 gene (pHO-1) by inhalation was evaluated in an ALI animal model. Resveratrol delivery using the PAM-Chol/Res micelles inhibited the nuclear translocation of nuclear factor-κB (NF-κB) and reduced pro-inflammatory cytokines in the lungs. pHO-1 delivery using PAM-Chol induced HO-1 expression and reduced pro-inflammatory cytokines. However, the highest anti-inflammatory effects were obtained with combined delivery of pHO-1 and resveratrol using the pHO-1/PAM-Chol/Res complex, as demonstrated in cytokine assays and immunohistochemical studies. Therefore, the PAM-Chol micelle is an efficient carrier of resveratrol and pHO-1 into the lungs and could be useful for the treatment of ALI by inhalation.

A ternary-complex of a suicide gene, a RAGE-binding peptide, and polyethylenimine as a gene delivery system with anti-tumor and anti-angiogenic dual effects in glioblastoma.

The receptor for advanced glycation end-products (RAGE) is involved in tumor angiogenesis. Inhibition of RAGE might be an effective anti-angiogenic therapy for cancer. In this study, a cationic RAGE-binding peptide (RBP) was produced as an antagonist of RAGE, and a ternary-complex consisting of RBP, polyethylenimine (2 kDa, PEI2k), and a suicide gene (pHSVtk) was developed as a gene delivery system with dual functions: the anti-tumor effect of pHSVtk and anti-angiogenic effect of RBP. As an antagonist of RAGE, RBP decreased the secretion of vascular-endothelial growth factor (VEGF) in activated macrophages and reduced the tube-formation of endothelial cells in vitro. In in vitro transfection assays, the RBP/PEI2k/plasmid DNA (pDNA) ternary-complex had higher transfection efficiency than the PEI2k/pDNA binary-complex. In an intracranial glioblastoma animal model, the RBP/PEI2k/pHSVtk ternary-complex reduced α-smooth muscle actin expression, suggesting that the complex has an anti-angiogenic effect. In addition, the ternary-complex had higher pHSVtk delivery efficiency than the PEI2k/pHSVtk and PEI25k/pHSVtk binary-complexes in an animal model. As a result, the ternary-complex induced apoptosis and reduced tumor volume more effectively than the PEI2k/pHSVtk and PEI25k/pHSVtk binary-complexes. In conclusion, due to its dual anti-tumor and anti-angiogenesis effects, the RBP/PEI2k/pHSVtk ternary-complex might be an efficient gene delivery system for the treatment of glioblastoma.

A curcumin-loaded polymeric micelle as a carrier of a microRNA-21 antisense-oligonucleotide for enhanced anti-tumor effects in a glioblastoma animal model.

A glioblastoma is a common primary brain tumor that expresses microRNA-21 (miR-21), which inhibits the expression of pro-apoptotic genes such as phosphatase and tensin homologue (PTEN) and programmed cell death 4 (PDCD4). Therefore, an antisense-oligonucleotide against miR-21 (miR21ASO) could have therapeutic effects for glioblastomas. In this study, curcumin was loaded into deoxycholic acid-conjugated polyethylenimine (DP) micelles. The curcumin-loaded DP micelle (DP-Cur) was evaluated as a carrier for the combined delivery of curcumin and miR21ASO. Gel retardation and heparin competition assays showed that DP-Cur formed stable complexes with miR21ASO. The anti-tumor effects of the combined delivery of curcumin and miR21ASO were evaluated in C6 glioblastoma cells. In vitro transfection showed that DP-Cur had an miR21ASO delivery efficiency similar to that of polyethylenimine (25 kDa, PEI25k) and DP. In the C6 cells, the delivery of miR21ASO using DP-Cur effectively reduced the miR21 level. The miR21ASO/DP-Cur complex induced apoptosis more effectively than the single delivery of curcumin or miR21ASO. The therapeutic effect of the miR21ASO/DP-Cur complex was also evaluated in an intracranial glioblastoma animal model. The miR21ASO/DP-Cur complex reduced the tumor volume more effectively than single therapy of curcumin or miR21ASO. Immunohistochemistry showed that PDCD4 and PTEN were induced in the miR21ASO/DP and miR21ASO/DP-Cur complex groups. Therefore, DP-Cur is an efficient carrier of miR21ASO and the combined delivery of miR21ASO and curcumin may be useful in the development of combination therapy for glioblastoma.

Production and application of HMGB1 derived recombinant RAGE-antagonist peptide for anti-inflammatory therapy in acute lung injury.

Acute lung injury (ALI) is an inflammatory lung disease caused by sepsis, infection, or ischemia-reperfusion. The receptor for advanced glycation end-products (RAGE) signaling pathway plays an important role in ALI. In this study, a novel RAGE-antagonist peptide (RAP) was produced as an inhibitor of the RAGE signaling pathway based on the RAGE-binding domain of high mobility group box-1 (HMGB1). Recombinant RAP was over-expressed and purified using nickel-affinity chromatography. In lipopolysaccharide- or HMGB1-activated RAW264.7 macrophage cells, RAP reduced the levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). RAP decreased the levels of cell surface RAGE and inhibited the nuclear translocation of nuclear factor-κB (NF-κB). These results imply that RAP decreases RAGE-mediated NF-κB activation and subsequent inflammatory reaction. For in vivo evaluation, RAP was delivered to the lungs of ALI-model animals via intratracheal administration. As a result, RAGE was down-regulated in the lung tissues by pulmonary delivery of RAP. Consequently, TNF-α, IL-6, and IL-1ß were also reduced in broncoalveolar lavage fluid and the lung tissues of RAP-treated animals. Hematoxylin and eosin staining indicated that inflammation was decreased in RAP-treated animals. Collectively, these results suggest that RAP may be a useful treatment for ALI.

Combined Delivery of a Lipopolysaccharide-Binding Peptide and the Heme Oxygenase-1 Gene Using Deoxycholic Acid-Conjugated Polyethylenimine for the Treatment of Acute Lung Injury.

A ternary complex comprising plasmid DNA, lipopolysaccharide-binding peptide (LBP), and deoxycholic acid-conjugated polyethylenimine (PEI-DA) is prepared for combinational therapy of acute lung injury (ALI). The LBP is designed as an anti-inflammatory peptide based on the lipopolysaccharide (LPS)-binding domain of HMGB-1. In vitro cytokine assays show that LBP reduces levels of proinflammatory cytokines by inhibiting LPS. PEI-DA is synthesized as the gene carrier by conjugation of deoxycholic acid to low-molecular weight polyethylenimine (2 kDa, PEI2k). PEI-DA has higher transfection efficiency than high-molecular weight polyethylenimine (25 kDa, PEI25k). The ternary complex of an HO-1 plasmid (pHO-1), PEI-DA, and LBP is prepared as a combinational system to deliver the therapeutic gene and peptide. The transfection efficiency of the ternary complex is higher than that of the pHO-1/PEI-DA binary complex. The ternary complex also reduces TNF-α secretion in LPS-activated Raw264.7 macrophage cells. Administration of the ternary complex into the lungs of an animal ALI model by intratracheal injection induces HO-1 expression and reduces levels of proinflammatory cytokines more efficiently than the pHO-1/PEI-DA binary complex or LBP alone. In addition, the ternary complex reduces inflammation in the lungs. Therefore, the pHO-1/PEI-DA/LBP ternary complex may be an effective treatment for ALI.

Anti-cancer effect of R3V6 peptide-mediated delivery of an anti-microRNA-21 antisense-oligodeoxynucleotide in a glioblastoma animal model.

MicroRNA-21 (miR-21) expression in glioblastoma inhibits the expression of pro-apoptotic genes, thereby promoting tumor growth. A previous study showed that the amphiphilic R3V6 peptide is an efficient carrier of the anti-miR-21 antisense oligodeoxynucleotide (antisense-ODN) into cells in vitro. In the current study, in vivo delivery of antisense-ODN using the R3V6 peptide was evaluated in a glioblastoma animal model. In vitro transfection showed that the R3V6 peptide delivered antisense-ODN more efficiently than polyethylenimine (25 kDa, PEI25k) in C6 glioblastoma cells. For in vivo evaluation, antisense-ODN/R3V6 complex was injected intratumorally into a C6 glioblastoma xenograft animal model. Tumor growth was suppressed by the injection of the antisense-ODN/R3V6 complex, compared with the antisense-ODN/PEI25k and scrambled-antisense-ODN (scr-antisense-ODN)/R3V6 complexes. Real-time RT-PCR showed that miR-21 levels were reduced most efficiently by the antisense-ODNR3V6 complex in tumors. Due to inhibition of miR-21, expression of the programed cell death 4 (PDCD4) gene was promoted in tumors, resulting in the induction of apoptosis of tumor cells. These results suggest that delivery of antisense-ODN using R3V6 peptides may be useful for the development of antisense-ODN therapy for glioblastoma.