Single stranded oligonucleotides inhibiting endocytosis
11345916 · 2022-05-31
Assignee
Inventors
- Peter Järver (Stockholm, SE)
- Anna-Lena Marie Spetz (Bromma, SE)
- Aleksandra Maria Dondalska (Solna, SE)
Cpc classification
C12N2310/18
CHEMISTRY; METALLURGY
C12N15/11
CHEMISTRY; METALLURGY
International classification
C12N15/113
CHEMISTRY; METALLURGY
Abstract
The invention provides a single-stranded oligonucleotide (ssON) for use in preventing or treating an influenza virus infection in a subject, wherein the single-stranded oligonucleotide is one that inhibits endocytosis. The invention also provides a single-stranded oligonucleotide (ssON) that comprises or consists of a polynucleotide having a sequence sharing at least 60% sequence identity with a sequence of any one of SEQ ID NOs: 13-21 listed in Table 1, or a complementary sequence thereof.
Claims
1. A single-stranded oligonucleotide (ssON) that comprises or consists of a polynucleotide (i) having a sequence of any one of SEQ ID NOs: 13-21, or a complementary sequence thereof, wherein up to five nucleotides are replaced by another nucleotide; and/or (ii) having a sequence sharing at least 85% sequence identity with a sequence of any one of SEQ ID NOs: 13-21 , or a complementary sequence thereof, wherein the ssON inhibits endocytosis, and wherein the ssON does not contain a CpG motif.
2. The ssON according to claim 1, wherein the ssON comprises or consists of a polynucleotide having a sequence of any one of SEQ ID NOs: 13-21, or a complementary sequence thereof.
3. The ssON according to claim 1, wherein the ssON comprises at least six phosphorothioate internucleotide linkages and at least six 2′-O-methyl modifications.
4. The ssON according to claim 1, wherein all internucleotide linkages in the ssON are phosphorothioate internucleotide linkages.
5. The ssON according to claim 1, wherein the length of the ssON is between 30 and 70 nucleotides.
6. The ssON according to claim 1, wherein not more than 16 consecutive nucleotides in the ssON are complementary with any human mRNA sequence and/or wherein the ssON is not self-complementary.
7. The ssON according to claim 1, wherein the ssON does not contain TTAGGG.
8. A pharmaceutical composition comprising the ssON according to claim 1, and a pharmaceutically acceptable carrier.
9. A composition comprising the ssON according to claim 1, and an additional therapeutic agent.
10. A method for preventing or treating an influenza virus infection in a subject, comprising administering a single-stranded oligonucleotide (ssON) to the subject, wherein the ssON is one that inhibits endocytosis; wherein the ssON does not contain a CpG motif; and wherein the ssON is at least 25 nucleotides in length.
11. The method according to claim 10, wherein the ssON is one that modulates Toll-like receptor signalling, preferably wherein the ssON is one which inhibits TLR3 signalling.
12. The method according to claim 10, wherein either (i) at least 90% of the internucleotide linkages in the ssON are phosphorothioate internucleotide linkages; or (ii) the ssON comprises at least four phosphorothioate internucleotide linkages and at least four 2′-O-methyl modifications.
13. The method according to claim 10, wherein the length of the ssON is between 25 and 70 nucleotides.
14. The method according to claim 10, wherein not more than 16 consecutive nucleotides in the ssON are complementary with any human mRNA sequence and/or wherein the ssON is not self-complementary.
15. The method according to claim 10, wherein the monosaccharides in the ssON are selected from the group consisting of 2′-deoxyribose and 2′-O-methylribose.
16. The method according to claim 10, wherein the ssON does not contain TTAGGG.
17. The method according to claim 10, wherein the ssON is administered as a monotherapy.
18. The method according to claim 10, wherein the subject is administered an additional therapeutic agent.
19. The method according to claim 10, wherein the subject is not administered poly (I:C).
Description
(1) Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:
(2)
(3) Human moDCs were treated for 45 min at +37° C. or +4° C. with endocytic uptake markers (Transferrin-red, Cholera toxin B-green, Dextran-blue and LDL-yellow) with or without addition of ssON 35 or ssON 35 alone (purple). Histograms show representative data from at least three donors. Dark coloured area (red, green, yellow, blue and purple) depict uptake of indicated molecules at +37° C. whereas addition of ssON is depicted with dashed line (light colour). Background, with experiment conducted at +4° C. is displayed as grey.
(4) (A, B) 10 μM ssON 35 PO (MedON02) partly inhibited uptake of CME and CDE dependent uptake (Transferrin, Cholera toxin B).
(5) (C, D) 0.5 μM ssON 35 PS (MedON01) completely inhibited uptake of CME and CDE dependent uptake (Transferrin, Cholera toxin B).
(6) (E) 0.5 μM ssON 35 PS (MedON01) did not influence MPC (Dextran). Complete overlap dark and light blue.
(7) (F) Fluorescently labelled ssON 35 PS had the same uptake at +37° C. and +4° C. Complete overlap dark and light purple.
(8) (G, H) Quantitative analysis of Transferrin, Cholera toxin B uptake in the presence of 10 μM ssON 35 PO, 0.5 μM ssON 35 PS at +37° C. or +4° C.
(9) (I) Quantitative analysis of fluorescently labelled ssON 35 PS uptake at +37° C. and +4° C.
(10) (J) 0.5 μM ssON 35 PS (MedON-01) completely inhibited uptake of LDL.
(11) Error bars are given in SEM. Non-parametric Mann-Whitney test was used to compare the data. P-value: not significant (n.s) P>0.05; *P≤5 0.05; **P≤5 0.01; ***P≤5 0.001.
(12)
(13) (A) 0.5 μM ssON 35 PS completely inhibited pI:C induced CD86 expression.
(14) (B) The ssON 35 PS mediated inhibition of CD86 expression in pI:C exposed moDC was concentration dependent (EC.sub.50 between 100-200 nM).
(15) (C) SsON 35 PS completely inhibited pI:C induced IL-6 secretion.
(16) (D) PS modification was not essential for the inhibitory effect.
(17) (E) Uptake of fluorescent pI:C in moDCs (F) in the presence of 0.5 μM ssON 35 PS.
(18) (G) The inhibition of pI:C induced CD86 expression was sequence independent (for sequences, see Table A).
(19) (H, I) The inhibition of pI:C induced CD86 expression was dependent on the ssON length (ssON PS 15-35).
(20) (J) Uptake of fluorescent ssON 15 PS in moDCs (K) in the presence 0.5 μM ssON 35 PS.
(21) (L) Inhibition of fluorescent ssON 15 PS uptake was dependent on ssON 35 PS concentration. Error bars are given in SEM. Non-parametric Mann-Whitney test was used to compare the data. P-value: not significant (n.s) P>0.05; *P≤5 0.05; **P≤5 0.01; ***P≤5-0.00.
(22)
(23) (A, B) CD86 expression and IL-6 secretion from moDCs exposed to pI:C and subsequently challenged with ssON 35 PS at time-points from 5 min to 24 h.
(24) (C, D) MoDCs treated in the opposite way, ssON 35 PS and subsequently challenged with pI:C.
(25) (E) PI:C induced cell death in moDCs was reversed by the addition of 0.5 μM ssON 35 PS (n>25).
(26) (F) Introduction of the complementary strand (ssON Compl PO) abolished the inhibitory effect on pI:C induced CD86 expression of 0.5 μM ssON 35 PS.
(27) (G) SsON mediated inhibition of IL-6 secretion in PBMCs was limited to agonists that are taken up by endocytosis.
(28) Error bars are given in SEM. Non-parametric Mann-Whitney test was used to compare the data. P-value: not significant (n.s) P>0.05; *P 5 0.05; **P 5 0.01; ***P 5 0.001.
(29)
(30) (A) Schematic view of TLR4 mediated cell signalling.
(31) (B, C) 0.5 μM ssON 35 PS had no influence on secretion of cytokines/chemokines that are downstream of NFkB mediated signalling pathways (IL-6 and IL-10).
(32) (D, E) 0.5 μM ssON 35 PS reduced secretion of cytokines/chemokines that are downstream of TRIF mediated signalling pathways (IL-29 and CXCL10).
(33) (F) 0.5 μM ssON 35 PS inhibited uptake of fluorescently labelled LPS. Histogram shows representative data from at least three donors.
(34) (G) Quantitative analysis of LPS uptake in the presence of 10 μM ssON 35 PO, 0.5 μM ssON 35 PS at +37° C. or +4° C.
(35) (H) Uptake of fluorescent LPS in moDCs (I) in the presence of 0.5 μM ssON 35 PS.
(36) Error bars are given in SEM. Non-parametric Mann-Whitney test was used to compare the data. P-value: not significant (n.s) P>0.05; *P 0.05; **P≤0.01; ***P≤0.001.
(37)
(38) Human moDCs were treated for 45 min at +37° C. or +4° C. with Transferrin (red) or LPS (orange) with or without addition of ssON 2′OMe PS (Table A). Histograms show representative data from at least three donors.
(39) (A, B) 0.5 μM ssON 2′OMe PS completely inhibited uptake of fluorescently labelled Transferrin and LPS.
(40) (C) SsON 2′OMe PS displayed the same efficacy as ssON 35 PS in blocking pI:C induced secretion of IL-6 from moDC.
(41) (D) Total RNA inhibited pI:C induced IL-6 secretion from moDCs in a concentration dependent manner.
(42) Error bars are given in SEM. Non-parametric Mann-Whitney test was used to compare the data. P-value: not significant (n.s) P>0.05; *P≤0.05; **P≤0.01; ***P≤0.001.
(43)
(44) (A) Analysis of RNA expression in skin following injection with pI:C in the presence or absence of ssON 35 PS by microarrays.
(45) (B) Volcano plot illustrating log 2 fold change and statistical significance across genes: genes with fold change>1 or <−1 and uncorrected p-value<0.05 highlighted in red (B1, B2).
(46) (C) Clustering of genes showing the highest differential expression comparing pI:C treatment alone or in combination with ssON 35 PS, as determined by the Volcano plot (B1-top, B2-below).
(47) Each row (in A, C,) represents a gene (probe); each column represents a sample.
(48) The expression level for each gene was standardized by subtracting row mean expression and dividing by the standard deviation. This scaled expression value, (row z-score), is plotted in red-blue color scale with red indicating high expression and blue indicating low expression. Unsupervised hierarchical clustering of genes was based on Spearman correlation and are shown with means±SEM.
(49) (D) Relative IL-6 mRNA expression values of individual macaque skin biopsies collected 24 hours post-stimulation.
(50) (E) Concentrations of IL-6 protein present in supernatants of enzymatically digested dermis were measured by Bio-Plex technology.
(51) Two skin biopsies (left and right side of the back) were obtained from each animal, where one of the biopsies were taken after injection of PBS and the other biopsy was taken from the site injected with the compound as indicated in the figures. Significant differences were assessed by non-parametric Kruskal-Wallis test and Dunn's post-test (* P<0.05, ** P<0.01 and *** P<0.001). Different treatment groups were compared using nonparametric Mann-Whitney unpaired test, as indicated (dash arrows).
(52)
(53) Human moDCs were treated for 45 min at +37° C. or +4° C. with fluorescently labelled PolyI:C-Cy3 with or without addition of MedON01 (concentrations range 500-62.5 nM). Uptake was quantified by flow cytometry (MFI). Quantitative analysis of PolyI:C uptake in the presence of 500-62.5 nM MedON01 at +37° C., control PolyI:C alone at +4° C. (baseline negative control) or positive control PolyI:C alone at 37° C.
(54)
(55)
(56) Human monocyte derived DCs were infected in vitro with Influenza H1N1 Cal2009 (IAV) using MOI 0.2 or MOI 2. MoDC were either untreated or pre-treated with MedON-01 (0.5 μM) for 2 hours before infection. Infection with IAV was then carried out during another 2 hours. Cells were washed and incubated for 24 hours either with MedON-01 added again or not (m=medium). The viral load in the culture supernatants were quantified by real time PCR. Data were obtained in four independent experiments using six different donors analyzed in triplicate. Statistical calculations were made using non-parametric paired Wilcoxon text.
(57)
(58) Human monocyte derived DCs were infected in vitro with Influenza H1N1 Cal2009 (IAV) using MOI 1. MoDC were either untreated or pre-treated with MedON's 01-21 shown as SEQ ID Table 1 (1 μM) for 2 hours before infection. Infection with IAV was then carried out during another 2 hours. Cells were washed and incubated for 24 hours either with MedON's added again or not (m=medium). The viral load in the culture supernatants were quantified by real time PCR. Data were obtained i using two different donors analyzed in triplicate.
(59)
(60) Balb/c mice were infected intranasal with Influenza H1N1 (Cal09). One group were untreated (
(61)
(62) Balb/c mice were infected intranasally with Influenza H1N1 (Cal09). One group were untreated (
(63)
(64) MoDCs were treated with the TLR3 agonist poly I:C (25 μg/ml) in the absence or presence of 0.5 μM MedONs and incubated at +37° C. 24 h post treatments, the supernatant was collected and assessed for secretion of the pro-inflammatory cytokine IL-6 by using ELISA. MedON-01 and MedON-13-20 are able to inhibit poly I:C mediated induction of IL-6.
(65)
(66) MoDCs were treated with the TLR3 agonist poly I:C (25 μg/ml) in the absence or presence of 0.5 μM MedONs and incubated at +37° C. 48 h post treatments, the cells were harvested and stained for immuno-maturation marker CD86. Surface expression of CD86 was quantified by flow cytometry. MedON-01 and MedON-13-20 are able to inhibit poly I:C mediated immune-maturation.
(67)
(68) (A) The inhibition of pI:C-induced CD86 expression (48 h) in moDC was not dependent on a canonical sequence (for sequences, see Table 2).
(69) (B, C) The inhibition of pI:C-induced CD86 expression (48 h) (B) and IL-6 production (24 h) (C) in moDC was dependent on the ssON length (ssON PS 15-35).
(70) (D) Uptake of fluorescent ssON-color Cy5 15 PS in moDC
(71) (E) in the presence 0.5 μM ssON 35 PS.
(72) (F) Inhibition of fluorescent ssON 15 PS uptake was dependent on ssON 35 PS concentration.
(73) (G) Introduction of the complementary strand (ssON Compl PO) abolished the inhibitory effect on pI:C-induced CD86 expression (48 h) of 0.5 μM ssON 35 PS.
(74) All data are from at least three donors in duplicate. Error bars are given in SEM. Nonparametric Mann-Whitney test was used to compare the data. P-value: not significant (n.s) P>0.05; *P≤0.05; **P≤0.01; ***P≤5 0.001.
(75)
(76) Human moDC were treated for 45 min at +37° C. or +4° C. with TF (Alexa 647) or LPS (Alexa 488) with or without addition of ssON 2′OMe RNA PS (Table 2).
(77) (A) Structures of repeating units of oligonucleotides and oligonucleotide analogues used in this study.
(78) (B, C) 0.5 μM ssON 2′OMe RNA PS inhibited uptake of fluorescently labelled TF and LPS.
(79) (D) SsON 2′OMe RNA with a native PO backbone is able to block TF uptake in moDC.
(80) (E) SsON 2′OMe RNA PS displayed the same efficacy as ssON 35 DNA PS in blocking pI:C-induced secretion of IL-6 from moDC.
(81) (F) Total RNA inhibited pI:C-induced IL-6 secretion from moDC in a concentration dependent manner.
(82) All data are from at least three donors in duplicate. Error bars are given in SEM. Nonparametric Mann-Whitney test was used to compare the data. P-value: not significant (n.s) P>0.05; *P≤0.05; **P≤0.01; ***P≤0.001.
(83)
(84) Colored circles mark genes included in specific categories (green=ISGs, blue=inflammasome related).
(85) (A-C) RNA-seq data from moDC 24 h post treatment (3 donors). MA-plots log 2 fold changes adjusted p-values below 0.05 in red. (A) PI:C treatment vs non-treated cells shows high upregulation of ISGs. (B) Combined treatment with pI:C and ssON 35 PS (0.5 μM) vs nontreated cells show minor differences. (C) SsON 35 PS treatment (0.5 μM) vs non-treated cells show minor differences.
(86) (D-F) Whole cell proteomic data from moDC 24 h post treatment (3 donors). MA-plots of whole cell proteomic data (protein log intensity ratio versus average intensity). (D) PI:C treatment vs non-treated cells show high upregulation of ISGs. (E) Combined treatment with pI:C and ssON 35 PS (0.5 μM) vs non-treated cells show minor differences. (F) SsON 35 PS treatment (0.5 μM) vs non-treated cells show minor differences.
(87)
(88) (A) RNA expression in skin following injection with dsRNA (pI:C) in the presence or absence of ssON 35 PS. Volcano plot illustrating log 2 fold-change and p<0.05 in red.
(89) (B) Clustering of genes showing the highest differential expression comparing pI:C+ssON with ssON 35 PS treatment alone (b, c). Gene expression is represented as gene-wise standardized expression (Z score) with p<0.05. Red and blue correspond to up- and downregulated genes, respectively. Unsupervised hierarchical clustering of genes based on Spearman-correlation.
(90) (C) Relative mRNA expression values from the microarray analyses of individual macaque skin biopsies 24 h post-stimulation (means±SEM).
(91) (D) Concentrations of indicated cytokine proteins present in supernatants of enzymatically digested skin biopsies (24 h post-stimulation in vivo) (means±SEM from individual animals).
(92) Lower right panel shows IL-10 production in a dose escalation experiment with ssON ranging from 85-680 μg per injection (n=2).
(93) Significant differences were assessed by non-parametric Kruskal-Wallis test and Dunn's post test (* P<0.05, ** P<0.01 and *** P<0.001). Different treatment groups were compared using nonparametric Mann-Whitney unpaired test, as indicated.
Example 1: Extracellular Single Stranded Oligonucleotides Regulate Endocytic Uptake And Downstream Toll-Like Receptor Responses
(94) Summary
(95) Endocytic pathways need to be tightly regulated in order to maintain fundamental cellular functions and control uptake of molecules destined for endosomes. However, there is a lack of knowledge about extracellular molecules that regulate endocytic uptake. In the present study we show that single stranded oligonucleotides (ssON) inhibit some endocytic pathways in human monocyte derived cells (moDC), while leaving other pathways unaffected. Both single stranded DNA and RNA conferred the endocytic inhibition. It was affected by concentration and ssON length. The ssON-mediated inhibition modulated cell signalling downstream of pattern recognition receptors (PRR) that localize within the affected endosomal pathways. Introduction of non-natural linkages and/or modified nucleosides was not essential for inhibition, but was tolerated and might be necessary in order to retain the effect over longer periods of time. We provide evidence that ssON exerted immunomodulatory effects after intradermal injections in macaques. These studies reveal a regulatory role for extracellular ssONs in the endocytic uptake of Toll-like receptor (TLR) ligands and provide a mechanistic explanation for their immunomodulation. The identified ssON-mediated interference of endocytosis (SOMIE) is a regulatory process that temporarily dampens TLR3/4/7 signaling, thereby averting excessive immune responses.
(96) Introduction
(97) Endocytic mechanisms control lipid and protein compositions of the plasma membrane, thereby regulating how cells interact and communicate with their surrounding environment. In order to appropriately respond to, and influence its environment, the cell has to tightly regulate the plasma membrane composition as well as the uptake of ligands into endosomes (Doherty and McMahon, 2009). For example, cell surface removal of receptors enable long-term sensitivity to specific ligands (Irannejad and Zastrow, 2014). However, endocytosis does not only negatively regulate interactions with the external world. These processes also control fundamental mechanisms such as mitosis, cell migration, and immune-regulating processes such as initiation of the innate immune responses and antigen presentation (Doherty and McMahon, 2009). Immune regulation stems from the fact that many pathogens use endocytic pathways to mediate their cellular internalization (Cossart and Helenius, 2014). Likewise, nucleic acids released from microbes or dying cells are taken up through endocytosis enabling activation of TLRs located in the endosomes (Kawai and Akira, 2010).
(98) Although a lot is known about the cargoes for different endocytic structures, the specific mechanisms by which these cargoes are engaged, internalized, and how their uptake is regulated, are less clear. There is an increasing stream of endocytosis pathways reported in the literature with varying degree of description and specificity. In the present study have assessed whether extracellular ssONs modulate three well defined pathways, Clathrin mediated endocytosis (CME), Caveolin dependent endocytosis (CDE), and Macropinocytosis (MPC) (Doherty and McMahon, 2009).
(99) CME is mediated by small (100 nm diameter) vesicles, which have a coat made up by the clathrin protein. Clathrin-coated vesicles (CCVs) are found in virtually all cells and form domains of the plasma membrane termed clathrin-coated pits. These CCVs are responsible for removing GCPRs from the outer plasma membrane, reduce their signalling, and desensitise cells for specific ligands. Further, these pits can concentrate receptors responsible for the receptor-mediated endocytosis of ligands such as low-density lipoprotein, transferrin, and several others (Kumari et al., 2010), including dsRNA and its analogue pI:C (Itoh et al., 2008).
(100) CDE is one of the most well studied non-clathrin-coated endocytosis pathways. It exists on the surface of many cell types, and consists of the protein caveolin and a bilayer enriched in cholesterol and glycolipids. Caveolae are smaller (approx. 50 nm in diameter) flask-shape pits in the membrane. The caveolar pathway is responsible for the endocytosis of ligands such as albumin, tetanus toxin, and cholera toxin subunit B (CTB) (Kumari et al., 2010).
(101) MPC, occurs from highly ruffled regions of the plasma membrane, which then pinch off into the cell to form a larger vesicle (0.5-5 μm in diameter) filled with a large volume of extracellular fluid and molecules. Uptake indicators include fluid phase markers such as dextran (Doherty and McMahon, 2009).
(102) Nucleic acid sensing TLRs localise distinctively to endosomes and stimulate moDC maturation in vitro (Isomura et al., 2005). TLR 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids in the endosomes. This localization of TLRs to distinct intracellular compartments plays a fundamental role in activating responses to foreign nucleic acids while maintaining tolerance to self-nucleic acids (Roers et al., 2016). TLR signalling constitutes a defense mechanism during viral infection in response to nucleic acids present in e.g. viral genomes or their intermediates during the replication cycle (Amarante and Watanabe, 2010; Vercammen et al., 2008). The dsRNA analogue pI:C is frequently used as a TLR3 agonist, and it has been shown to rely on CME to enter the endocytic pathway (Itoh et al., 2008).
(103) In order to assess whether extracellular ssONs have the ability to modulate endocytic pathways, we here used a cellular model system that allows for both fluorescently based uptake studies using endocytic markers (e.g. Transferrin CTB and Dextran), and downstream cellular pathways from ‘signalling endosomes’ (Scita and Di Fiore, 2010; Sorkin and Zastrow, 2009). Cell signalling from endosomes occurs for receptors, such as NOTCH-family members, tumour necrosis factor receptor 1 and some TLRs (reviewed by Sorkin et al (Sorkin and Zastrow, 2009)). The TLRs that are located in endosomes (TLR3, TLR7, TLR8, and TLR9) are activated by ONs present in e.g. viral or bacterial genomes as well as viral intermediates during replication (Matsumoto et al., 2011). However, host-produced ONs also have the ability to initiate TLR signalling when released from damaged cells or tissues (Amarante and Watanabe, 2010; Pelka et al., 2016). While ON-sensing TLRs reside in and signal from specific endosomal compartments, TLR4 that senses bacterial lipopolysaccharides (LPS) has two distinct signalling pathways; one MyD88 dependent pathway that signals from the plasma membrane, and one TRIF dependent pathway that is reliant on CME and only can signal after endocytosis has occurred (Kagan et al., 2008; Rajaiah et al., 2015; Tatematsu et al., 2016).
(104) Dendritic cells (DCs) are antigen-presenting cells that integrate stimuli from the environment leading to immune activation or induction and maintenance of immune tolerance. DCs consist of developmentally and functionally distinct subsets that differentially regulate T cell responses thereby acting as messengers between the innate and the adaptive immune systems (Merad et al., 2013). DCs subsets are present in tissues that are in contact with the external environment, such as the skin and the inner lining of the respiratory system as well as intestines. Because of their role as sensors of the external environment, DCs exhibit a wide variety of endocytic pathways. Further, as messengers between the innate and the adaptive immune systems they respond to several triggering molecules that are taken up and processed in the endocytic pathways, such as ON-sensing TLRs. Conversely, DNA that contains repetitive TTAGGG motifs originating from mammalian telomeres can inhibit immune reactions (Gursel et al., 2003). Synthetic ss-DNA molecules with immunosuppressive functions are being studied in pre-clinical models and they vary in size, sequence and nucleotide backbone but there is often a lack of understanding on their mechanism of action (Bayik et al., 2016).
(105) It is well established that ONs do not passively cross the plasma membrane and transfection reagents or other membrane permeable approaches are required for intracellular delivery. (Sharma and Watts, 2015; Silva et al., 2015). However, free ONs have been suggested to be taken up through integrins and/or scavenger receptors (Juliano and Carver, 2015). Whether there is a difference in cellular uptake between ssONs and dsONs is to our knowledge not known. Here, we compared the endocytic capacity of ssONs with varying length and sequences. We showed that 35 bases long ssONs were not endocytosed by moDCs, and instead inhibited endocytosis of transferrin, CTB, and the dsRNA analogue polyinosinic-polycytidylic acid (pI:C) but spared uptake of dextran. Introduction of non-natural phosphorothioate linkages (PS) increased the inhibitory effect, but naturally occurring native phosphodiester (PO) ssONs and RNA purified from cells pI:C induced IL-6 production in short-term cultures.
(106) We previously reported that a CpG containing ssON inhibited TLR3 signalling in primary human monocyte derived cells (moDC) that express TLR3/4/8, but lack TLR7/9 (Sköld et al., 2012). In the present study, we synthesized a panel of ssONs that lack CpG motifs, to identify the requirements for inhibition of dsRNA-mediated activation of moDC (Table A). We discovered that ssONs not only inhibited TLR3 activation but also activation of TLR7. Further, we show that ssON modulated signaling from TLR4 that was dependent on endosomal uptake, while leaving signaling from the plasma membrane unaffected. We provide evidence that certain ssON temporarily shut down CME without causing major harm to the cell, as shown by viability assay, cytokine production, RNAseq and whole cell proteomic analyses. Finally, we demonstrate that ssONs can shape the immune responses induced by dsRNA locally in the skin of macaques. These findings show that extracellular ssON's can inhibit CME and CDE, providing a mechanism to temporarily shut down uptake of cargo to endosomes, with subsequent alterations of the inflammatory signatures.
(107) Results
(108) Extracellular ssONs Inhibited Endocytosis of Transferrin and CTB
(109) Here we investigated whether extracellular ssONs can inhibit uptake of commonly used markers of endocytic pathways in human moDCs (without any transfection method, or delivery system). Human moDCs were treated for 45 minutes at +37° C. or +4° C. with endocytic uptake markers (Transferrin, CTB, and Dextran as well as LDL) with or without addition of 0.5 μM ssONs (sequences in Table A). At +37° C. Transferrin, CTB and Dextran as well as LDL were taken up as measured by flow cytometry (
(110) Length Requirement for Extracellular ssONs Capacity to Inhibit Uptake of TLR3 Ligands and Subsequent TLR3 Activation in moDC
(111) The dsRNA analogue pI:C, is a TLR3 agonist that is taken up through CME (Itoh et al., 2008). We next asked whether ssON 35 PS can block pI:C mediated maturation of moDCs and if so whether it occurs by inhibition of endocytic uptake (
(112) We then asked if the observed inhibition of endocytosis and downstream TLR3 activation could be linked to and specific ssON sequence. We compared three different ssON sequences (ssON 35 PS, ssON GtA PS, and ssON Compl PS; Table A) they all displayed the same inhibitory effect in moDCs treated with pI:C for 48 hours (
(113) After concluding that the inhibitory effect was not strictly dependent on ssON sequence, we next assessed if the ssON length could influence the efficacy. We found that shorter ssONs did not possess the same inhibitory effect as longer ones with a cut off between 20 and 25 bases long ssONs (
(114) Altogether these data showed that ssON can inhibit endocytic uptake and downstream effects of the TLR3 agonist pI:C in a concentration dependent manner and that the effects were not linked to a specific ssON sequence or motif. The ssON length did affect the inhibitory effects, and had a cut-off between 20-25 bases.
(115) Kinetics of ssON Mediated Inhibition of TLR Signalling
(116) If extracellular ssONs are able to inhibit certain endocytic pathways, the biokinetics should follow previous endosomal uptake studies in moDCs (Choi et al., 2013; Smole et al., 2015). We therefore assessed the kinetics of both activation and inhibition of moDC maturation by exposing cells to either TLR3 agonist pI:C or inhibiting ssON 35 PS and subsequently pulsed cells with ssON 35 PS/pI:C over a period of time ranging from 5 minutes to 24 hours. Expression of co-stimulatory CD86 and secretion of IL-6 were measured as signs of pI:C induced moDC maturation. Cellular exposure to pI:C followed by pulsing with ssON 35 PS showed that the inhibiting effect was present up to 50 minutes, and was then rapidly reduced and vanished about 2 h post initial pI:C treatment (
(117) SsON Prevented pI:C Mediated Cell Death in Human moDCs
(118) PI:C can induce apoptosis in human cells (Sun et al., 2011). To assess if ssONs can prevent this, moDCs were treated with pI:C with or without the addition of ssON 35 PS and apoptosis was measured by Live/Dead cell stain kit using flow cytometry. Cells treated with only pI:C had a reduced viability by approximately 20%. The frequency of cell death was reduced back to the level of untreated cells by the addition of 0.5 μM ssON 35 PS, 48 hours post-initial treatment (
(119) Inhibition of pI:C Uptake Occurred for ssONs but not dsONs
(120) We provide data here showing that extracellular ssONs ability to inhibit endocytic uptake was dependent on ON length, but independent of its sequence (
(121) The Inhibitory Effect of ssONs Occurred for TLR3 and TLR7 Located in Endosomes but Spared Effects by Membrane Permeable TLR Agonist
(122) As ssON-sensing TLRs are dependent on endocytosis and signalling occurs from signalling endosomes (Sorkin and Zastrow, 2009), we next assessed whether ssONs have the ability to block TLR-induced moDC maturation from a membrane permeable TLR agonist. R848 is a membrane permeable TLR7/8 agonist, and is thus not dependent on endocytosis to activate TLR signalling (Govindaraj et al., 2011). CL307 is a TLR7 agonist that is coupled to spermin, and hence dependent on endocytosis for its cellular uptake (Soulet et al., 2002). MoDCs lack the expression of ssON-sensing TLR7, thus these experiments were performed in peripheral blood mononuclear cells (PBMCs). PBMCs were treated with pI:C (TLR3 agonist), CL307 (TLR7 agonist), or R848 (membrane permeable TLR7/8 agonist) in the absence or presence of ssON 35 PS (
(123) SsON Mediated Inhibition of TLR4 Signalling was Dependent on Endosomal Uptake
(124) TLR4 responds to LPS stimulation and has two distinct signalling pathways (Kagan et al., 2008; Rajaiah et al., 2015; Tatematsu et al., 2016). One is mediated through MyD88 and occurs at the plasma membrane leading to NFkB activation. The second pathway is mediated through TRIF, stimulating a strong interferon response. The TRIF mediated signalling occurs from clathrin-coated endosomes, and is hence dependent on CME (
(125) Inhibition of Endosomal Uptake can be Achieved with Both DNA and RNA
(126) To elucidate if the ability to inhibit endocytic uptake is exclusive for DNA, or if RNA also possesses this activity, we synthesised 35 mer ssON RNA and RNA analogues (Table A). A native unmodified RNA 35-mer had no effect on endocytic uptake in moDCs (data not shown). However, the ON RNA analogue 2′OMe and total RNA purified from moDCs had the ability to inhibit endosomal uptake (
(127) Administration of ssON 35 PS Modulated dsRNA-Mediated Inflammation in the Skin of Macaques
(128) To assess whether the stabilized ssON 35 PS can mediate effects in vivo, we measured the global innate response to pI:C in the presence or absence of ssON 35 PS by transcriptional profiling of skin biopsies taken from the site of injection. We found that intradermal injection of pI:C resulted in induction of multiple innate immune response genes as compared to injection of PBS (
(129) Experimental Procedures
(130) Single Stranded Oligonucleotides
(131) Fully PS-substituted ssON 35 PS and 2′OMe PS were made by made by Integrated DNA Technologies. Other modified oligonucleotides were purchased from Eurofins. For sequences, see Table A. Total RNA was purified from moDCs using RNeasy total RNA purification kit, according to manufacturer's instructions (Qiagen).
(132) TLR Ligands and Labeled Endosomal Markers
(133) TLR3 ligand high molecular weight pI:C (25 μg/mL unless otherwise stated), TLR7 ligand CL307 and TLR7/8 ligand R848 were all purchased from Invivogen. Endosomal markers; Transferrin-Alexa647, Cholera toxin B-Alexa488, Dextran-Texas red 10 kDa, and LPS-Alexa488 were all purchased from Life Technologies.
(134) Uptake Studies in MoDCs
(135) Human Monocytes were negatively selected from buffy coats using the RosetteSep Monocyte Enrichment Kit (1 mL/10 mL buffy coat; StemCell Technologies) and differentiated into moDC, with GM-CSF (250 ng/mL; PeproTech) and IL-4 (6.5 ng/mL; R&D Systems) for 6 days at a density of 5×10.sup.5 cells/mL in RPMI 1640 completed with 10% FCS, 1 mM sodium pyruvate, 10 mM HEPES, 2 mM L-glutamine, and 1% streptomycin and penicillin (all from Invitrogen Life Technologies). MoDCs were exposed to endosomal markers and ssON on ice in complete 10% RPMI media (or serum free media for PO ON uptake studies), and then transferred to 37° C. for 45 minutes. After incubation, the cells were put back on ice, washed and fixed and uptake was monitored by flow cytometry. CME was monitored by uptake of 50 μg/ml Alexa647 labeled Transferrin. CDE was monitored by 1 μg/ml Alexa488 labeled cholera toxin B, and MPC was monitored by 0.5 mg/ml Texas-Red labeled Dextran. Endocytic uptake of TLR4 was monitored by 1 μg Alexa488 labeled LPS. Uptake of LDL was measured by quantifying uptake of DiL labelled LDL (Molecular probes).
(136) MoDC Maturation Studies
(137) Human Monocytes were differentiated as described above. MoDCs were exposed to pI:C and ssON and maturation was assessed 48 hours later using monoclonal antibodies (Abs) targeting CD1a and the moDC maturation markers, CD86, CD83, and CD80 (all Abs from BD Biosciences). Flow cytometry sample data were acquired on a Fortessa (BD Biosciences) and the analysis was performed with FlowJo software (TreeStar).
(138) Cytokine/Chemokine Secretion Studies
(139) Supernatants were collected from cells at given time points post treatment (pI:C, CL307, R848 (Invivogen) or LPS (Sigma)) and secretion of Cytokines/Chemokines was measured by standard ELISA according to manufacturer's instructions (Mabtech; IL-6, IL-10, and IL-29. Life Technologies: CXCL10). Protein amount was monitored by 3,3′,5,5′-Tetramethylbenzidine (TMB) absorbance at 450 nm, and given in pg/ml or relative absorbance units.
(140) Wide-Field Microscopy
(141) MoDCs were adhered in phenol red-free complete RPMI medium to poly-L-lysine coated glass slides for 2-4 h. Cells were treated with indicated ligands at 37° C. for 45 minutes in the presence of absence of ssON 35 PS. Non-bound ligands were washed away and the cells were covered with complete medium and acquired at 37° C. in a wide-field Cell Observer microscope using the 40× lens. Images were acquired and analyzed using the SlideBook 6 program (Intelligent imaging innovations).
(142) Kinetic Analysis of TLR3 Inhibition
(143) To elucidate the kinetics behind the observed inhibition of TLR3 signalling, moDCs were exposed to 25 μg/ml pI:C/0.5 μM ssON 35 PS and then pulsed with either 0.5 μM ssON 35 PS or pI:C respectively over a time period from 5 minutes to 24 hours. Cells were analyzed by flow cytometry 48 hours post initial exposure for expression of moDC maturation markers CD80 and CD86. Additionally, supernatant was collected from cells 48 hours post treatment and secretion of pro-inflammatory IL-6 was measured by ELISA (Mabtech).
(144) Viability Test
(145) Viable cells were selected from total cell populations 48 hours post initial treatment with 25 μg/ml pI:C/0.5 μM ssON 35 PS. Viable cells were assessed using a Live/Dead fixable near-IR dead cell stain kit according to manufacturer's protocol (Life Technologies). Sample data was acquired using a Fortessa (BD Biosciences) and analysed using FlowJo software (TreeStar Inc.).
(146) MoDC Maturation Studies with dsON
(147) SsON 35 PS was pre-incubated at 5 μM with complementary ssON 35 PO at 1-10 μM in PBS at 55° C. and then left at ambient temperature for 30 minutes. ON mixture was added to MoDC in a final concentration of 0.5 μM ssON 35 PS in combination with 25 μg/ml pI:C. MoDC maturation was assessed as described above.
(148) Animals and Injections
(149) Adult cynomolgus macaques (Macaca fascicularis) (n=18), were handled in accordance with European guidelines for NHP care (EU Directive N 63/2010). This study was approved and accredited under statement number 12-013 by the Ethical Animal Committee of the CEA “Comité d'Ethique en Expérimentation Animale” registered by the French Research Ministry under number 44. Animals were handled under sedation and intradermal injections were done in the upper left and right back flank with 170 μg of pI:C alone or with 170 μg of ssON in 100 μL of PBS, or PBS alone. Skin biopsies (8 mm in diameter) were collected from anesthetized animals 24 hours after injection.
(150) Cytokine Secretion Assays in Macaque
(151) To evaluate cytokine and chemokine production from macaque skin biopsies directly ex vivo, aliquots of filtered-dermis supernatants were collected and measured with the MILLIPLEX MAP NHP Cytokine Magnetic Bead Panel (Millipore) on a Bio-Plex device (Bio-Rad), according to manufacturer's instructions.
(152) Microarray Analysis
(153) Whole skin RNA were extracted from macaque skin biopsies, stored at least 24 hours at 4° C. in RNA Later, using Tissue Ruptor® followed by RNeasy Plus Universal Kit (QIAgen), according to manufacturer's instructions. Total RNA was quality checked on Agilent 2100 Bioanalyzer. RNA quantity was measured using NanoDrop ND-1000 Spectrophotometer. Cyanine-3 (Cy3) labeled cRNA was prepared from 200ng Total RNA using the Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, Calif.). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer. 1.65 μg of Cy3-labelled cRNA was fragmented at 60° C. for 30 minutes in a reaction volume of 55 μL containing 1× Agilent fragmentation buffer and 2× Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 55 μL of 2× Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Rhesus Macaque Gene Expression Microarrays v2 for 17 h at 65° C. in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 min at room temperature with GE Wash Buffer 1 (Agilent) and 1 min with 37° C. GE Wash buffer 2 (Agilent). Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 4×44K array slides (Scan Area 61×21.6 mm, Scan resolution 5 μm, Dye channel is set to Green, PMT is set to 100%). The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. The signals were background correction by the Robust Multi-array Average (RMA) method and quantile-normalized. For the generation of heat maps and cluster analysis, the data was log.sub.2 transformed and mean centered.
(154) Statistical Analysis
(155) Non-parametric Mann-Whitney test was used to compare the presented data. Number of donors and repeated experiments is stated in each figure legend. P-value: not significant (n.s) P>0.05; * P≤0.05; ** P≤0.01; *** P≤0.001.
(156) TABLE-US-00002 TABLE A SEQ ID Med ON Name Sequence Length NO No ssON 35 PO GAAGTTTTGAGGTTTTGAAGTTGTTGGTGGTGGTG 35 2 02 ssON 35 PS G*A*A*G*T*T*T*T*G*A*G*G*T*T*T*T*G*A*A*G*T*T*G*T*T*G*G*T*G*G*T* 35 1 01 ssON 30 PS G*G*T*G 30 3 03 ssON 25 PS A*G*T*T*T*T*G*A*G*G*T*T*T*T*G*A*A*G*T*T*G*T*T*G*G*T*G*G*T*G 25 4 04 ssON 20 PS T*T*T*G*A*G*G*T*T*T*T*G*A*A*G*T*T*G*T*T*G*G*T*G*G 20 5 05 ssON 15 PS T*G*A*G*G*T*T*T*T*G*A*A*G*T*T*G*T*T*G*G 15 6 06 G*G*T*T*T*T*G*A*A*G*T*T*G*T*T ssON GtA PS A*A*A*A*T*T*T*T*A*A*A*A*T*T*T*T*A*A*A*A*T*T*A*T*T*A*A*T*A*A*T* 35 24 024 A*A*T*A ssON Compl PO CACCACCACCAACAACTTCAAAACCTCAAAACTTC 35 7 07 ssON Compl PS C*A*C*C*A*C*C*A*C*C*A*A*C*A*A*C*T*T*C*A*A*A*A*C*C*T*C*A*A*A*A 35 7 07 ssON 2′OMe PS *C*T*T*C 35 8 08 totRNA G*A*A*G*U*U*U*U*G*A*G*G*U*U*U*U*G*A*A*G*U*U*G*U*U*G*G*U*G*G* >200 U*G*G*U*G Random All sequences written 5′ to 3′ * = PS italic = RNA _ = 2′OMe
(157) Experimental Methods for Examples 2-8
(158) Synthesis of Oligonucleotides Synthetic, endotoxin-free, oligonucleotides were synthesized according to methods known in the art, as disclosed in e.g. Artificial DNA: Methods and Applications (Khudyakov, Y. E. & Howard A. Fields, H. A., Eds.) CRC Press, 2002 (ISBN 9780849314261).
(159) Human In Vitro Derived DCs
(160) Monocytes were negatively selected from buffy coats using the RosetteSep™ Monocyte Enrichment Cocktail (StemCell Technologies) and differentiated into DC, as described previously (Sköld, A. E. et al 2012 Blood 120:768-777) at a density of 5×10.sup.5 cells/mL in RPMI 1640 completed with 10% FCS, 1 mM sodium pyruvate, 10 mM HEPES, 2 mM L-glutamine, and 1% streptomycin and penicillin (all from Invitrogen Life Technologies), with GM-CSF (250 ng/mL; PeproTech) and IL-4 (6.5 ng/mL; R&D Systems) for 6 or 7 days. 1×10.sup.5 MoDCs were exposed to Cy3 labeled ploy I:C (polyI:C was labeled using Mirusbio LabelIT® Nucleic Acid Labeling Kit 3600 according to manufacturer's protocol) and MedON oligonucleotides on in 100 μl complete 10% RPMI media, at 37° C. or +4° C. for 45 minutes. After incubation, the cells were washed with ice cold PBS, fixed and uptake was monitored by flow cytometry. Sample data were acquired on a FACSCalibur™ or Fortessa™ (BD Biosciences); the analysis was performed with FlowJo™ software (TreeStar).
(161) Influenza Infection of DCs
(162) Monocyte-derived dendritic cells (MoDCs), differentiated for 6-7 days in the presence of GM-CSF and rIL-4, were pretreated with ssONs (see Table 1; 0.5 μM) for 2 hours and then exposed to different MOI (multiplicity of infection) of influenza H1N1 Cal07/09. MoDCs (500.000/ml) were exposed to influenza at MOIs of 0.2 or 2 for two hours. The cells were thereafter washed and cultured with ssONs (see Table 1; 0.5 μM), for 24 hours. RNA was isolated from the cell culture supernatants and the viral load was measured by QT-PCR. The supernatants were harvested at the indicated time points and kept at −80° C. until RNA purification was performed. QIAamp® Viral RNA mini kit (QIAGEN) was used for viral RNA purification in supernatants, following manufacturer's protocol. Viral load was determined by qRT-PCR (SuperScript® III Platinum® One-Step Quantitative RT-PCR System, Life Technologies) using IAV Cal07/09 as internal standard. Taqman Real-Time PCR using primers and probe specific for IAV Cal07/09 HA segment.
(163) The following primer sequences (5′-3′) specific for IAV HA segment were used:
(164) TABLE-US-00003 (SEQ ID NO: 26) Forward: GGCTGCTTTGAATTTTACCACAA; (SEQ ID NO: 27) Reverse: TTTGGGTAGTCATAAGTCCCATTTT;
as well as the probe FAM-TGCGATAACACGTGCATGGAAAGTGTC-TAMRA (SEQ ID NO: 28)
(165) MoDC Maturation Studies
(166) Human Monocytes were differentiated as described above. MoDCs were exposed to polyI:C and MedONs and maturation was assessed 48 hours later using monoclonal antibodies (Abs) targeting CD1a and the moDC maturation markers CD86 (all Abs from BD Biosciences). Flow cytometry sample data were acquired on a Fortessa (BD Biosciences) and the analysis was performed with FlowJo software (TreeStar).
(167) Cytokine/Chemokine Secretion Studies
(168) Supernatants were collected from cells at given time points post treatment polyI:C (Invivogen) and MedONs. Secretion of IL-6 was measured by standard ELISA according to manufacturer's instructions (Mabtech). Protein amount was monitored by 3,3′,5,5′-Tetramethylbenzidine (TMB) absorbance at 450 nm, and given in pg/ml or relative absorbance units.
(169) In Vivo Influenza a Infection of Mice
(170) BALB/c mice were placed into groups of n=5 and housed in a fully acclimatized room. 5×10.sup.5 PFU influenza H1N1 A/Eng/195/2009 virus in 100 μl were administered nasally to anesthetized mice. In indicated group ssON (25 μg) (MedON-01) were co-administered with the virus. In a third group, ssON (25 μg in 25 μl) in PBS were administered nasally to anesthetized mice day 3 post-virus infection. Mice body weights were monitored every day for productive infection as determined by weight loss. To determine lung viral loads, mice were culled 4 days post infection.
(171) Determination of Lung Viral Titres and Cytokines
(172) After collection of bronchoalveolar lavage (BAL), lungs were removed and homogenized by passage through 100 μm cell strainers, then centrifuged at 200×g for 5 minutes. Supernatants were removed and stored for ELISA analysis and the cell pellet treated with red blood cell lysis buffer (ACK; 0.15 M ammonium chloride, 1 M potassium hydrogen carbonate, and 0.01 mM EDTA, pH 7.2) before centrifugation at 200×g for 5 minutes. The remaining cells were resuspended in RPMI 1640 medium with 10% fetal calf serum, and viable cell numbers determined by trypan blue exclusion.
(173) Viral load in vivo was assessed using Trizol extracted RNA from frozen lung tissue disrupted in a TissueLyzer (Qiagen, Manchester, UK). RNA was converted into cDNA and quantitative RT-PCR was carried out using bulk viral RNA, for the influenza M gene and mRNA using 0.1
(174) TABLE-US-00004 μM forward primer: (SEQ ID NO: 29) 5′-AAGACAAGACCAATYCTGTCACCTCT-3′, 0.1 μM reverse primer: (SEQ ID NO: 30) 5′-TCTACGYTGCAGTCCYCGCT-3′, and 0.2 μM probe: (SEQ ID NO: 31) 5′-FAM-TYACGCTCACCGTGCCCAGTG-TAMRA-3′,
(175) on a Stratagene Mx3005p (Agilent technologies, Santa Clara, Calif., USA). M-specific RNA copy number was determined using an influenza M gene standard plasmid.
(176) Statistical Analysis
(177) Statistical analyses were performed with Prism 5.0™ (Graph-Pad Software Inc.) using nonparametric Kruskal-Wallis unpaired test followed by Dunn's post-test (*P<0.05, **P<0.01 and ***P<0.001) or one-way ANOVA. When indicated different treatment groups were compared using non-parametric Mann-Whitney unpaired tests.
Example 2: MedON-01 Inhibits the Uptake of PolyI:C
(178) Human monocyte-derived cells were treated for 45 min at +37° C. or +4° C. with endocytic, fluorescently labeled, uptake marker (PolyI:C) with or without addition of MedON-01. At +37° C. PolyI:C was taken up as measured by flow cytometry (
Example 3: The Capacity to Inhibit Uptake of PolyI:C is not Strictly Dependent on Sequence but Requires a Certain Length and Chemical Modification of the Oligonucleotide
(179) Human monocyte-derived cells were treated for 45 min at +37° C. or +4° C. with endocytic, fluorescently labeled, uptake marker (PolyI:C) with or without addition of MedON-01-23. At +37° C. PolyI:C was taken up as measured by flow cytometry (
Example 4: Inhibition of Influenza a by Pre-Treatment with MedON-01
(180) Human monocyte derived DCs were infected in vitro with Influenza H1N1 Cal2009 (IAV) using MOI 0.2 or MOI 2. MoDC were either untreated or pre-treated with MedON-01 shown as SEQ ID NO: 1 (0.5 μM) for 2 hours before infection. Infection with IAV was then carried out during another 2 hours. Cells were washed and incubated for 24 hours either with MedON-01 (SEQ ID NO: 1) added again or not (m=medium). The viral load in the culture supernatants were quantified by real time PCR. Data were obtained in four independent experiments using six different donors analyzed in triplicate. Statistical calculations were made using non-parametric paired Wilcoxon text.
(181) The results (
Example 5: The Capacity to Inhibit Influenza a Infection is Sequence Independent but Requires a Certain Length and Chemical Modification of the Oligonucleotide
(182) Human monocyte derived DCs were infected in vitro with Influenza H1N1 Cal2009 (IAV) using MOI 1. MoDC were either untreated or pre-treated with MedON's shown (see
(183) The results (
Example 6: Inhibition of Influenza a Viral Load by MedON-01 Treatment In Vivo
(184) Balb/c mice were infected intranasal with Influenza H1N1 (Cal09). One group were untreated (
(185) As shown in
(186) Mice were weighed every day, which is a sensitive measure of their well-being during virus infections. The mice that received the prophylactic treatment with MedON-01 were able to maintain their weight (
Example 7: TNF-α and IL-6 Production in MedON-01 Treated Influenza A Infected Mice
(187) Balb/c mice were infected intranasally with Influenza H1N1 (Cal09). One group were untreated (
(188) The results (
Example 8: Effect of ssONs on Secretion of IL-6 by MoDCs and Expression of CD86
(189) MoDCs were treated with the TLR3 agonist poly I:C (25 μg/ml) in the absence or presence of 0.5 μM MedONs and incubated at +37° C. 48 h post treatments, the cells were harvested and stained for immuno-maturation marker CD86. MedON-01 and MedON-13-20 are able to inhibit poly I:C mediated immune-maturation. The results are shown in
(190) MoDCs were treated with the TLR3 agonist poly I:C (25 μg/ml) in the absence or presence of 0.5 μM MedONs and incubated at +37° C. 24 h post treatments, the supernatant was collected and assessed for secretion of the pro-inflammatory cytokine IL-6. MedON-01 and MedON-13-20 are able to inhibit poly I:C mediated induction of IL-6. The results are shown in
(191) Further Experimental Data
(192) Experimental Methods for Further Experimental Data
(193) Primary Human Cells
(194) Human Buffy coats were acquired from Karolinska Institutet, Stockholm, Sweden. All experimental work with human peripheral blood cells were carried out in accordance with Swedish guidelines and regulations. All experimental protocols were approved by the local ethical committee in Stockholm (“Regionala etikprövningsnämnden i Stockholm”, Ethical permit Dnr 2006/229-31/3). According to regulations in Sweden, experimental in vitro work with cells from buffy coats does not require informed consent. Human monocytes were generated using the RosetteSep Monocyte Enrichment Kit (1 mL/10 mL buffy coat; StemCell Technologies) and differentiated into moDC, with GM-CSF (250 ng/mL; PeproTech) and IL-4 (6.5 ng/mL; R&D Systems) for 6 days in 37° C., 5% CO2 at a density of 5×105 cells/mL in RPMI 1640 completed with 10% FCS, 1 mM sodium pyruvate, 10 mM HEPES, 2 mM Lglutamine, and 1% streptomycin and penicillin (all from Invitrogen Life Technologies) as previously described (Sköld et al (2012). Differentiation of the cells was monitored by CD1a expression. PBMCs were isolated from buffy coats after Ficoll separation (Stemcell).
(195) Oligonucleotides
(196) Fully PS-substituted ssON 35 PS and 2′OMe PS/PO were made by Integrated DNA Technologies. Other modified oligonucleotides were purchased from Eurofins. For sequences, see Table 2. Total RNA was purified from moDC using RNeasy total RNA purification kit, according to manufacturer's instructions (Qiagen).
(197) TLR Ligands and Labelled Endosomal Markers
(198) TLR3 ligand pI:C (25 μg/mL unless otherwise stated), TLR7 ligand CL307 (1 μg/mL) and TLR7/8 ligand R848 (1 μg/mL) were all purchased from Invivogen. LPS (100 ng/ml) was purchased from Sigma. Transferrin-Alexa647, Dextran-Texas red 10 kDa, and LPS-Alexa488 were all purchased from Molecular probes. Cy3 labelled pI:C was generated using an oligonucleotide Label IT-kit (Mirus), according to manufacturer's instructions.
(199) Uptake Studies in moDCs
(200) MoDC were exposed to endosomal markers, with or without addition of ssON, on ice in complete 10% RPMI media (or serum free media for PO ON uptake studies), and then transferred to 37° C. for 45 min. Cells were washed with cold PBS and fixed (Cytofix, BD Bioscience). Fluorescent signal was monitored by flow cytometry (Fortessa, BD Biosciences). Data were analyzed with FlowJo software (Tree Star, version 9.6.4). CME was monitored by: 25 μg/ml Transferrin-Alexa647, 5 μg/ml LDL-Dil or 1 to 5 μg/ml pI:C-Cy3. MPC: 0.5 mg/ml 10 kDa Dextran-Texas-Red. Endocytic uptake of TLR4: 1 μg LPS-Alexa488. Transfection of pI:C-Cy3 was performed with LyoVec (Invivogen) in a ratio 1:6 according to manufacturer's instructions. For microscopy, moDC were adhered poly-L-lysine coated glass slides for 2-4 h. Cells were treated with ligands at 37° C. for 45 min in the presence or absence of 0.5 μM ssON 35 PS. Cells were washed and acquired at 37° C. in a wide-field Cell Observer microscope (Zeiss) using the 40× lens. Images were acquired and analyzed using the SlideBook 6 program (Intelligent imaging innovations). Alternatively, cells were treated with Poly I:C-Cy3 and Poly I:C-Cy3 with LyoVec the presence or absence of 0.5 μM ssON 35 PS at 37° C. for 40 min, after cell adherence. Cells were washed with PBS and stained with Wheat Germ agglutinin Alexa633 (Invitrogen) for 10 min. Cells were washed prior to fixation in 3.7% Formaldehyde (Sigma). Images were acquired in a LSM800 airy scan confocal microscope (Zeiss) using the 63× oil lens and the images were analyzed using the Zen blue software (Zeiss).
(201) MoDC Maturation Studies
(202) MoDC were exposed to pI:C (25 μg/ml), CL307 (1 μg/ml), R848 (1 μg/ml) LPS (100 ng/ml), or pI:C/LyoVec (1 μg/ml) (Invivogen) with or without the addition of ssON, and assessed 48 h later by flow cytometry using monoclonal antibodies (Abs) targeting CD1a and the moDC maturation markers CD86, CD83, and CD80 (CD1a-Bv510, CD86-APC, CD83-FITC, CD80-PE; all from BD Biosciences). Where chloroquine was used, cells were pre-incubated with 10 μM chloroquine for 1 h at 37° C. Dead cells were excluded using Live/Dead fixable near-IR dead cell stain kit (Life Technologies).
(203) Cytokine/Chemokine Secretion Studies
(204) Supernatants were collected at given time points post treatment and secretion of cytokines/chemokines was measured by standard ELISA according to manufacturer's instructions (Mabtech; IL-6, IL-10, and IL-29. Life Technologies: CXCL10) by a SpectraMax i3x, Molecular devices.
(205) TLR3 Activation Studies in HEK Cells
(206) TLR3 transfected HEK-blue™ cells (Invivogen) were seeded at 20 000 cells/well in 384 well plate (Corning) and left to attach for 1 h. Cells were treated with 1 μg/ml pI:C, specified concentrations of ssON 35 PS and incubated over night. TLR3 activity was measured at 640 nm in HEK-blue™ detection medium (Invivogen) with the Envision Plate Reader (Perklin Elmer), and given as % of pI:C treated cells without ssON 35 PS.
(207) Nuclease Degradation of ssON 35 PO and PS
(208) Nuclease degradation was performed similarly to Ciafre et al. and carried out in serum free RPMI media. DNase I (Qiagen) was used at a ratio of 1 U/μg DNA. The reaction was carried out at 37° C. for 0-24 h at a concentration of 10 μM ssON 35 PO/PS. Aliquots were removed at given time-points, frozen at −80° C. and later analyzed on a 2% agarose gel. Electrophoresis was performed in TBE buffer for 1 h at 100V. The gel was stained with ethidium bromide (0.5 μg/ml) and photographed using a gel doc ez imager (Biorad).
(209) Kinetic Analysis of TLR3 Inhibition
(210) MoDC were exposed to 25 μg/ml pI:C/0.5 μM ssON 35 PS and then pulsed with either 0.5 μM ssON 35 PS or 25 μg/ml pI:C respectively over a time period from 5 min to 24 h. Cells were analyzed by flow cytometry 48 h post initial exposure for expression of maturation markers CD80 and CD86 in CD1a positive cells. Additionally, supernatants were collected 48 h post treatment and IL-6 was measured by ELISA (Mabtech).
(211) Viability Test
(212) Viable cells were assessed using a Live/Dead fixable near-IR dead cell stain kit 48 h post initial treatment with 25 μg/ml pI:C/0.5 μM ssON 35 PS according to manufacturer's protocol (Life Technologies). Data was acquired by Flow cytometry. WST-1 assay was preformed according to the manufacturer's protocol (Sigma) and measured on SpectraMax i3x.
(213) MoDC Maturation Studies with dsON
(214) SsON 35 PS was pre-incubated at 5 μM with complementary ssON 35 PO at 1-10 μM in PBS at 55° C. and then left at ambient temperature for 30 min. ON mixture was added to moDC in a final concentration of 0.5 μM ssON 35 PS in combination with 25 μg/ml pI:C. MoDC maturation was assessed as described above.
(215) RNA Sequencing and Differential Expression Analysis
(216) Total RNA was purified from moDC using RNeasy total RNA purification kit, according to manufacturer's instructions (Qiagen) and submitted to the National Genomics Infrastructure Sweden Stockholm (NGI) for sequencing. Bioanalyzer traces and concentration values were obtained following the guidelines in the Sample requirements for genomics documentation available at the NGI website. The RNA sequencing was performed with the TruSeq RiboZero kit from Illumina.
(217) Mass Spectrometry Based Proteomics Analysis of moDC
(218) 2×8 samples were collected at given timepoints, after moDC stimulation with either pI:C (25 μg/ml) or ssON 35 PS (0.5 μM), and snap frozen. For a complete Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed using an Agilent 1200 nano-LC system coupled online to a Q Exactive Orbitrap (Thermo Fischer Scientific). The software Proteome Discoverer vs.1.4.0.288 including Sequest-Percolator for improved identification (Käll, L., et al (2007)) was used to search the data against the human Ensembl database (version 37) for protein identification (false discovery rate (FDR) of <1%).
(219) Hierarchical Clustering and Network Generation
(220) The analyses were performed using the software Morpheus (https://software.broadinstitute.org/morpheus/index.html) on log 2 transformed data. Network analysis to identify directly interconnected genes within the hierarchical clusters was performed using MetaCore™ version 6.22 (Thomson Reuters), and results displayed using Cytoskape version 3.2.1 (Shannon P. et al (2003).
(221) Animals and Injections
(222) Adult cynomolgus macaques (Macaca fascicularis) (n=18, both females and males), were handled in accordance with European guidelines for NHP care (EU Directive N 63/2010). This study was approved and accredited under statement number 12-013 by the Ethical Animal Committee of the CEA “Comité d'Ethique en Experimentation Animale” registered by the French Research Ministry under number 44. Animals were handled under sedation and intradermal injections were done in the upper left and right back flank with 170 μg of pI:C alone or with 170 μg of ssON 35 PS in 100 μL of PBS, or PBS alone. Alternatively, a dose escalation with ssON 35 PS was performed as indicated in figure legends. Skin biopsies (8 mm in diameter) were collected from anesthetized animals 24 h after injection.
(223) NHP Tissue Collection and Flow Cytometry
(224) Cells were extracted from fresh skin biopsies collected 24 h after injections. Epidermal and dermal cells were stained with a mix of monoclonal antibodies (HLA-DRV500, CD123-PECy7, CD45-V450, CD11c-APC, CD14-APC-H7 from BD Bioscience; CD66-APC, CD66-FITC from Miltenyi; CD1a from DAKO; CD163-PcPCy5.5 from Biolegend) and acquired by flow cytometry.
(225) Histochemistry
(226) Live CD66+CD45+NHP cells were FACS-sorted (FACSAria, BD Biosciences) and put on Superfrost® Plus gold slides (Thermo Scientific) by cytospin and stained using Giemsa according to clinical routine procedures at the Clinical Pathology Laboratory at Karolinska University Hospital Huddinge.
(227) Cytokine Secretion Assays
(228) Aliquots of filtered-dermis supernatants isolated ex vivo, without further stimulation, were measured with the MILLIPLEX MAP NHP Cytokine Magnetic Bead Panel (Millipore, France) on a Bio-Plex device (Bio-Rad, France).
(229) Microarray Analysis
(230) Whole skin RNA was extracted from macaque skin biopsies. For detailed sample preparation and protocol, see supplementary Material and Methods section. RNA was hybridized to Agilent Rhesus Macaque Gene Expression Microarrays v2 for 17 h at 65° C. in a rotating Agilent hybridization oven. Microarrays were washed and scanned on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 4×44K array slides (Scan Area 61×21.6 mm, Scan resolution 5 μm, Dye channel is set to Green, PMT is set to 100%). Images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters to obtain a background adjusted signal (gProcessedSignal) for each gene. The signals were subsequently quantile normalized to increase inter-sample comparability.
(231) Pathway Analysis
(232) Ingenuity Pathway Analysis software (Build version 456367M, Content version 39480507 release date 20170914) (Ingenuity Systems) was used to identify canonical signalling pathways. To calculate significance of enrichment (Fisher's exact test, performed within the software), the reference molecule set was Ingenuity Knowledge Base (Genes only). Three input molecule sets depending on analysis; (i) proteome clusters of proteins with correlating expression (mass spectrometry data, human moDC in vitro) selected based on hierarchical clustering (ii) RNAseq data (human moDC in vitro) filtered by absolute log 2 fold change above 2 and (iii) microarray data from macaque skin biopsies filtered by absolute log 2 fold change above 5. Gene Ontology (GO) enrichment analyses were performed using the Gorilla (Gene Ontology enRIchment anaLysis and visuaLizAtion tool) web tool (Eden E. et al (2009)).
(233) Quantification and Statistical Analysis
(234) Non-parametric Kruskal-Wallis unpaired test followed by Dunn's post-test or Mann-Whitney test was used to compare the presented data in
(235) Further Experimental Data
(236) Length Requirement of at Least 25 Bases for ssON's Capacity to Inhibit TLR3 Activation
(237) CpG motifs in DNA can bind to TLR9, leading to activation of TLR9+ cells such as plasmacytoid dendritic cells and B cells. We previously showed that a CpG containing ssON (35 bases) inhibited TLR3 activation in moDC lacking both TLR7 and TLR9 (Sköld et al (2012)). In the present study, we have exclusively used ssON without CpG motifs to address their capacity to inhibit TLR3 activation. To get further insight as to whether the observed inhibition of endocytosis and downstream TLR3 activation could be linked to a particular ssON sequence, three different sequences were compared (Table 2). SsON GtA 35 PS was based on the parent sequence ssON 35 PS, but all the guanosine (G) bases were substituted for adenosine (A), while ssON Compl 35 PS is the complementary sequence to ssON 35 PS. All displayed the same inhibitory effect in moDC treated with pI:C for 48 h (
(238) We next investigated whether the inhibitory effect is induced by other classes of extracellular nucleic acids, or if it requires ssON. In order to assess if the introduction of double-stranded (ds)DNA influences endocytic uptake, ssON 35 PS was kept at a constant concentration (0.5 μM), while increasing concentrations of a non-stabilized complementary strand (ssON Compl 35 PO) were introduced (
(239) Both ssDNA and ssRNA Inhibit Clathrin-Dependent Endocytic Pathways
(240) To elucidate if the ability to inhibit endocytosis is exclusive for ssDNA, or if ssRNA also possess similar inhibitory capability, we synthesized a 35mer ON, comprised of the non-toxic, modification 2′-O-Methyl RNA (2′OMe) often found in ribosomal RNA and small nuclear RNAs (
(241) No Major Changes in the Transcriptome or Proteome after Inhibition of Clathrin-Mediated Endocytosis in moDC
(242) To gain insight to the cellular response occurring after the shut-down of the clathrin-mediated endocytosis in moDC, we performed combined transcriptomic and proteomic studies after exposure to ssON (
(243) The proteomic analyses similarly showed up-regulation of ISGs, but not inflammasome-related proteins in pI:C-stimulated moDC after 24 h (
(244) Administration of ssON 35 PS Modulates dsRNA-Mediated Inflammation in the Skin of Macaques
(245) To assess whether ssON 35 PS can mediate effects in vivo, we measured the local infiltration of cells and their innate immune signatures after intradermal injection of pI:C in the presence or absence of ssON 35 PS in macaque skin biopsies taken from the site of injection. Multicolor flow cytometry was used to phenotype cells isolated from epidermal and dermal layers (Adam et al (2015) and Epaulard et al (2014)) (
(246) To assess the global innate response to pI:C in the presence or absence of ssON, transcriptional profiling was performed on the skin biopsies. IPA analyses revealed that several pathways for innate immunity, such as “Communication between Innate and Adaptive Immune Cells”, “TREM1 signaling”, “Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses”, and “Crosstalk between Dendritic cells and Natural Killer cells”, were engaged after injection of pI:C (
(247) IFN-γ regulate many chemokines (Antonelli et al (2014)). PI:C injection indeed resulted in increased expression of both IFN-γ and IL-6, while the combined pI:C and ssON treatment reduced this expression in concordance with modulation of chemokine expression (
(248) TABLE-US-00005 TABLE 2 Primary structure of oligonucleotides. Name Sequence Length ssON 35 PO GAAGTTTTGAGGTTITGAAGTTGTTGGTGGTGGTG 35 ssON 35 PS G*A*A*G*T*T*T*T*G*A*G*G*T*T*T*T*G*A*A*G*T*T*G*T*T*G*G*T*G*G*T*G*G*T*G 35 ssON 30 PS A*G*T*T*T*T*G*A*G*G*T*T*T*T*G*A*A*G*T*T*G*T*T*G*G*T*G*G*T*G 30 ssON 25 PS T*T*T*G*A*G*G*T*T*T*T*G*A*A*G*T*T*G*T*T*G*G*T*G*G 25 ssON 20 PS T*G*A*G*G*T*T*T*T*G*A*A*G*T*T*G*T*T*G*G 20 ssON 15 PS G*G*T*T*T*T*G*A*A*G*T*T*G*T*T 15 ssON GtA PS A*A*A*A*T*T*T*T*A*A*A*A*T*T*T*T*A*A*A*A*T*T*A*T*T*A*A*T*A*A*T*A*A*T*A 35 ssON Compl PO CACCACCACCAACAACTICAAAACCTCAAAACTTC 35 ssON Compl PS C*A*C*C*A*C*C*A*C*C*A*A*C*A*A*C*T*T*C*A*A*A′A*C*C*T*C*A*A*A*A*C*T*T*C 35 ssON 2′OMe PO GAAGUUUUGAGGUUUUGARGUUGUUGGUGGUGGUG 35 ssON 2′OMe PS G*A*A*G*U*U*U*U*G*A*G*G*U*U*U*U*G*A*A*G*U*U*G*U*U*G*G*U*G*G*U*G*G*U*G 35 All sequences written 5′ to 3′ * = PS italic = RNA _ = 2′OMe
(249) Discussion of Experimental Findings
(250) Herein, we revealed that certain ssON can abolish uptake of ligands and thereby inhibit the activation of TLR3, 4 and 7 signaling endosomes, which supports and extends our previous publication that a CpG ssON (35 bases) inhibited TLR3 signaling (Sköld et al (2012)). We here also demonstrated that ssON block the uptake of TF, LDL, LPS and pI:C as shown by microscopy and quantified by flow cytometry. Functional inhibition of TLR3, 4 and 7 signaling was shown by measuring cytokine secretion and expression of co-stimulatory molecules from stimulated moDC and PBMC. We further assessed global changes by performing RNAseq and whole cell proteomic analyses, which support the block in endosomal signaling. Characterization of structural requirements for inhibition of clathrin-mediated endocytosis revealed that either ssDNA or ssRNA, but not dsDNA conferred the inhibition. The capability was not strictly dependent on the ssON sequence, but required that it was composed of at least 25 bases to have a full inhibitory effect. Hence, the inhibitory effect was not dependent on CpG motifs. We further provide evidence that ssON 35 PS modulated TLR3 activation in vivo in macaques by measuring local responses in the skin after injection.
(251) The transient shut down of clathrin-mediated endocytosis did not severely hamper the cells. This was supported by the finding that cytokine secretion occurred in the presence of ssON provided that pI:C was taken up prior to the addition of ssON. Further, we show that ssON did not induce any apparent toxicity, and that it can rescue cells from pI:C-induced cell death. RNAseq and whole cell proteomic analyses supported this conclusion and no major changes occurred in the transcriptome or proteome after ssON treatment alone, which was measured for up to 24 h post stimulation. Injection of ssON in macaques did not induce inflammation, instead a dampening of dsRNA (pI:C)-mediated inflammation was observed. We also show that macropinocytosis (measured by uptake of dextran), was not affected by ssON, allowing continued uptake of nutrients in fluid phase.
(252) Our findings that either ssDNA or ssRNA of at least 25 nucleotides have the capacity to temporarily (provided that they are not degraded) shut down clathrin-mediated endocytosis opens up intriguing questions in host-viral interactions, RNA biology and autoimmunity. Our data have implications for 1) viral endocytosis 2) cellular uptake of miRNA 3) role of stabilizing modifications of endogenous RNA 4) immune regulatory function of noncoding RNAs 5) development of ssON based therapeutics (anti-sense ONs, splice switching ONs, immunosuppressive ONs, siRNA, CpG adjuvant).
(253) Altogether, these studies reveal a regulatory role for extracellular ssON in the endocytic uptake of TLR ligands and we have termed this regulatory process SOMIE (SsON-Mediated Interference of Endocytosis).
(254) Infection by pathogens and cellular damages lead to an accumulation of extracellular nucleic acids that can be degraded by RNases and DNases, but also internalized into endosomes for subsequent TLR activation. To avoid excessive endosomal TLR activation under circumstances of heavy load on these systems, it is crucial to tightly control innate immune responses, thereby mitigating potential self-destructive events. An increasing number of molecules such as A20, CYLD, USP4, USP25, OTUB1/2, DUBA, and MYSM1 have been implicated in the dampening of intracellular signaling cascades triggered by PRR (Panda et al (2015)). However, there is a growing appreciation that the effects exerted by these enzymes cannot solely explain the rigorous regulation of PRR activation. Here we demonstrate that ssON, both native ssDNA and ssRNA 2′OMe, temporarily interfere with CME and downstream endosomal TLR activation. SOMIE serves as a regulatory process to temporarily limit activation of the innate immune system. Treatment with stabilized ssON 35 PS displayed a more prolonged block of endocytosis, while the MPC remained unaffected, showing that the inhibitory effect was specific for certain endocytic pathways. Phagocytosis is known to be controlled by both activating (prophagocytic; ‘eat me’) and inhibitory (‘don't eat me’) receptors. The inhibitory signal-regulatory protein (SIRP)α provides a blockade of phagocytosis after binding to the ligand CD47, a checkpoint of phagocytosis (Veilette et al (2018)).
(255) Endocytosis of cargo was quantified by flow cytometry and visualized by microscopy, while functional activation of TLRs were measured by induction of costimulatory molecules CD80 and CD86 in moDC and secretion of cytokines. To reveal if ssON specifically affect activation from TLRs localized in endosomal compartments, we selected TLR agonists with distinct properties and assessed if ssON 35 PS inhibited secretion of IL-6. The TLR7/8 agonist R848 is membrane permeable (Govindaraj et al (2011)) and was not affected by the addition of ssON. In comparison, the activity of the endocytosis-dependent TLR7 agonist CL307 was significantly inhibited by ssON 35 PS. Furthermore, we studied LPS-sensing TLR4 that has two distinct signaling pathways in moDC. SsON 35 PS selectively decreased LPS-mediated secretion of IL-29/CXCL10, which is primarily downstream of TRIF and dependent on endocytosis, while it left the MyD88-dependent IL-6/IL-10 secretion unaffected. It remains to be further elucidated in which cell types TLR4 are coupled to both the Myd88 and TRIF signaling pathways and to reveal potential species differences.
(256) Kinetic analyses revealed that the inhibitory capability of ssON 35 PS decreased if added more than 50 min post stimulation with pI:C and was almost negligible after 2 h. It therefore seems likely that ssON cannot inhibit ongoing TLR3 signaling. When treating moDC in the reverse fashion, starting with a single treatment of ssON 35 PS, pI:C-induced maturation was completely blocked for up to 24 h. PS substituted ssON have a half-life in serum-containing cell culture media that exceed 24 h (Ciafrè et al (1995)). PI:C, on the other hand, is rapidly degraded in serum containing media (Levy et al (1975)) and was not able to trigger TLR3 in moDC after pretreatment with ssON 35 PS. It can be envisioned that the interference with endocytic activity allows moDC to respond to cargo recently taken up, without receiving excessive additional stimulus or contra-orders prior to fulfilling the response to the first endocytosed cargo. In addition, the finding that moDC can mature and produce IL-6 production in the presence of ssON, provided that the cargo (pI:C) was first allowed to be taken up, suggests that a temporal shut-down of clathrin-mediated endocytosis does not severely impair the cell. Transcriptomic and proteomic moDC data provided further evidence that ssON effectively blunted the pI:Cmediated response and that no major changes occurred in ssON treated cells for up to 24 h.
(257) We synthesized a panel of ON (Table 2) to determine the structural requirements for SOMIE. The SOMIE effect was not strictly dependent on the sequence or motif because the complementary strand to ssON 35 PS and a mutated ssON, in which all G were changed to A, showed similar efficacy. The interference with endosomal TLR activation by ssON was potent with an IC50 of about 150 nM. This led us to speculate that we are monitoring a biologically conserved function. However, to our knowledge the source of ssDNA in the cell is limited and primarily found in exosomes (Balaj et al (2011)), while ssRNA on the other hand are abundant. We demonstrated that a 35mer fully PS substituted RNA analogue 2′OMe ssON has the ability to block pI:C-induced moDC maturation and that total RNA (totRNA) inhibited pI:C-induced secretion of IL-6. Taken together, the SOMIE activity is not strictly dependent on ssON sequence, can occur with both ssDNA and ssRNA, and stability modifications (i.e. PS) are not essential for inhibition, but needed for retained activity over time. It remains to be further elucidated whether the metabolism of oligonucleotides and differential addition of stabilizing base modifications, such as 2′OMe or locked nucleic acid (LNA), can influence susceptibility to autoimmunity by setting the threshold of endosomal TLR activation.
(258) The mammalian cell contains a plethora of both coding and non-coding ONs with varying lengths (Cech et al (2014)). Immuno-regulatory micro RNAs (miRNA) consist of 18-23 bases ssON (Mehta et al (2016)). However, cells also contain ssON that are longer in size (Cech et al (2014)). During cell death with release of cellular content, it is conceivable that both coding and non-coding DNA and RNA of varying length can be released into the extracellular space, becoming accessible to degradation by DNases and RNases. It can be postulated that the activity of these enzymes will greatly affect the size composition of ONs in the extracellular space, and ultimately affect triggering of nucleic acid sensing PRR. We here show that there is a length requirement of at least 20-25 bases to exert SOMIE activity. SsON (25-35 bases) inhibited uptake of shorter ssON (15 bases), while ssON 15 PS did not display SOMIE activity. This is consistent with recent data suggesting that miRNA (18-23 bases) released into the circulation and extracellular space during ischemia may instead trigger cytokine production via endosomal TLR7/MyD88 signaling (Feng et al (2017)). The revealed length requirement for SOMIE allude to an unknown function of longer non-coding RNAs and shows that miRNA are too short to provide endocytic checkpoint control.
(259) Our finding that ssON of at least 20-25 bases interfere with endocytosis provides a rational as to why ssON used as antisense, or exon skipping ssON with gymnotic delivery, should be shorter than 20 bases to allow cellular uptake through endocytic pathways. Similarly, CpG ssON aimed for TLR9 agonistic actions should be less than 20 bases (Scheiermann et al (2014)), to avoid inhibition of endocytosis, which may inadvertently cause off-target effects in cells lacking TLR9 (Sköld et al (2012). It is conceivable that the size and the relative ds:ss composition of ON in the extracellular space will depend on the local microbial milieu, with an overflow of ON during infections. To our knowledge, there is currently no technique available to enable measurement of the intracellular or extracellular concentrations of naturally occurring ON with varying length. Hence, to what extent and the timeframes for the observed ssON-mediated TLR inhibition occurring by natural ligands cannot be truly estimated at the present time. Nevertheless, we provide evidence that ssON 35 PS exerted immunomodulatory effects in vivo after intradermal injection in macaques. Skin biopsies showed dampened pI:C-mediated inflammation in the dermis of macaques after injection of ssON and reduced secretion of IL-6 was confirmed in supernatants obtained from skin biopsies ex vivo (without additional in vitro stimulation). Notably, ssON induced dose-dependent IL-10 secretion in vivo, suggesting that inoculation with ssON may shift the direction of local immune responses. It remains to be elucidate which cell type(s) in the skin produce IL-10 after ssON treatment. We did not find any signs of IL-10 production in moDC in the genomic and proteomic studies after stimulation with ssON. We hypothesize that the local environment and cellular cross-talk might be pivotal for induction of IL-10 and a wide range of cell populations can be induced to produce human IL-10, including T cells (Tregs, Th1, Th2, and Th17), subsets of DC, macrophages, neutrophils, B cells, mast cells, fibroblasts, and keratinocytes (Berti et a; (2017)).
(260) Immunosuppressive ssON have been suggested to dampen or prevent diseases characterized by pathologic immune stimulation and autoimmunity (Dong et al (2004); Klinman et al (2005); Peter et al (2008)). Several immunosuppressive ssON were previously shown to contain sequences or motifs required for direct binding, to for example, TLR7 or TLR9 (Bayik et al (2016) and Sarvestani et al (2015)), or repeats (TTAGGG) that interact with STAT1 and STAT4 (Shirota et al (2015)). We, and others, have previously suggested that TLR3 signaling can be inhibited by ssON in order to dampen over-reactive responses contributing to TLR3 linked pathogenesis (Skold et al (2012) and Ranjith-Kumar et al (2008)). Here we provide evidence that ssON might have a broader immune regulatory role than previously anticipated by acting as gatekeepers for uptake of cargo into endosomes, thereby providing a fluctuating threshold for potential autoimmune reactions triggered by endosomal TLR3/4/7 activation.
REFERENCES
(261) Adam, L., Rosenbaum, P., Cosma, A., Le Grand, R. & Martinon, F. Identification of skin immune cells in non-human primates. J. Immunol. Methods 426, 42-49 (2015). Akira, S. & Takeda, K. Toll-like receptor signalling. Nat. Rev. Immunol. 4, 499-511 (2004). Amarante, M. K., Oda, J. M. M., Reiche, E. M. V., Morimoto, H. K., Aoki, M. N., Watanabe, M. A. E., 2011. Human endogenous RNAs: Implications for the immunomodulation of Toll-like receptor 3. Exp Ther Med 2, 925-929. doi:10.3892/etm.2011.303 Amarante, M. K., Watanabe, M. A. E., 2010. Toll-like receptor 3: involvement with exogenous and endogenous RNA. Int. Rev. Immunol. 29, 557-573. doi:10.3109/08830185.2010.525723 Amit, I. et al. Unbiased reconstruction of a mammalian transcriptional network mediating pathogen responses. Science 326, 257-263 (2009). Antonelli, A. et al. Chemokine (C-X-C motif) ligand (CXCL) 10 in autoimmune diseases. Autoimmun Rev 13, 272-280 (2014). Balaj, L., Lessard, R., Dai, L., Cho, Y.-J., Pomeroy, S. L., Breakefield, X. O., Skog, J., 2011. Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences. Nature Communications 2, 180. doi:10.1038/ncomms1180 Barouch, D. H. et al. Rapid Inflammasome Activation following Mucosal SIV Infection of Rhesus Monkeys. Cell 165, 656-667 (2016). Bayik, D., Gursel, I., Klinman, D. M., 2016. Structure, mechanism and therapeutic utility of immunosuppressive oligonucleotides. Pharmacol. Res. 105, 216-225. doi:10.1016/j.phrs.2015.11.010 Bennett, R. L. et al. RAX, the PKR activator, sensitizes cells to inflammatory cytokines, serum withdrawal, chemotherapy, and viral infection. Blood 108, 821-829 (2006). Berti, F. C. B., Pereira, A. P. L., Cebinelli, G. C. M., Trugilo, K. P. & Brajão de Oliveira, K. The role of interleukin 10 in human papilloma virus infection and progression to cervical carcinoma. Cytokine Growth Factor Rev. 34, 1-13 (2017). Cavassani, K. A. et al. TLR3 is an endogenous sensor of tissue necrosis during acute inflammatory events. J. Exp. Med. 205, 2609-2621 (2008). Cech, T. R. & Steitz, J. A. The noncoding RNA revolution-trashing old rules to forge new ones. Cell 157, 77-94 (2014). Choi, C. H. J., Hao, L., Narayan, S. P., Auyeung, E., Mirkin, C. A., 2013. Mechanism for the endocytosis of spherical nucleic acid nanoparticle conjugates. PNAS 110, 7625-7630. doi:10.1073/pnas.1305804110 Ciafrè, S. A., Rinaldi, M., Gasparini, P., Seripa, D., Bisceglia, L., Zelante, L., Farace, M. G., Fazio, V. M., 1995. Stability and functional effectiveness of phosphorothioate modified duplex DNA and synthetic ‘mini-genes’. Nucleic Acids Res. 23, 4134-4142. Cossart, P., Helenius, A., 2014. Endocytosis of viruses and bacteria. Cold Spring Harb Perspect Biol 6, a016972-a016972. doi:10.1101/cshperspect.a016972 de Bouteiller, O. et al. Recognition of double-stranded RNA by human toll-like receptor 3 and downstream receptor signaling requires multimerization and an acidic pH. J Biol Chem. 280, 38133-38145 (2005). Doherty, G. J., McMahon, H. T., 2009. Mechanisms of endocytosis. Annu. Rev. Biochem. 78, 857-902. doi:10.1146/annurev.biochem.78.081307.110540 Dong, L., Ito, S.-I., Ishii, K. J., Klinman, D. M., 2004. Suppressive oligonucleotides protect against collagen-induced arthritis in mice. Arthritis Rheum. 50, 1686-1689. doi:10.1002/art.20263 Duan, Y., Zhang, S., Wang, B., Yang, B. & Zhi, D. The biological routes of gene delivery mediated by lipid-based non-viral vectors. Expert Opin Drug Deliv 6, 1351-1361 (2009). Eden, E., Navon, R., Steinfeld, I., Lipson, D. & Yakhini, Z. GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists. BMC Bioinformatics 10, 48 (2009). Epaulard, O. et al. Macrophage- and neutrophil-derived TNF-α instructs skin langerhans cells to prime antiviral immune responses. J. Immunol. 193, 2416-2426 (2014). Feng, Y. et al. Extracellular MicroRNAs Induce Potent Innate Immune Responses via TLR7/MyD88-Dependent Mechanisms. J. Immunol. 199, 2106-2117 (2017). Gitlin, L. et al. Essential role of mda-5 in type I IFN responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus. Proc. Natl. Acad. Sci. 103, 8459-8464 (2006). Govindaraj, R. G., Manavalan, B., Basith, S., Choi, S., 2011. Comparative analysis of species-specific ligand recognition in Toll-like receptor 8 signaling: a hypothesis. PLoS ONE 6, e25118. doi:10.1371/journal.pone.0025118 Gursel, I., Gursel, M., Yamada, H., Ishii, K. J., Takeshita, F., Klinman, D. M., 2003. Repetitive elements in mammalian telomeres suppress bacterial DNA-induced immune activation. The Journal of Immunology 171, 1393-1400. Husebye, H. et al. Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity. EMBO J. 25, 683-692 (2006). Irannejad, R., Zastrow, von, M., 2014. GPCR signaling along the endocytic pathway. Curr. Opin. Cell Biol. 27, 109-116. doi:10.1016/j.ceb.2013.10.003 Isomura, I., Yasuda, Y., Tsujimura, K., Takahashi, T., Tochikubo, K., Morita, A., 2005. Recombinant cholera toxin B subunit activates dendritic cells and enhances antitumor immunity. Microbiol. Immunol. 49, 79-87. Itoh, K., Watanabe, A., Funami, K., Seya, T., Matsumoto, M., 2008. The clathrin-mediated endocytic pathway participates in dsRNA-induced IFN-beta production. J. Immunol. 181, 5522-5529. Juliano, R. L., Carver, K., 2015. Cellular uptake and intracellular trafficking of oligonucleotides. Advanced Drug Delivery Reviews 87, 35-45. doi:10.1016/j.addr.2015.04.005 Kagan, J. C., Su, T., Horng, T., Chow, A., Akira, S., Medzhitov, R., 2008. TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-beta. Nature Publishing Group 9, 361-368. doi:10.1038/ni1569 Kato, H. et al. Cell type-specific involvement of RIG-I in antiviral response. Immunity 23, 19-28 (2005). Kawai, T., Akira, S., 2010. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nature Publishing Group 11, 373-384. doi:10.1038/ni.1863 Käll, L., Canterbury, J. D., Weston, J., Noble, W. S. & MacCoss, M. J. Semisupervised learning for peptide identification from shotgun proteomics datasets. Nat. Methods 4, 923-925 (2007). Klein, D. C. G. et al. CD14, TLR4 and TRAM Show Different Trafficking Dynamics During LPS Stimulation. Traffic 16, 677-690 (2015). Klinman, D. M., Gursel, I., Klaschik, S., Dong, L., Currie, D., Shirota, H., 2005. Therapeutic potential of oligonucleotides expressing immunosuppressive TTAGGG motifs. Ann. N. Y. Acad. Sci. 1058, 87-95. doi:10.1196/annals.1359.015 Kumari, S., Mg, S., Mayor, S., 2010. Endocytosis unplugged: multiple ways to enter the cell. Cell Res. 20, 256-275. doi:10.1038/cr.2010.19 Levy, H. B., Baer, G., Baron, S., Buckler, C. E., Gibbs, C. J., Iadarola, M. J., London, W. T., Rice, J., 1975. A modified polyriboinosinic-polyribocytidylic acid complex that induces interferon in primates. J. Infect. Dis. 132, 434-439. Matsumoto, M., Oshiumi, H., Seya, T., 2011. Antiviral responses induced by the TLR3 pathway. Rev. Med. Virol. 21, 67-77. doi:10.1002/rmv.680 Mehta, A., Baltimore, D., 2016. MicroRNAs as regulatory elements in immune system logic. Nat Rev Immunol 16, 279-294. doi:10.1038/nri.2016.40 Merad, M., Sathe, P., Helft, J., Miller, J., Martha, A., 2013. The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annu. Rev. Immunol. 31, 563-604. doi:10.1146/annurev-immunol-020711-074950 Panda, S., Nilsson, J. A. & Gekara, N. O. Deubiquitinase MYSM1 Regulates Innate Immunity through Inactivation of TRAF3 and TRAF6 Complexes. Immunity 43, 647-659 (2015). Pandey, S., Kawai, T. & Akira, S. Microbial sensing by Toll-like receptors and intracellular nucleic acid sensors. Cold Spring Harb Perspect Biol 7, a016246 (2014). Pelka, K., Shibata, T., Miyake, K., Latz, E., 2016. Nucleic acid-sensing TLRs and autoimmunity: novel insights from structural and cell biology. Immunol. Rev. 269, 60-75. doi:10.1111/imr.12375 Peter, M., Bode, K., Lipford, G. B., Eberle, F., Heeg, K., Dalpke, A. H., 2008. Characterization of suppressive oligodeoxynucleotides that inhibit Toll-like receptor-9-mediated activation of innate immunity. Immunology 123, 118-128. doi:10.1111/.1365-2567.2007.02718.x Politz, J. C., Taneja, K. L., Singer, R. H., 1995. Characterization of hybridization between synthetic oligodeoxynucleotides and RNA in living cells. Nucleic Acids Res. 23, 4946-4953. Rajaiah, R., Perkins, D. J., Ireland, D. D. C., Vogel, S. N., 2015. CD14 dependence of TLR4 endocytosis and TRIF signaling displays ligand specificity and is dissociable in endotoxin tolerance. PNAS 112, 8391-8396. doi:10.1073/pnas.1424980112 Ranjith-Kumar, C. T., Duffy, K. E., Jordan, J. L., Eaton-Bassiri, A., Vaughan, R., Hoose, S. A., Lamb, R. J., Sarisky, R. T., Kao, C. C., 2008. Single-stranded oligonucleotides can inhibit cytokine production induced by human toll-like receptor 3. Mol. Cell. Biol. 28, 4507-4519. doi:10.1128/MCB.00308-08 Roers, A., Hiller, B., Hornung, V., 2016. Recognition of Endogenous Nucleic Acids by the Innate Immune System. Immunity 44, 739-754. doi:10.1016/j.immuni.2016.04.002 Rosadini, C. V., Zanoni, I., Odendall, C., Green, E. R., Paczosa, M. K., Philip, N. H., Brodsky, I. E., Mecsas, J., Kagan, J. C., 2015. A Single Bacterial Immune Evasion Strategy Dismantles Both MyD88 and TRIF Signaling Pathways Downstream of TLR4. Cell Host and Microbe 18, 682-693. doi:10.1016/j.chom.2015.11.006 Sacre, S., Lo, A., Gregory, B., Stephens, M., Chamberlain, G., Stott, P., Brennan, F., 2015. Oligodeoxynucleotide inhibition of Toll-like receptors 3, 7, 8 and 9 suppresses cytokine production in a human rheumatoid arthritis model. Eur. J. Immunol. n/a-n/a. doi:10.1002/eji.201546123 Sarvestani, S. T. et al. Sequence-dependent off-target inhibition of TLR7/8 sensing by synthetic microRNA inhibitors. Nucleic Acids Res. 43, 1177-1188 (2015). Scita, G., Di Fiore, P. P., 2010. The endocytic matrix. Nature 463, 464-473. doi:10.1038/nature08910 Scheiermann, J. & Klinman, D. M. Clinical evaluation of CpG oligonucleotides as adjuvants for vaccines targeting infectious diseases and cancer. Vaccine 32, 6377-6389 (2014). Shalek, A. K. et al. Single-cell RNA-seq reveals dynamic paracrine control of cellular variation. Nature 510, 363-369 (2014). Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498-2504 (2003). Sharma, V. K., Watts, J. K., 2015. Oligonucleotide therapeutics: chemistry, delivery and clinical progress. Future Med Chem 7, 2221-2242. doi:10.4155/fmc.15.144 Shirota, H., Gursel, I., Gursel, M., Klinman, D. M., 2005. Suppressive Oligodeoxynucleotides Protect Mice from Lethal Endotoxic Shock. The Journal of Immunology 174, 4579-4583. doi:10.4049/jimmunol.174.8.4579 Silva, A. C., Lopes, C. M., Sousa Lobo, J. M., Amaral, M. H., 2015. Nucleic Acids Delivery Systems: A Challenge for Pharmaceutical Technologists. Curr. Drug Metab. 16, 3-16. Sköld, A. E., Hasan, M., Vargas, L., Saidi, H., Bosquet, N., Le Grand, R., Smith, C. I. E., Spetz, A.-L., 2012. Single-stranded DNA oligonucleotides inhibit TLR3-mediated responses in human monocyte-derived dendritic cells and in vivo in cynomolgus macaques. Blood 120, 768-777. doi:10.1182/blood-2011-12-397778 Smole, U., Radauer, C., Lengger, N., Svoboda, M., Rigby, N., Bublin, M., Gaier, S., Hoffmann-Sommergruber, K., Jensen-Jarolim, E., Mechtcheriakova, D., Breiteneder, H., 2015. The major birch pollen allergen Bet v 1 induces different responses in dendritic cells of birch pollen allergic and healthy individuals. PLoS ONE 10, e0117904. doi:10.1371/journal.pone.0117904 Soifer, H. S., Koch, T., Lai, J., Hansen, B., Hoeg, A., Oerum, H., Stein, C. A., 2012. Silencing of gene expression by gymnotic delivery of antisense oligonucleotides. Methods Mol. Biol. 815, 333-346. doi:10.1007/978-1-61779-424-7_25 Sorkin, A., Zastrow, von, M., 2009. Endocytosis and signalling: intertwining molecular networks. Nat. Rev. Mol. Cell Biol. 10, 609-622. doi:10.1038/nrm2748 Soulet, D., Covassin, L., Kaouass, M., Charest-Gaudreault, R., Audette, M., Poulin, R., 2002. Role of endocytosis in the internalization of spermidine-C(2)-BODIPY, a highly fluorescent probe of polyamine transport. Biochem. J. 367, 347-357. doi:10.1042/BJ20020764 Stein, C. A., Hansen, J. B., Lai, J., Wu, S., Voskresenskiy, A., Hog, A., Worm, J., Hedtjarn, M., Souleimanian, N., Miller, P., Soifer, H. S., Castanotto, D., Benimetskaya, L., (Drum, H., Koch, T., 2010. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. Nucleic Acids Res. 38, e3-e3. doi:10.1093/nar/gkp841 Sun, R., Zhang, Y., Lv, Q., Liu, B., Jin, M., Zhang, W., He, Q., Deng, M., Liu, X., Li, G., Li, Y., Zhou, G., Xie, P., Xie, X., Hu, J., Duan, Z., 2011. Toll-like receptor 3 (TLR3) induces apoptosis via death receptors and mitochondria by up-regulating the transactivating p63 isoform alpha (TAP63alpha). J. Biol. Chem. 286, 15918-15928. doi:10.1074/jbc.M110.178798 Takeda, K., Kaisho, T. & Akira, S. Toll-like receptors. Annu. Rev. Immunol. 21, 335-376 (2003). Tatematsu, M., Yoshida, R., Morioka, Y., Ishii, N., Funami, K., Watanabe, A., Saeki, K., Seya, T., Matsumoto, M., 2016. Raftlin Controls Lipopolysaccharide-Induced TLR4 Internalization and TICAM-1 Signaling in a Cell Type-Specific Manner. J. Immunol. 196, 3865-3876. doi:10.4049/jimmunol.1501734 Veillette, A. & Chen, J. SIRPα-CD47 Immune Checkpoint Blockade in Anticancer Therapy. Trends in Immunology 39, 173-184 (2018). Vercammen, E., Staal, J., Beyaert, R., 2008. Sensing of viral infection and activation of innate immunity by toll-like receptor 3. Clin. Microbiol. Rev. 21, 13-25. doi:10.1128/CMR.00022-07 Weber, C., Müller, C., Podszuweit, A., Montino, C., Vollmer, J., Forsbach, A., 2012. Toll-like receptor (TLR) 3 immune modulation by unformulated small interfering RNA or DNA and the role of CD14 (in TLR-mediated effects). Immunology 136, 64-77. doi:10.1111/j.365-2567.2012.03559.x Zhu, F.-G., Reich, C. F., Pisetsky, D. S., 2002. Inhibition of murine macrophage nitric oxide production by synthetic oligonucleotides. J. Leukoc. Biol. 71, 686-694.
(262) TABLE-US-00006 TABLE 1 Primary structure of oligonucleotides. All sequences are written 5′ to 3′. All oligonucleotides were fully phosphorothioated (except MedON-02, 22, 23) and consist of deoxynucleotides (except MedON-08). Name MedON# Sequence (3′-5′) * = PS _ = 2′Ome ssON 35 PS MedON01 G*A*A*G*T*T*T*T*G*A*G*G*T*T*T*T*G*A*A*G*T*T*G*T*T*G*G*T*G*G*T*G*G*T*G ssON 35 PO MedON02 GAAGTTTTGAGGTTTTGAAGTTGTTGGTGGTGGTG ssON 30 PS MedON03 A*G*T*T*T*T*G*A*G*G*T*T*T*T*G*A*A*G*T*T*G*T*T*G*G*T*G*G*T*G ssON 25 PS MedON04 T*T*T*G*A*G*G*T*T*T*T*G*A*A*G*T*T*G*T*T*G*G*T*G*G ssON 20 PS MedON05 T*G*A*G*G*T*T*T*T*G*A*A*G*T*T*A*T*T*G*G ssON 15 PS MedON06 G*G*T*T*T*T*G*A*A*G*T*T*G*T*T ssON 35 PS MedON07 C*A*C*C*A*C*C*A*C*C*A*A*C*A*A*C*T*T*C*A*A*A*A*C*C*T*C*A*A*A*A*C*T*T*C Complementary ssON 35 2′OMe PS MedON08 G*A*A*G*U*U*U*U*G*A*G*G*U*U*U*U*G*A*A*G*U*U*G*U*U*G*G*U*G*G*U*G*G*U*G ssON A-rich PS MedON09 A*A*A*A*T*A*A*A*A*T*A*A*A*A*T*A*A*A*A*T*A*A*A*A*T*A*A*A*A*T*A*A*A*A*T ssON T-rich PS MedON10 T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T ssON C-rich PS MedON11 C*C*C*C*T*C*C*C*C*T*C*C*C*C*T*C*C*C*C*T*C*C*C*C*T*C*C*C*C*T*C*C*C*C*T ssON C-rich PS MedON12 G*G*G*A*A*G*G*G*A*A*G*G*G*A*A*G*G*G*A*A*G*G*G*A*A*G*G*G*A*A*G*G*G*A*A G-ssON 35 0% PS MedON13 C*A*C*T*C*C*A*C*T*C*A*A*T*C*C*A*T*C*A*C*C*A*C*A*C*T*C*T*C*T*C*A*T*C*T G-ssON 35 8% PS MedON14 G*A*C*T*C*C*A*C*T*C*A*A*T*C*C*A*T*C*A*C*C*A*C*A*C*T*C*T*G*T*G*A*T*C*T G-ssON 35 15% PS MedON15 G*A*C*T*C*C*A*C*T*G*A*A*T*C*C*A*T*C*A*C*C*A*C*A*G*T*C*T*G*T*G*A*T*C*T G-ssON 35 23% PS MedON16 G*A*G*T*C*C*A*C*T*G*A*A*T*C*C*A*T*G*A*C*C*A*C*A*G*T*C*T*G*T*G*A*T*G*T G-ssON 35 29% PS MedON17 G*A*G*T*C*C*A*G*T*G*A*A*T*C*C*A*T*G*A*C*C*A*G*A*G*T*C*T*G*T*G*A*T*G*T G-ssON 35 37% PS MedON18 G*A*G*T*C*C*A*G*T*G*A*A*T*G*G*A*T*G*A*C*C*A*A*G*G*T*G*T*G*T*G*A*T*G*T G-ssON 35 49% PS MedON19 G*A*G*T*G*G*A*G*T*G*A*A*T*G*G*A*T*G*A*G*G*A*G*A*G*T*G*T*G*T*G*A*T*G*T ssON 35 AT PS MedON020 A*A*T*T*A*A*T*T*A*T*A*A*T*A*T*A*T*A*T*A*A*T*A*T*T*A*A*T*A*A*T*A*A*T*A ssON 70 PS MedON21 G*A*A*G*T*T*T*T*G*A*G*G*T*T*T*T*G*A*A*G*T*T*G*T*T*G*G*T*G*G*T*G*G*T*G* ssON 35 3ePS MedON22 G*A*A*G*T*T*T*T*G*A*G*G*T*T*T*T*G*A*A*G*T*T*G*T*T*G*G*T*G*G*T*G*G*T*G G*A*A*GTTTTGAGGTTTTGAAGTTGTTGGTGGTG*G*T*G ssON 35 3e0Me MedON23 G*A*A*GTTTTGAGGTTTTGAAGTTGTTGGTGGTG*G*T*G ssON GtA PS MedON24 A*A*A*A*T*T*T*T*A*A*A*A*T*T*T*T*A*A*A*A*T*T*A*T*T*A*A*T*A*A*T*A*A*T*A Name Length Comment SEQ ID No ssON 35 PS 35 Also named: non- 1 CpG ssON 35 PO 35 2 ssON 30 PS 30 3 ssON 25 PS 25 4 ssON 20 PS 20 5 ssON 15 PS 15 6 ssON 35 PS 35 7 Complementary ssON 35 2′OMe PS 35 RNA analogue 8 ssON A-rich PS 35 9 ssON T-rich PS 35 10 ssON C-rich PS 35 11 ssON C-rich PS 35 12 G-ssON 35 0% PS 35 13 G-ssON 35 8% PS 35 14 G-ssON 35 15% PS 35 15 G-ssON 35 23% PS 35 Also named: 16 generic ssON 35 PS G-ssON 35 29% PS 35 17 G-ssON 35 37% PS 35 18 G-ssON 35 49% PS 35 19 ssON 35 AT PS 35 20 ssON 70 PS 70 double ssON 3 PS 21 ssON 35 3ePS 35 22 ssON 35 3e0Me 35 23 ssON GtA PS 35 24