esiRNA Research Articles

CEPT1-Mediated Phospholipogenesis Regulates Endothelial Cell Function and Ischemia-Induced Angiogenesis Through PPARα.

De novo phospholipogenesis, mediated by choline-ethanolamine phosphotransferase 1 (CEPT1), is essential for phospholipid activation of transcription factors such as peroxisome proliferator-activated receptor α (PPARα) in the liver. Fenofibrate, a PPARα agonist and lipid-lowering agent, decreases amputation incidence in patients with diabetes. Because we previously observed that CEPT1 is elevated in carotid plaque of patients with diabetes, we evaluated the role of CEPT1 in peripheral arteries and PPARα phosphorylation (Ser12). CEPT1 was found to be elevated in diseased lower-extremity arterial intima of individuals with peripheral arterial disease and diabetes. To evaluate the role of Cept1 in the endothelium, we engineered a conditional endothelial cell (EC)-specific deletion of Cept1 via induced VE-cadherin-CreERT2-mediated recombination (Cept1Lp/LpCre +). Cept1Lp/LpCre + ECs demonstrated decreased proliferation, migration, and tubule formation, and Cept1Lp/LpCre + mice had reduced perfusion and angiogenesis in ischemic hind limbs. Peripheral ischemic recovery and PPARα signaling were further compromised by streptozotocin-induced diabetes and ameliorated by feeding fenofibrate. Cept1 endoribonuclease-prepared siRNA decreased PPARα phosphorylation in ECs, which was rescued with fenofibrate but not PC16:0/18:1. Unlike Cept1Lp/LpCre + mice, Cept1Lp/LpCre + Ppara -/- mice did not demonstrate hind-paw perfusion recovery after feeding fenofibrate. Therefore, we demonstrate that CEPT1 is essential for EC function and tissue recovery after ischemia and that fenofibrate rescues CEPT1-mediated activation of PPARα.

Combination of chemotherapy and physical plasma elicits melanoma cell death via upregulation of SLC22A16

Malignant melanoma is an aggressive cancer that develops drug resistance leading to poor prognosis. Efficient delivery of chemotherapeutic drugs to the tumor tissue remains a major challenge in treatment regimens. Using murine (B16) and human (SK-MEL-28) melanoma cells, we investigated traditional cytotoxic agents in combination with cold physical plasma-derived oxidants. We report synergistic cytotoxicity of doxorubicin and epirubicin, and additive toxicity of oxaliplatin with plasma exposure in coefficient of drug interaction analysis. The combination treatment led to an increased DNA damage response (increased phosphorylation of ATM, γ-H2AX foci, and micronuclei formation). There was also an enhanced secretion of immunogenic cell death markers ATP and CXCL10 in cell culture supernatants following combination treatment. The observed synergistic effects in tumor cells was due to enhanced intracellular doxorubicin accumulation via upregulation of the organic cationic transporter SLC22A16 by plasma treatment. The doxorubicin uptake was reversed by pretreating cells with antioxidants or calcium influx inhibitor BTP2. Endoribonuclease-prepared siRNAs (esiRNA)-mediated knockdown of SLC22A16 inhibited the additive cytotoxic effect in tumor cells. SK-MEL 28 and THP-1 monocytes co-culture led to greater THP-1 cell migration and SK-MEL-28 cytotoxicity when compared with controls. Taken together, we propose pro-oxidant treatment modalities to sensitize chemoresistant melanoma cells towards subsequent chemotherapy, which may serve as therapeutic strategy in combination treatment in oncology.

Downregulation of PIK3CA via antibody-esiRNA-complexes suppresses human xenograft tumor growth.

Precision cancer therapy requires on the one hand detailed knowledge about a tumor’s driver oncogenes and on the other hand an effective targeted therapy that specifically inhibits these oncogenes. While the determination of genomic landscape of a tumor has reached a very precise level, the respective therapy options are scarce. The application of small inhibitory (si) RNAs is a promising field of investigation. Here, we present the effective in vivo-treatment of colorectal cancer (CRC) xenograft tumors with antibody-complexed, endoribonuclease-prepared small inhibitory (esi)RNAs. We chose heterogeneous endoribonuclease-prepared siRNA pools (esiRNAs) against the frequently mutated genes PIK3CA and KRAS and coupled them to the anti-EGFR antibody cetuximab, which was internalized specifically into the tumor cells. esiRNA pools have been shown to exhibit superior specificity in target gene knockdown compared to classic siRNAs. We identified a significant decrease in tumor growth upon this treatment due to decreased tumor cell proliferation. The ex vivo-analysis of the xenograft CRC tumors revealed the expected downregulation of the intended direct targets PIK3CA and KRAS on protein level. Moreover, known downstream targets for EGFR signaling such as p-ERK, p-AKT, and c-MYC were decreased as well. We therefore propose the use of antibody-esiRNA complexes as a novel experimental treatment option against key components of the EGFR signaling cascade.

PO-130 β8 integrin critically contributes to pancreatic cancer cell radiochemoresistance and intracellular vesicle trafficking under stress conditions

Introduction Pancreatic ductal adenocarcinoma (PDAC) is one of the five most lethal malignancies in the world and has a 5 year relative overall survival rate of less than 5%. Thus, there is a great need for functional targeting strategies. As cell-matrix adhesion is essential for the survival, invasion and therapy resistance, we sought to identify the function of 117 focal adhesion proteins (FAP) in PDAC cell radiochemoresistance. Intriguingly, β8 integrin turned out to be one of the most potential novel targets in PDAC. Material and methods We performed a 3D endoribonuclease-prepared siRNA (esiRNA)-based high throughput screening (3DHTesiS) in PDAC cell cultures (established and patient-derived (PDC)) grown in laminin-rich extracellular matrix (IrECM). In addition to characterising β8 integrin expression, distribution and co-localization with other cellular organelles such as golgi apparatus, clonogenic survival assays were performed upon esiRNA-mediated knockdown, X-ray irradiation (6 Gy single dose) and gemcitabine. Fiji software was used to determine Peason’s correlation coefficient, vesicle distribution and expression patterns upon irradiation or gemcitabine. An inhibitor screen was conducted to identify pathway involved in changes of β8 integrin localization upon treatment. Results and discussions We identified a series of novel targets including β8 integrin. Without cytotoxicity, β8 integrin depletion elicited radiochemosensitization in PDAC, PDCs cell lines and reduced sphere formation and 3D invasion into collagen-I. Intriguingly, we found β8 integrin located in perinuclear area where it colocalized with the cis-Golgi matrix protein GM130. Upon irradiation and gemcitabine, β8 integrin dissociated from the perinuclear region and spread throughput the cytosol without enhanced localization to exosomes; a process abrogated by antimycin A or oligomycin pre-treatment. Conclusion Our findings, generated in 3D lrECM PDAC cell ccultures, suggest β8 integrin as a novel determinant of PDAC radiochemoresistance. Moreover, β8 integrin may, although not found in the cell membrane to facilitate cell adhesion, a critical role in intracellular vesicle trafficking under stress conditions. Ongoing work will unravel the underlying mechanisms how β8 integrin is controlling cytoplasmic and nuclear survival pathways.

500 Exosomal microRNAs as putative predictive biomarkers for targeted therapy in stage IV cutaneous malignant melanoma

495 Hyperactive NRAS downstream signaling induces specific transcriptome changes eesiRNA based identification of new therapeutic targets in NRAS mutant melanoma identifies the noncoding RNA 7SL as a major proliferation enhancer I Vujic, M Vujic, M Sanlorenzo, C Posch, A Preschitz, R Esteve-Puig, K Lai, W Ho, K Rappersberger and S Ortiz-Urda 1 Dermatology, KA Rudolfstiftung, Vienna, Austria and 2 Dermatology, University of California, San Francisco, San Francisco, CA Benign naevi and malignant melanomas both can have oncogenic mutations in the NRAS gene but naevi only rarely progress to cancer. Such NRAS mutations should lead to specific transcriptome changes. The knowledge of these changes can A) identify new therapeutic targets and B) explain why some naevi have NRAS mutations but stay benign. Here, we introduce NRAS(Q61) mutant plasmids in a pool of human melanocytes to overactivate NRAS downstream pathways. We perform deep RNASeq and compare transcriptome changes in melanocytes with and without NRAS mutation. We list differentially expressed coding and noncoding transcripts by filtering our results with transcriptomes from 2 NRAS mutant melanoma cell lines and 89 NRAS mutant patient tumors. Next we use esiRNA (Endoribonuclease-prepared siRNA) libraries to knock down these transcripts and perform proliferation assays to identify potential targets. For most promising candidates we perform further siRNA knockdowns, cell based assays and take a closer look at their mechanistics. Our approach identified 237 transcripts, of which 2 coding and 6 noncoding transcripts played an important role in the proliferation of NRAS mutant melanoma. The knockdown of each of these transcripts led to cell proliferation decreases of 30-60%. We focused on the noncoding RNA RN7SL1 and identified its up-stream regulation (MAPK pathway) and its downstream effectors (p53). In conclusion, we identify new therapeutic targets which might be used in the battle against NRAS mutant melanoma. 496 Radiation therapy in melanoma patients with poor clinical characteristics e a collaborative experience with 107 patients C Posch, C Steffal, A Haider, B Flechl, T Kann, F Weihsengruber, A Schratter-Sehn and K Rappersberger 1 Dermatology, The Rudolfstiftung Hospital, Vienna, Austria and 2 Radiooncology, Kaiser-Franz-JOsef Hospital, Vienna, Austria Radiation therapy (RTX) is an important part of melanoma management. Even though it is effective for local disease control, the lack of improved overall survival often dampens enthusiasm for this therapeutic modality. We describe our collaborative experience treating 107 stage III and stage IV melanoma patients managed at a dermatology department and a radio-oncology center using therapeutic or adjuvant radiation. Our cohort comprised a subset of melanoma patients with unfavorable clinical characteristics (median Breslow thickness: 2.8mm (range 0.5-18mm); clinical stage at diagnosis: III+IV n1⁄495). The median age at diagnosis was 60.3 (13.6-90.0) years the median age at start of irradiation was 66.2 (20.691.5) years. The axillary area was the most frequent anatomical site for irradiation (n1⁄430) followed by bone irradiation (n1⁄420). The majority of patients received additional systemic therapy including immunotherapy (n1⁄460), chemotherapy (n1⁄450) and targeted therapy (n1⁄47). Average overall survival (OS) was calculated with 57.3 (SD 52.1) months. Average overall survival after RTX (OSaRTX) was 17.1 (SD 18.7) months. Loco-regional recurrence (LRR) of melanoma was observed in 17 irradiated areas. No difference in OS or OSaRTX and locoregional recurrence was found: OS without LRR 57.6 (SEM 5.7) years; OS with LRR 55.6 (SEM 9.2) years; OSaRTX without LRR 16.2 (SEM 1.8) years, OSaRTX with LRR 20.6 (SEM 3.8) years (all p>0.05). LDH serum levels did not correlate with local recurrence (r1⁄40.07). Subgroup analyses revealed that OSaRTX for axillary irradiation was 26.3 (SD 24.6) months. RTX was well tolerated with the majority of acute adverse events being grade 1 (CTCAE v4.02). 11 patients experienced late irradiation adverse effects (all grade 1; RTOG/EORTC score). From our data we conclude that RTX is a well-tolerated therapeutic modality with the potential for high local disease control, particularly in the axillary region.

A genome-wide screening uncovers the role of CCAR2 as an antagonist of DNA end resection.

There are two major and alternative pathways to repair DNA double-strand breaks: non-homologous end-joining and homologous recombination. Here we identify and characterize novel factors involved in choosing between these pathways; in this study we took advantage of the SeeSaw Reporter, in which the repair of double-strand breaks by homology-independent or -dependent mechanisms is distinguished by the accumulation of green or red fluorescence, respectively. Using a genome-wide human esiRNA (endoribonuclease-prepared siRNA) library, we isolate genes that control the recombination/end-joining ratio. Here we report that two distinct sets of genes are involved in the control of the balance between NHEJ and HR: those that are required to facilitate recombination and those that favour NHEJ. This last category includes CCAR2/DBC1, which we show inhibits recombination by limiting the initiation and the extent of DNA end resection, thereby acting as an antagonist of CtIP.

Bioinformatic analyses of sense and antisense expression from terminal inverted repeat transposons in Drosophila somatic cells

ABSTRACTUnderstanding regulation of transposon movement in somatic cells is important as mobile elements can cause detrimental genomic rearrangements. Generally, transposons move via one of 2 mechanisms; retrotransposons utilize an RNA intermediate, therefore copying themselves and amplifying throughout the genome, while terminal inverted repeat transposons (TIR Tns) excise DNA sequences from the genome and integrate into a new location. Our recently published work indicates that retrotransposons in Drosophila tissue culture cells are actively transcribed in the antisense direction. Our data support a model in which convergent transcription of retrotransposons from intra element transcription start sites results in complementary RNAs that hybridize to form substrates for Dicer-2, the endogenous small interfering (esi)RNA generating enzyme. Here, we extend our previous analysis to TIR Tns. In contrast to retrotransposons, our data show that antisense TIR Tn RNAs result from transcription of intronic TIR Tns oriented antisense to their host genes. Also, disproportionately less esiRNAs are generated from TIR transcripts than from retrotransposons and transcription of very few individual TIR Tns could be confirmed. Collectively, these data support a model in which TIR Tns are regulated at the level of Transposase production while retrotransposons are regulated with esiRNA post-transcriptional mechanisms in Drosophila somatic cells.

Targeting Human Long Noncoding Transcripts by Endoribonuclease-Prepared siRNAs

Broad sequencing enterprises such as the FANTOM or ENCODE projects have substantially extended our knowledge of the human transcriptome. They have revealed that a large portion of genomic DNA is actively transcribed and have identified a plethora of novel transcripts. Many newly identified transcripts belong to the class of long noncoding RNAs (lncRNAs), which range from a few hundred bases to multiple kilobases in length and harbor no protein-coding potential. Although the biological activity of some lncRNAs is understood, the functions of most lncRNAs remain elusive. Tools that allow rapid and cost-effective access to functional data of lncRNAs are therefore essential. Here, we describe the construction and validation of an endoribonuclease-prepared siRNA (esiRNA) library designed to target 1779 individual human lncRNAs by RNA interference. We present a compendium of lncRNA expression data for 11 human cancer cell lines. Furthermore, we show that the resource is suitable for combined knockdown and localization analysis. We discuss challenges in sequence annotation of lncRNAs with respect to their often low and cell type–specific expression and specify esiRNAs that are suitable for targeting lncRNAs in commonly used human cell lines.

Genome-Wide RNAi High-Throughput Screen Identifies Proteins Necessary for the AHR-Dependent Induction of CYP1A1 by 2,3,7,8-Tetrachlorodibenzo-p-dioxin

The aryl hydrocarbon receptor (AHR) has a plethora of physiological roles, and upon dysregulation, carcinogenesis can occur. One target gene of AHR encodes the xenobiotic and drug-metabolizing enzyme CYP1A1, which is inducible by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) via the AHR. An siRNA library targeted against over 5600 gene candidates in the druggable genome was used to transfect mouse Hepa-1 cells, which were then treated with TCDD, and subsequently assayed for CYP1A1-dependent ethoxyresorufin-o-deethylase (EROD) activity. Following redundant siRNA activity (RSA) statistical analysis, we identified 93 hits that reduced EROD activity with a p value ≤ .005 and substantiated 39 of these as positive hits in a secondary screening using endoribonuclease-prepared siRNAs (esiRNAs). Twelve of the corresponding gene products were subsequently confirmed to be necessary for the induction of CYP1A1 messenger RNA by TCDD. None of the candidates were deficient in aryl hydrocarbon nuclear translocator expression. However 6 gene products including UBE2i, RAB40C, CRYGD, DCTN4, RBM5, and RAD50 are required for the expression of AHR as well as for induction of CYP1A1. We also found 2 gene products, ARMC8 and TCF20, to be required for the induction of CYP1A1, but our data are ambiguous as to whether they are required for the expression of AHR. In contrast, SIN3A, PDC, TMEM5, and CD9 are not required for AHR expression but are required for the induction of CYP1A1, implicating a direct role in Cyp1a1 transcription. Our methods, although applied to Cyp1a1, could be modified for identifying proteins that regulate other inducible genes.

Construction and advantage of endoribonuclease III-prepared siRNAs (esiRNA) library

Objective To establish KIF4A 3'UTR esiRNA library and analyze the advantage of method in studying the function of KIF4A in SGC-7901 cells.Methods The GST-fusion protein of Escherichia coli endoribonuclease Ⅲ (GST-RNase Ⅲ) was used to digest the double strand RNA (dsRNA),which was transcribed in vitro from a 502bp template of KIF4A genome (T7-KIF4A 3'UTR).The KIF4A esiRNA library generated from the above method and chemically synthesized KIF4A siRNA were then used to transfect SGC-7901 cells at 10 nmol/L and 20 nmol/L.Real-time quantitative PCR and Western Blot were used to detect the mRNA and protein level of KIF4A,respectively.Results The KIF4A esiRNA library was effectively established from dsRNA digestion using GST-RNase Ⅲ.KIF4A expression was significantly reduced in SGC-7901 cells transfected with KIF4A esiRNA or siRNA.In addition,the inhibitory effect of KIF4A esiRNA was more effective than that of chemically synthesized siRNA.Conclusion KIF4A esiRNA library which was obtained using biological method can more effectively inhibited the expression of KIF4A more effectively in SGC-7901 cells than chemically synthesized siRNA.Therefore,esiRNA library can be used as a new and more effective method in studying the function of KIF4A in SGC-7901 cells. Key words: RNA interference; esiRNA library; KIF4A

Functional Mutation of SMAC/DIABLO, Encoding a Mitochondrial Proapoptotic Protein, Causes Human Progressive Hearing Loss DFNA64

SMAC/DIABLO is a mitochondrial proapoptotic protein that is released from mitochondria during apoptosis and counters the inhibitory activities of inhibitor of apoptosis proteins, IAPs. By linkage analysis and candidate screening, we identified a heterozygous SMAC/DIABLO mutation, c.377C>T (p.Ser126Leu, refers to p.Ser71Leu in the mature protein) in a six-generation Chinese kindred characterized by dominant progressive nonsyndromic hearing loss, designated as DFNA64. SMAC/DIABLO is highly expressed in human embryonic ears and is enriched in the developing mouse inner-ear hair cells, suggesting it has a role in the development and homeostasis of hair cells. We used a functional study to demonstrate that the SMAC/DIABLO(S71L) mutant, while retaining the proapoptotic function, triggers significant degradation of both wild-type and mutant SMAC/DIABLO and renders host mitochondria susceptible to calcium-induced loss of the membrane potential. Our work identifies DFNA64 as the human genetic disorder associated with SMAC/DIABLO malfunction and suggests that mutant SMAC/DIABLO(S71L) might cause mitochondrial dysfunction.

A polyacrylamide microbead-integrated chip for the large-scale manufacture of ready-to-use esiRNA

Endoribonuclease-prepared siRNAs (esiRNAs) have the advantages of cost effectiveness and lower off-target effects than chemically synthesized siRNA. However, the current manufacture of esiRNA is a complex process, requiring an expensive instrument and demanding skills to accomplish the transfer, purification, quantification and normalization of liquid samples. These performances significantly hamper the application of esiRNAs on a large-scale level. In this study, we present a polymer microbead-integrated chip capable of the large-scale manufacture of esiRNA in a convenient and robust manner. This chip is able to perform the amplification, transcription and enzymatic digestion of targets on polymer scaffold, thus simplifying the transfer and purification manipulation process. What is also noted, this chip can readily tailor and normalize the amount of esiRNA product by controlling the number of DNA probes and the cycle of the amplification reaction. Thus the esiRNA, also referred to as gel-esiRNA, can be immediately applied to loss-of-function study without any further treatment. The silencing specificity and efficiency of gel-esiRNAs were assessed on transcriptional, translational or cell functional levels. All data of real-time PCR, Western blot assay, or FACS clearly supported that the gel-esiRNA produced specific gene silencing as effectively as the one generated following the conventional approach. We believe that this approach would provide a more robust and cost-effective choice to manufacture esiRNAs, thus promising both more intensive and extensive applications of these heterogeneous RNA strands.

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs. Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10. In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein(1,2,3). RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs. Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown(10). In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

High-content imaging

High-content imaging

Stromal Cell-Derived Factor-1α–Directed Chemoattraction of Transiently CXCR4-Overexpressing Bone Marrow Stromal Cells into Functionalized Three-Dimensional Biomimetic Scaffolds

Three-dimensional (3D) bone substitute material should not only serve as scaffold in large bone defects but also attract mesenchymal stem cells, a subset of bone marrow stromal cells (BMSCs) that are able to form new bone tissue. An additional crucial step is to attract BMSCs from the surface into deeper structures of 3D porous bone substitute scaffolds. Here we show that transient overexpression of CXCR4 in human BMSCs induced by mRNA transfection enhances stromal cell-derived factor-1alpha (SDF-1alpha)-directed chemotactic capacity to invade internal compartments of porous 3D bone substitute scaffolds in vitro and in vivo. In vitro native BMCSs invaded up to 500 mum into SDF-1alpha-releasing 3D scaffolds, whereas CXCR4-overexpressing BMSCs invaded up to 800 mum within 5 days. In addition, 60% downregulation of endogenous SDF-1 transcription in BMSCs by endoribonuclease-prepared siRNA before CXCR4 mRNA transfection enhanced SDF-1alpha-directed migration of human BMSCs by 50%. Implantation of SDF-1alpha-releasing scaffolds seeded with transiently CXCR4-overexpressing BMSCs resulted in an increase of invasion into internal compartments of the scaffolds in a mouse model. In vivo native BMCS invaded up to 250 mum into SDF-1alpha-releasing 3D scaffolds, whereas CXCR4-overexpressing BMSC invaded up to 500 mum within 5 days. Thus, the SDF-1alpha/CXCR4 chemoattraction system can be used to efficiently recruit BMSCs into SDF-1alpha-releasing 3D scaffolds in vitro and in vivo.

Inhibition of hepatitis B virus gene expression and replication by endoribonuclease-prepared siRNA

Endoribonuclease-prepared siRNA (esiRNA) is an alternative tool to chemical synthetic siRNA for gene silencing. Since esiRNAs are directed against long target sequences, the genetic variations in the target sequences will have little influence on their effectiveness. The ability of esiRNAs to inhibit hepatitis B virus (HBV) gene expression and replication was tested. EsiRNAs targeting the coding region of HBV surface antigen (HBsAg) and the nucleocapsid (HBcAg) inhibited specifically the expression of HBsAg and HBcAg when cotransfected with the respective expression plasmids. Both esiRNAs reduced the HBV transcripts and replication intermediates in transient transfected cells and cells with HBV genomes integrated stably. Compared with synthetic siRNA, esiRNA targeting HBsAg was less effective than the selected synthetic siRNA in terms of the inhibition of HBV gene expression and replication. However, while the ability of synthetic siRNAs for specific gene silencing was impaired strongly by the nucleotide substitutions within the target sequences. The efficiency of gene silencing by esiRNAs was not influenced by sequence variation. The transfection of esiRNA did not induce interferon-stimulated genes (ISGs) like STAT1 and ISG15, indicating the absence of off-target effects. In general, esiRNAs strongly inhibited HBV gene expression and replication and may have an advantage against HBV strains which are variable genetically.

Endoribonuclease-Prepared Short Interfering RNAs Induce Effective and Specific Inhibition of Human Immunodeficiency Virus Type 1 Replication

Short interfering RNAs (siRNAs) targeting viral or cellular genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, optimal HIV-1 gene silencing by siRNA requires precise complementarity with most of the target sequence. The emergence of mutations in the targeted gene could lead to rapid viral escape from the siRNA. In the present study, Escherichia coli endoribonuclease III (RNase III) or mammalian Dicer was used to cleave double-stranded RNA into endoribonuclease-prepared siRNA (esiRNA). esiRNAs generate a variety of siRNAs which can efficiently and specifically target multiple sites in the cognate RNA. esiRNAs targeting the region encoding the HIV-1 reverse transcriptase (RT) reduced viral replication by 90%. The inhibition was dose dependent and sequence specific because several irrelevant esiRNAs did not inhibit HIV-1 replication. Importantly, esiRNAs obtained from the prototypic RT sequence of the HXB2 strain and from highly mutated RT sequences showed similar degrees of viral inhibition, suggesting that the heterogeneous population of esiRNAs could overcome individual mismatches in the RT sequence. Finally, esiRNAs generated by Dicer cleavage were five times more potent than those generated by bacterial RNase III digestion. These results show that esiRNAs are potent HIV-1 inhibitors. Moreover, sequence targets do not need to be highly conserved to reach a high level of viral replication inhibition.

RNA interference against hepatitis B virus with endoribonuclease-prepared siRNA despite of the target sequence variations

RNA interference (RNAi) has proven to be very powerful in inhibiting hepatitis B virus (HBV) replication by cell culture and mouse model studies. We have previously reported that endoribonuclease-prepared short interfering RNAs (esiRNAs) were able to inhibit HBV replication more efficiently than synthesized siRNAs. Here we tested the hypothesis that esiRNAs are able to inhibit gene expression with limited mutations within the target region. Target sequences with different similarities to esiHBVP (esiRNA targeting the DNA polymerase and S antigen of Hepatitis B virus) were amplified and cloned into the 3′ untranslated region of HBsAg, respectively. When the obtained expression vectors were co-transfected with esiHBVP into CHO cells, HBsAg expression was suppressed with same efficiency regardless of the target sequence similarities. In HepG2 cells, esiHP9 based on one of the amplified sequence that sharing 87% similarity to the target region suppressed HBsAg expression effectively and dose dependently. In vivo experiment showed that a single dose of 5 μg esiHP9 was able to reduce HBsAg and HBeAg level in the mouse sera by 88 and 77% despite of its 87% similarity to the target sequence, which was as good as esiHBVP that is 100% similar to the target sequence. All the data suggest that esiRNA can tolerate limited target sequence variations without losing its inhibitory capacity. It would be very helpful to suppress virus replication by RNAi despite of their high mutation rate.

EsiRNAs inhibit Hepatitis B virus replication in mice model more efficiently than synthesized siRNAs

RNA interference (RNAi) has been proved to be a promising strategy to combat Hepatitis B virus (HBV) infection by way of cell culture and animal model studies. In this work, esiRNAs (endoribonuclease-prepared siRNAs) targeting all of the four open reading frames (ORFs) of HBV genome were prepared. In vitro experiment showed that esiHBVP suppressed HBsAg expression most effectively. Its capacity to suppress HBV replication in vivo was then tested. A single dose of 1 μg esiHBVP was able to reduce HBsAg and HBeAg level in the mouse serum by 90 and 89% one day after injection, while the same amount of chemically synthesized siRNA only reduced that by 33 and 45%. Immunostaining of HBcAg showed that esiHBVP inhibited HBcAg expression more potently than chemically synthesized siRNA. Quantification of HBV DNA in the mouse serum showed 1 μg eiHBVP treatment reduced serum HBV DNA copy number to 18% that of the untreated control, while 1 μg siRNA treatment only reduced that to 63%. In conclusion, the data presented here proved that esiRNA is much more efficient in suppressing HBV replication than chemically synthesized siRNA, and it might be a better therapeutic agent to fight against HBV infection.