Joanna Bereta

In recent years, CRISPR interference (CRISPRi) technology of gene silencing has emerged as a promising alternative to RNA interference (RNAi) surpassing the latter in terms of efficiency and accuracy. Here, we describe the construction of a set of transposon vectors suitable for constitutive or tetracycline (doxycycline)-inducible silencing of genes of interest via CRISPRi method and conferring three different antibiotic resistances, using vectors available via Addgene repository. We have analyzed the performance of the new vectors in the silencing of mouse Adam10 and human lncRNA, NORAD. The empty vector variants can be used to efficiently silence any genes of interest.

RNA interference is one of the common methods of studying protein functions. In recent years critical reports have emerged indicating that off-target effects may have a much greater impact on RNAi-based analysis than previously assumed. We studied the influence of Adam10 and Adam17 silencing on MC38CEA cell response to proinflammatory stimuli. Eight lentiviral vector-encoded shRNAs that reduced ADAM10 expression, including two that are specific towards ADAM17, caused inhibition of cytokine-induced Nos2 expression presumably via off-target effects. ADAM10 silencing was not responsible for this effect because: (i) CRISPR/Cas9 knockdown of ADAM10 did not affect Nos2 levels; (ii) ADAM10 inhibitor increased rather than decreased Nos2 expression; (iii) overexpression of ADAM10 in the cells with shRNA-silenced Adam10 did not reverse the effect induced by shRNA; (iv) shRNA targeting ADAM10 resulted in decrease of Nos2 expression even in ADAM10-deficient cells. The studied shRNAs influenced transcription of Nos2 rather than stability of Nos2 mRNA. They also affected stimulation of Ccl2 and Ccl7 expression. Additionally, we used vectors with doxycycline-inducible expression of chosen shRNAs and observed reduced activation of NF-κB and, to a lesser extent, AP-1 transcription factors. We discuss the requirements of strict controls and verification of results with complementary methods for reliable conclusions of shRNA-based experiments.

In parallel with the expansion of RNA interference techniques evidence has been accumulating that RNAi analyses may be seriously biased due to off-target effects of gene-specific shRNAs. Our work points to another possible source of misinterpretations of shRNA-based data – off-target effects of non-targeting shRNA. We found that one such control for the MISSION ^® library (commercialized TRC library), SHC016, is cytotoxic. Using a lentiviral vector with inducible expression of SHC016 we proved that this shRNA induces apoptosis in murine cells and, depending on p53 status, senescence or mitotic catastrophe in human tumor cell lines. We identified SNRPD3, a core spliceosomal protein, as a major SHC016 target in several cell lines and confirmed in A549 and U251 cell lines that CRISPRi-knockdown of SNRPD3 mimics the effects of SHC016 expression. Our finding disqualifies non-targeting SHC016 shRNA and adds a new premise to the discussion about the sources of uncertainty of RNAi results.

Neuregulin 2 (NRG2) belongs to the EGF family of growth factors. Most of this family members require proteolytic cleavage to liberate their ectodomains capable of binding and activating their cognate ErbB receptors. To date, most of the studies investigating proteolytic processing of neuregulins focused on NRG1, which was shown to undergo ectodomain shedding by several ADAM proteases and BACE1 and the remaining fragment was further cleaved by γ-secretase. Recently, NRG2 attracted more attention due to its role in the neurogenesis and modulation of behaviors associated with psychiatric disorders. In this study, we used genetic engineering methods to identify proteases involved in proteolytic processing of murine NRG2. Using non-neuronal cell lines as well as cultures of primary hippocampal neurons, we demonstrated that the major proteases responsible for releasing NRG2 ectodomain are ADAM10 and BACE2. Co-expression of NRG2 and BACE2 in neurons of certain brain structures including medulla oblongata and cerebellar deep nuclei was confirmed via immunohistochemical staining. The cleavage of NRG2 by ADAM10 or BACE2 generates a C-terminal fragment that serves as a substrate for γ-secretase. We also showed that murine NRG2 is subject to post-translational modifications, substantial glycosylation of its extracellular part, and phosphorylation of the cytoplasmic tail.

Background: The functionalization of a nanoparticle surface with PEG (polyethylene glycol) is an approach most often used for extending nanomaterial circulation time, enhancing its delivery and retention in the target tissues, and decreasing systemic toxicity of nanocarriers and their cargos. However, because PEGylated nanomedicines were reported to induce immune response including production of anti-PEG antibodies, activation of the complement system as well as hypersensitivity reactions, hydrophilic polymers other than PEG are gaining interest as its replacement in nanomaterial functionalization. Here, we present the results of in vivo evaluation of polyelectrolyte nanocapsules with biodegradable, polyelectrolyte multilayer shells consisting of poly-l-lysine (PLL) and poly-l-glutamic (PGA) acid as a potential drug delivery system. We compared the effects of nanocapsules functionalized with two different “stealth” polymers as the external layer of tested nanocapsules was composed of PGA (PGA-terminated nanocapsules, NC-PGA) or the copolymer of poly-l-lysine and polyethylene glycol (PEG-terminated nanocapsules, NC-PEG). Methods: Nanocapsules pharmacokinetics, biodistribution and routes of eliminations were analysed postmortem by fluorescence intensity measurement. Toxicity of intravenously injected nanocapsules was evaluated with analyses of blood morphology and biochemistry and by histological tissue analysis. DNA integrity was determined by comet assay, cytokine profiling was performed using flow cytometer and detection of antibodies specific to PEG was performed by ELISA assay. Results: We found that NC-PGA and NC-PEG had similar pharmacokinetic and biodistribution profiles and both were eliminated by hepatobiliary and renal clearance. Biochemical and histopathological evaluation of long-term toxicity performed after a single as well as repeated intravenous injections of nanomaterials demonstrated that neither NC-PGA nor NC-PEG had any acute or chronic hemato-, hepato- or nephrotoxic effects. In contrast to NC-PGA, repeated administration of NC-PEG resulted in prolonged increased serum levels of a number of cytokines. Conclusion: Our results indicate that NC-PEG may cause undesirable activation of the immune system. Therefore, PGA compares favorably with PEG in equipping nanomaterials with stealth properties. Our research points to the importance of a thorough assessment of the potential influence of nanomaterials on the immune system.

Background: Curcumin is a natural polyphenol with anti-inflammatory, chemopreventive and anticancer activity. However, its high hydrophobicity and poor bioavailability limit its medical application. The development of nanocarriers for curcumin delivery is an attractive approach to overcome its low bioavailability and fast metabolism in the liver. We synthesized a blood compatible alginate-curcumin conjugate, AA-Cur, which formed colloidally stable micelles of approximately 200 nm and, as previously shown, exerted strong cytotoxicity against mouse cancer cell lines. Here we analyze in vivo toxicity and antitumor activity of AA-Cur in two different mouse tumor models. Method: Potential toxicity of intravenously injected AA-Cur was evaluated by: i) analyses of blood parameters (morphology and biochemistry), ii) histology, iii) DNA integrity (comet assay), and iv) cytokine profiling (flow cytometry). Antitumor activity of AA-Cur was evaluated by measuring the growth of subcutaneously inoculated colon MC38-CEA- or orthotopically injected breast 4T1 tumor cells in control mice vs mice treated with AA-Cur. Results: Injections of four doses of AA-Cur did not reveal any toxicity of the conjugate, thus indicating the safety of its use. AA-Cur elicited moderate anti-tumor activity toward colon MC38-CEA or breast 4T1 carcinomas. Conclusion: The tested conjugate of alginate and curcumin, AA-Cur, is non-toxic and safe, but exhibits limited anticancer activity.

ADAM17 is a cell membrane metalloproteinase responsible for the release of ectodomains of numerous proteins from the cell surface. Although ADAM17 is often overexpressed in tumours and at sites of inflammation, little is known about the regulation of its expression. Here we investigate the role of NF-κB and Elk-1 transcription factors and upstream signalling pathways, NF-κB and ERK1/2 in ADAM17 expression in mouse brain endothelial cells stimulated with pro-inflammatory factors (TNF, IL-1β, LPS) or a phorbol ester (PMA), a well-known stimulator of ADAM17 activity. Notably, NF-κB inhibitor, IKK VII, interfered with the IL-1β- and LPS-mediated stimulation of ADAM17 expression. Furthermore, Adam17 promoter contains an NF-κB binding site occupied by p65 subunit of NF-κB. The transient increase in Adam17 mRNA in response to PMA was strongly reduced by an inhibitor of ERK1/2 phosphorylation, U0126. Luciferase reporter assay with vectors encoding the ERK1/2 substrate, Elk-1, fused with constitutively activating or repressing domains, indicated Elk-1 involvement in Adam17 expression. The site-directed mutagenesis of potential Elk-1 binding sites pointed to four functional Elk-1 binding sites in Adam17 promoter. All in all, our results indicate that NF-κB and Elk-1 transcription factors via NF-κB and ERK1/2 signalling pathways contribute to the regulation of mouse Adam17 expression.

Background: Toxicity of nanomaterials is one of the most important factors limiting their medical application. Evaluation of in vitro nanotoxicity allows for the identification and elimination of most of the toxic materials prior to animal testing. The current knowledge of the possible side effects of biodegradable nanomaterials, such as liposomes and polymeric organic nanoparticles, is limited. Previously, we developed a potential drug delivery system in the form of nanocapsules with polyelectrolyte, biodegradable shells consisting of poly-L-lysine and poly-L-glutamic acid (PGA), formed by the layer-by-layer adsorption technique. Methods: Hemolysis assay, viability tests, flow cytometry analysis of vascular cell adhesion molecule-1 expression on endothelium, analysis of nitric oxide production, measurement of intracellular reactive oxygen species levels, detection of antioxidant enzyme activity, and analysis of DNA damage with comet assay were performed to study the in vitro toxicity of nanocapsules. Results: In this work, we present the results of an in vitro analysis of toxicity of five-layer positively charged poly-L-lysine–terminated nanocapsules (NC5), six-layer negatively charged PGA-terminated nanocapsules (NC6) and five-layer PEGylated nanocapsules (NC5-PEG). PGA and polyethylene glycol (PEG) were used as two different “stealth” polymers. Of all the polyelectrolyte nanocapsules tested for blood compatibility, only cationic NC5 showed acute toxicity toward blood cells, expressed as hemolysis and aggregation. Neither NC6 nor NC5-PEG had proinflammatory activity evaluated through changes in the expression of NF-κB–dependent genes, iNOS and vascular cell adhesion molecule-1, induced oxidative stress, or promoted DNA damage in various cells. Conclusion: Our studies clearly indicate that PGA-coated (negatively charged) and PEGylated polyelectrolyte nanocapsules do not show in vitro toxicity, and their potential as a drug delivery system may be safely studied in vivo.

We re-evaluate the evidence presented for the detection of mRNA in cells and tissues with Spherical Nucleic Acids.