Reverse Transfection Research Articles

Development of a recombinant non-replicating Newcastle disease virus

ABSTRACT Newcastle disease virus (NDV) is an avian orthoavulavirus I that causes high morbidity and mortality in birds, depending on the pathotype of the virus. Several clinical studies have shown that NDV is a promising oncolytic virus for cancer therapies, yet its application to clinics is restricted due to the pathogenicity of this virus in poultry. Hence, it is important to produce a recombinant NDV (rNDV) with reduced virulence to make it a fit candidate as an oncolytic virus. In this study, we aimed to produce a rNDV with a truncated ant-igenome via reverse genetics to render it non-replicating (nr). The genes responsible for replication were removed from the NDV genome. The rescued nr-NDV was verified, propagated, and purified from tissue culture medium. The virus failed to propagate in embryonated eggs, confirming its non-replicating property. The virus retained its oncolytic activities similar to that of the replicating rAF-GFP as observed in a cell viability assay when a breast cancer cell line, MCF-7, was treated with the virus. In conclusion, this study has successfully generated a system to produce a non-replicating NDV which can be used as a viral vector for heterologous protein delivery, as a safer alternative compared to the virulent strains as an oncolytic virus. RESEARCH HIGHLIGHTS Development of nr-NDV. Reverse transfection was applied for the recovery of nr-NDV. Propagation of nr-NDV was done by sub-passaging transfected BSR T7/5 cells. Safety profile was done to prove that the nr-NDV is non-replicating.

Development of ionizable lipid nanoparticles and a lyophilized formulation for potent CRISPR-Cas9 delivery and genome editing

CRISPR-Cas genome editing technology holds great promise for wide-ranging biomedical applications. However, the development of efficient delivery system for CRISPR-Cas components remains challenging. Herein, we synthesized a series of ionizable lipids by conjugation of alkyl-acrylate to different amine molecules and further assembled ionizable lipid nanoparticles (iLNPs) for co-delivery of Cas9 mRNA and sgRNA. Among all the iLNP candidates, 1A14-iLNP with lipids containing spermine as amine head, demonstrated the highest cellular uptake, endosomal escape and mRNA expression in vitro. Co-delivery of Cas9 mRNA and sgRNA targeting EGFP by 1A14-iLNP achieved the highest EGFP knockout efficiency up to 70% in HeLa-EGFP cells. In addition, 1A14-iLNP displayed passive liver-targeting delivery of Cas9 mRNA in vivo with good biocompatibility. Moreover, we developed a simple method of lyophilization-mediated reverse transfection of CRISPR-Cas9 components for efficient genome editing. Therefore, the developed 1A14-iLNP and the lyophilization formulation, represent a potent solution for CRISPR-Cas9 delivery, which might broaden the future of biomedical applications of both mRNA and CRISPR-based therapies.

Modifying peptide/lipid-associated nucleic acids (PLANAs) for CRISPR/Cas9 ribonucleoprotein delivery

With the first reports on the possibility of genome editing by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas)9 surfacing in 2005, the enthusiasm for protein silencing via nucleic acid delivery experienced a resurgence following a period of diminished enthusiasm due to challenges in delivering small interfering RNAs (siRNA), especially in vivo. However, delivering the components necessary for this approach into the nucleus is challenging, maybe even more than the cytoplasmic delivery of siRNA. We previously reported the birth of peptide/lipid-associated nucleic acids (PLANAs) for siRNA delivery. This project was designed to investigate the efficiency of these nanoparticles for in vitro delivery of CRISPR/Cas9 ribonucleoproteins. Our initial experiments indicated higher toxicity for PLANAs with the more efficient reverse transfection method. Therefore, polyethylene glycol (PEG) was added to the composition for PEGylation of the nanoparticles by partially replacing two of the lipid components with the PEG-conjugated counterparts. The results indicated a more significant reduction in the toxicity of the nanoparticle, less compromise in encapsulation efficiency and more PEGylation of the surface of the nanoparticles using DOPE-PEG2000 at 50 % replacement of the naïve lipid. The cell internalization and transfection efficiency showed a comparable efficiency for the PEGylated and non-PEGylated PLANAs and the commercially available Lipofectamine™ CRISPRMAX™. Next Generation Sequencing of the cloned cells showed a variety of indels in the transfected cell population. Overall, our results indicate the efficiency and safety of PEGylated PLANAs for in vitro transfection with CRISPR/Cas9 ribonucleoproteins. PEGylation has been studied extensively for in vivo delivery, and PEGylated PLANAs will be candidates for future in vivo studies.

An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios.

Due to its relative simplicity and ease of use, transient transfection of mammalian cell lines with nucleic acids has become a mainstay in biomedical research. While most widely used cell lines have robust protocols for transfection in adherent two-dimensional culture, these protocols often do not translate well to less-studied lines or those with atypical, hard-to-transfect morphologies. Using mouse pluripotent stem cells grown in 2i/LIF media, a widely used culture model for regenerative medicine, this method outlines an optimized, rapid reverse transfection protocol capable of achieving higher transfection efficiency. Leveraging this protocol, a three-plasmid poly-transfection is performed, taking advantage of the higher-than-normal efficiency in plasmid delivery to study an expanded range of plasmid stoichiometry. This reverse poly-transfection protocol allows for a one-pot experimental method, enabling users to optimize plasmid ratios in a single well, rather than across several co-transfections. By facilitating the rapid exploration of the effect of DNA stoichiometry on the overall function of delivered genetic circuits, this protocol minimizes the time and cost of embryonic stem cell transfection.

Ginsenoside Rh2 enhances immune surveillance of natural killer (NK) cells via inhibition of ERp5 in breast cancer

BackgroundOne critical component of the immune system that prevents breast cancer cells from forming distant metastasis is natural killer (NK) cells participating in immune responses to tumors. Ginsenoside Rh2 (GRh2) as one of the major active ingredients of ginseng has been employed in treatment of cancers, but the function of GRh2 in modulating the development of breast cancer remains elusive. PurposeThis study was to dissect the effect of GRh2 against breast cancer and its potential mechanisms associated with NK cells, both in vitro and in vivo. MethodsMDA-MB-231 and 4T1 cells were used to establish in situ and hematogenous mouse models. MDA-MB-231 and MCF-7 were respectively co-cultured with NK92MI cells or primary NK cells in vitro. Anti-tumor efficacy of GRh2 was verified by immunohistochemistry (IHC), Cell Counting Kit-8 (CCK8), high resolution micro-computed tomography (micro-CT) scanning of lungs and hematoxylin and eosin (H&E) staining. Lactate dehydrogenase (LDH) cytotoxicity assay, flow cytometry, in vivo depletion of NK cells, enzyme-linked immunosorbent assay (ELISA), western blot, quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunofluorescence and cell transfection were performed for investigating the anti-tumor mechanisms of GRh2. Molecular docking, microscale thermophoresis (MST) and cellular thermal shift assay (CETSA) were employed to determine the binding between endoplasmic reticulum protein 5 (ERp5) and GRh2. ResultsWe demonstrated that GRh2 exerted prominent impacts on retarding the growth and metastasis of breast cancer through boosting the cytotoxic function of NK cells, as validated by the elevated release of perforin, granzyme B and interferon-γ (IFN-γ). Mechanistical studies revealed that GRh2 was capable of diminishing the expression of ERp5 and GRh2 directly bound to ERp5 in MDA-MB-231 cells as well as on a recombinant protein level. GRh2 prevented the formation of soluble MICA (sMICA) and upregulated the expression level of MICA in vivo and in vitro. Importantly, the reduced lung metastasis of breast cancer by GRh2 was almost abolished upon the depletion of NK cells. Moreover, GRh2 was able to insert into the binding pocket of ERp5 directly. ConclusionWe firstly demonstrated that GRh2 played a pivotal role in augmenting NK cell activity by virtue of modulating the NKG2D-MICA signaling axis via directly binding to ERp5, and may be further optimized to a therapeutic agent for the treatment of breast cancer.

Oligodendrogenesis and myelination tracing in a CRISPR/Cas9-engineered brain microphysiological system.

Oligodendrocytes (OLs) are the myelin-forming cells of the central nervous system (CNS). Although OLs can be differentiated from human-induced pluripotent stem cells (hiPSCs), the in vitro modeling of axon myelination in human cells remains challenging. Brain microphysiological systems (bMPS, e.g. organoids) are complex three-dimensional (3D) cultures that offer an ideal system to study this process as OLs differentiate in a more in vivo-like environment; surrounded by neurons and astrocytes, which support the myelination of axons. Here, we take advantage of CRISPR/Cas9 technology to generate a hiPSC line in which proteolipid protein 1 (PLP1), an OLs marker, is tagged with super-fold GFP (sfGFP). While generating the PLP1-sfGFP reporter, we used reverse transfection and obtained higher Knock-In (KI) efficiency compared to forward transfection (61-72 vs. 46%). After validation of the KI and quality control of the PLP1-sfGFP line, selected clones were differentiated into bMPS, and the fidelity, specificity, and function of the tagged PLP protein were verified in this model. We tracked different stages of oligodendrogenesis in the verified lines based on PLP1-sfGFP+ cells' morphology, and the presence of PLP1-sfGFP surrounding axons during bMPS' differentiation. Finally, we challenged the bMPS with cuprizone and quantified changes in both the percentage of PLP1-sfGFP expressing cells and the intensity of GFP expression. This work demonstrates an efficient method for generating hiPSC KI lines and the description of a new 3D model to study OL differentiation, migration, and maturation both during in vitro neurodevelopment as well as in response to environmental chemicals or disease-associated stressors.

A Reverse Transfection System with Cationized Gelatin Nanospheres Incorporating Molecular Beacon as a Tool to Visualize Cell Function.

The objective of this research is to design a reverse transfection system with cationized gelatin nanospheres (cGNS) incorporating a molecular beacon (MB) to visualize a cell function. The cGNS were prepared by the conventional coacervation method. The MB as an imaging probe was incorporated into the cGNS to prepare imaging complexes (cGNSMB). The conventional transfection of 2D culture was performed by incubating MC3T3 cells in the medium containing cGNSMB. The reverse transfection was done by incubating cells on the substrate which had been precoated with both gelatin and cGNSMB. Significantly higher internalization efficiency and fluorescence intensity of cGNSMB were observed in the reverse transfection system than in the conventional one. To apply this system for visualization of 3D cell aggregate, gelatin microspheres (GMS) were prepared, while cGNSMB were bound on the GMS to prepare the GMS-cGNSMB of a cell scaffold. Then the cells were incubated with GMS-cGNSMB to form 3D cell aggregates. On the other hand, as a control, the conventional transfection of 3D culture was performed by incubating the cell aggregates formed with the medium containing cGNSMB. Homogeneous fluorescence of MB from the inside to the outside of aggregates was observed for the reverse transfection group. However, for the conventional transfection, the fluorescence was observed only around the surface of cell aggregates. It is concluded that the reverse transfection system with cGNS incorporating MB is promising to visualize the cell function of a higher transfection efficiency for the 2D culture and in a homogeneous manner for the 3D culture.

Reverse Transfection of Functional RNA Rings into Cancer Cells Followed by in Vitro Irradiation.

RNA nanoparticles are promising therapeutic platforms to improve radiotherapy since they can be functionalized with multiple small interfering RNAs (RNAi) to simultaneously silence critical radioresistance genes. Here we describe the transfer of RNA rings to mammalian cancer cells through reverse transfection, followed by in vitro irradiation and biological assays as surrogates' endpoints for radiotherapy efficacy.

Chitosan/Hyaluronic Acid/MicroRNA-21 Nanoparticle-Coated Smooth Titanium Surfaces Promote the Functionality of Human Gingival Fibroblasts.

PurposeForming a compact biological seal between the gingiva and the implant interface around the percutaneous parts of an implant is one of the key issues in preventing peri-implantitis.MethodsIn this study, since microRNA-21 (miR-21) has been approved to promote fibroblast proliferation and collagen formation in skin fibrosis, we prepared miR-21-loaded chitosan (CS)/tripolyphosphate (TPP)/hyaluronic acid (HA) nanoparticles (CTH NPs) and cross-linked them to smooth Ti surfaces with 0.2% gel solution for reverse transfection, after which isolated human gingival fibroblasts were cultured on the miR-21-functionalized Ti substrates.ResultsAn optimal CS:TPP:HA ratio (1:0.15:0.1) and N/P ratio (20:1) were chosen to produce appropriate nanoparticles. Finally, the CTH/miR-21 nanoparticle-coated smooth Ti surfaces demonstrated increased fibroblast adhesion, proliferation and expression of extracellular matrix-related genes along with similar cytotoxicity and cell spreading on the miR-21-functionalized Ti surface and the unmodified smooth Ti surface.ConclusionThe chitosan-based nanoparticles might be an efficient nonviral miRNA vector to form a stable biological seal in percutaneous areas of Ti for clinical use.

Proteasome dysfunction disrupts adipogenesis and induces inflammation via ATF3

ObjectiveRegulation of proteasomal activity is an essential component of cellular proteostasis and function. This is evident in patients with mutations in proteasome subunits and associated regulators, who suffer from proteasome-associated autoinflammatory syndromes (PRAAS). These patients display lipodystrophy and fevers, which may be partly related to adipocyte malfunction and abnormal thermogenesis in adipose tissue. However, the cell–intrinsic pathways that could underlie these symptoms are unclear. Here, we investigate the impact of two proteasome subunits implicated in PRAAS, Psmb4 and Psmb8, on differentiation, function and proteostasis of brown adipocytes. MethodsIn immortalized mouse brown pre-adipocytes, levels of Psmb4, Psmb8, and downstream effectors genes were downregulated through reverse transfection with siRNA. Adipocytes were differentiated and analyzed with various assays of adipogenesis, lipogenesis, lipolysis, inflammation, and respiration. ResultsLoss of Psmb4, but not Psmb8, disrupted proteostasis and adipogenesis. Proteasome function was reduced upon Psmb4 loss, but partly recovered by the activation of Nuclear factor, erythroid-2, like-1 (Nfe2l1). In addition, cells displayed higher levels of surrogate inflammation and stress markers, including Activating transcription factor-3 (Atf3). Simultaneous silencing of Psmb4 and Atf3 lowered inflammation and restored adipogenesis. ConclusionsOur study shows that Psmb4 is required for adipocyte development and function in cultured adipocytes. These results imply that in humans with PSMB4 mutations, PRAAS-associated lipodystrophy is partly caused by disturbed adipogenesis. While we uncover a role for Nfe2l1 in the maintenance of proteostasis under these conditions, Atf3 is a key effector of inflammation and blocking adipogenesis. In conclusion, our work highlights how proteasome dysfunction is sensed and mitigated by the integrated stress response in adipocytes with potential relevance for PRAAS patients and beyond.

Accelerated transgene expression of pDNA/polysaccharide complexes by solid-phase reverse transfection and analysis of the cell transfection mechanism

Accelerated transgene expression of pDNA/polysaccharide complexes by solid-phase reverse transfection and analysis of the cell transfection mechanism

Selenomethionine-Modified Polyethylenimine-Based Nanoparticles Loaded with miR-132-3p Inhibitor-Biofunctionalized Titanium Implants for Improved Osteointegration.

Titanium and its alloys have been widely used as bone implants, but for reduced treatment span, improvements are urgently needed to achieve faster and better osteointegration. In this study, we found that miR-132-3p inhibited bone-marrow-derived stem cell (BMSC) osteogenic differentiation via targeting BMP2, and that inhibiting miR-132-3p could significantly improve the osteogenic capability of BMSCs. Moreover, we fabricated a biocompatible selenomethionine (SEMET)-modified polyethylene glycol (PEG)/polyethylenimine (PEI) nanoparticle (SeNP) cross-linked with 0.2% gelatin solutions and delivered miR-132-3p inhibitor to biofunctionalize alkali heat-treated titanium implants, resulting in the development of a novel coating for reverse transfection. The biological performances of PEG/PEI/miR-132-3p inhibitor and SeNP/miR-132-3p inhibitor-biofunctionalized titanium were compared. The biological effects, including cell viability, cytotoxicity, adhesion, cellular uptake, and osteogenic capacity of SeNP/miR-132-3p inhibitor-biofunctionalized titanium implants, were then assessed. Results showed that SeNPs presented appropriate morphology, diameter, and positive zeta potential for efficient gene delivery. The transfection efficiency of the SeNP/miR-132-3p inhibitor was comparable to that of the PEG/PEI/miR-132-3p inhibitor, but the former induced less reactive oxygen species (ROS) production and lower apoptosis rates. Confocal laser scanning microscopy (CLSM) demonstrated that SeNP/miR-132-3p inhibitor nanoparticles released from the titanium surfaces and were taken up by adherent BMSCs. In addition, the release profile showed that transfection could obtain a long-lasting silencing effect for more than 2 weeks. The cell viability, cytotoxicity, and cell spreading of SeNP/miRNA-132-3p inhibitor-biofunctionalized titanium were comparable with those of untreated titanium and the SeNP/miRNA-132-3p inhibitor negative control (NC)-biofunctionalized titanium but resulted in higher ALP activity and osteogenic gene expression levels. In vivo animal studies further certified that SeNP/miRNA-132-3p inhibitor nanoparticles from titanium surfaces promoted osteointegration, which was revealed by microcomputed tomography (micro-CT) and histological observations. Taken together, these findings suggested that selenomethionine-modified PEI-based nanoparticles could achieve better biocompatibility. Moreover, titanium implants biofunctionalized by SeNP/miRNA-132-3p inhibitor nanoparticles might have significant clinical potential for more effective osteointegration.

RAAV Production and Titration at the Microscale for High-Throughput Screening.

In the literature, there are few high-throughput screens or even methods for high-throughput screens of recombinant adeno-associated virus (rAAV) production despite potential benefits to research and production. In this study, a generalizable high-throughput relative rAAV titration method is examined within the context of an siRNA screen as siRNA knockdown is a common means of pathway engineering in bioproduction. Crude samples generated from transfected HEK293T/17 cultures were subjected to quantitative PCR (qPCR) and used to transduce COS7 cells to assess relative differences in genomic and infectious rAAV titer, respectively, at the 384-well scale, evaluating both supernatant and lysed samples. To evaluate relevant differences in titer for conditions that could be used in an actual screen, cultures subjected to an siRNA reverse transfection and subsequent rAAV forward transfection were also tested. The delayed forward rAAV triple-plasmid transfection was not seen to affect the siRNA activity of tested controls, while siRNA transfection was shown to measurably impact rAAV titer. Effective differentiation between infectious titer levels was dependent upon the choice of sample dilution, but trends between qPCR and infectious titer assays were consistent across sample sets.

Effect of using amino acids in the freeze-drying of siRNA lipoplexes on gene knockdown in cells after reverse transfection.

Recently, small interfering RNA (siRNA)/cationic liposome complexes (siRNA lipoplexes) have become a crucial research tool for studying gene function. Easy and reliable siRNA transfection with a large set of siRNAs is required for the successful screening of gene function. Reverse (Rev)-transfection with freeze-dried siRNA lipoplexes is validated for siRNA transfection with a large set of siRNAs in a multi-well plate. In our previous study, it was shown that Rev-transfection with siRNA lipoplexes freeze-dried in disaccharides or trisaccharides resulted in long-term stability with a high level of gene-knockdown activity. In the present study, the effects of amino acids used as cryoprotectants in the freeze-drying of siRNA lipoplexes on gene knockdown via Rev-transfection were assessed. A total of 15 types of amino acids were used to prepare freeze-dried siRNA lipoplexes, and it was found that the freeze-drying of siRNA lipoplexes with amino acid concentrations >100 mM strongly suppressed targeted gene expression regardless of the amino acid type; however, some amino acids strongly upregulated or downregulated gene expression in the cells transfected with negative control siRNA. Amongst the amino acids tested, the presence of asparagine showed specific gene-knockdown activity, forming large cakes after freeze-drying and retaining a favorable siRNA lipoplex size after rehydration. These findings provide valuable information regarding amino acids as cryoprotectants for Rev-transfection using freeze-dried siRNA lipoplexes for the efficient delivery of siRNA into cells.

Development of a T7 RNA polymerase expressing cell line using lentivirus vectors for the recovery of recombinant Newcastle disease virus

The development of a T7 RNA polymerase (T7 RNAP) expressing cell line i.e. BSR T7/5 cells marks an improvement of reverse genetics for the recovery of recombinant Newcastle disease virus (rNDV). BSR T7/5 is developed by transient transfection of plasmid encoding T7 RNAP gene for rNDV rescue. However, the gene expression decreases gradually over multiple passages and eventually hinders the rescue of rNDV. To address this issue, lentiviral vector was used to develop T7 RNAP-expressing HEK293-TA (HEK293-TA-Lv-T7) and SW620 (SW620-Lv-T7) cell lines, evidenced by the expression of T7 RNAP after subsequent 20 passages. rNDV was rescued successfully using HEK293-TA-Lv-T7 clones (R1D3, R1D8, R5B9) and SW620-Lv-T7 clones (R1C11, R3C5) by reverse transfection, yielding comparable virus rescue efficiency and virus titres to that of BSR T7/5. This study provides new tools for rNDV rescue and insights into cell line development and virology by reverse genetics.

Designing and evaluation of MERS-CoV siRNAs in HEK-293 cell line

BackgroundThe MERS-CoV was identified for the first time from Jeddah, Saudi Arabia in 2012 from a hospitalized patient. This virus has now been spread to 27 countries with a total of 858 deaths and 2494 confirmed cases and has become a serious concern for the human population. Camels are well known for the transmission of the virus to the human population. MethodsIn this report, we have discussed the designing, prediction, and evaluation of potential siRNAs against the orf1ab gene of MERS-CoV. The online software was used to predict and design the siRNAs and finally, total twenty-one siRNA were filtered out from four hundred and sixty-two sIRNAs as per their scoring and specificity criteria. We have used only ten siRNAs to evaluate their cytotoxicity and efficacy by reverse transfection approach in HEK-293-T cell lines. ResultsBased on the results and data generated; no cytotoxicity was observed for any siRNAs at various concentrations in HEK-293-T cells. The ct value of real-time PCR showed the inhibition of viral replication in siRNA-1, 2, 4, 6, and 9. The data generated provided the preliminary information and encouraged us to evaluate the remaining siRNAs separately as well as in combination to analyses the replication of MERS-CoV inhibition in other cell lines. ConclusionBased on the results obtained; it is concluded that the prediction of siRNAs using online software resulted in the filtration of potential siRNAs with high accuracy and strength. This technology can be used to design and develop antiviral therapy not only for MERS-CoV but also against other viruses.

In silico prediction and experimental validation of siRNAs targeting ORF1ab of MERS-CoV in Vero cell line.

The Middle East Respiratory Syndrome Coronavirus is well known to cause respiratory syndrome and this virus was identified and isolated for the first time from Jeddah, Saudi Arabia in 2012 from infected patient. In this report, we have conducted the in-silico prediction, designing and evaluation of siRNAs targeting Middle East Respiratory Syndrome Coronavirus orf1ab gene to inhibit the virus replication. By using bioinformatics software, total twenty-one functional, off-target reduced siRNA were selected from four hundred and sixty-two siRNAs based on their greater potency and specificity. We have evaluated only seven siRNAs to analyze their performance and efficacy as antivirals by reverse transfection approach in Vero cells. There was no cytotoxicity of siRNAs at various concentrations was observed in Vero cells. Based on the real-time PCR results, better inhibition of viral replication was observed in the siRNA-1 and 4 as compared to other siRNAs. The results generated from this work provided suitable information about the efficacy of siRNAs which encouraged us to further evaluate the remaining siRNAs to determine their inhibitory effect on the virus replication. We concluded that the insilico prediction and designing resulted in the screening of potential siRNAs with better efficiency, and strength. This can be used to develop oligonucleotide-based antiviral therapeutics against MERS-CoV in the near future.

In Vitro Validation of Micro-RNAs (miRNAs) Associated to Treatment Failure in Diffuse Large B-Cell Lymphoma (DLBCL)

Introduction: Micro-RNAs (miRNAs), have been shown to be one of the main regulators of gene expression and other biological processes. Recently, attention is focused on their potential role as prognostic predictors in different types of cancer, including diffuse large B cell lymphoma (DLBCL). We previously reported several miRNAs associated to progression/refractoriness in DLBCL (miR-1244, miR-193b-5p and miR-1231). The aim of this study was to validate these resultsin vitro, exploring the molecular pathways involved. Material and methods: The DLBCL cell line, U-2932 (activated B-cell (ABC) subtype) (ACC 633) was obtained from DSMZ (Germany). Transient reverse transfection of DLBCL cells for 48 or 72 h with mirVana miRNA mimics or inhibitors (miR-1244, miR-193b-5p and miR-1231) or with a miRNA negative control (100 nM) was performed using Viromer®GREEN reagent (Lipocalyx). In some experiments, after 24 h of transfection cells were treated with vehicle (1% DMSO) or with increasing concentrations of CHOP (cyclophosphamide, adriamycin, vincristine sulfate, and prednisone) (Sigma-Aldrich) for 48 h. The ratio of the four drugs was 80 mg/ 5.33 mg/ 0.16 mg/ 5.77 mg. After 48 h of transfection or treatment, cell viability was measured using the CellTiter 96®AQueous One Solution and total RNA was isolated using the RNeasy Mini Kit from Qiagen. Gene expression profiling (GEP) was performed using GeneChip human Clarion S of Affymetrix-ThermoFisher. Results: Reverse transfection of U-2932 with the miRNA mimic negative control (100 nM) for 48 h did not alter the cell viability compared to non-transfected (NT) cells, showing that the transfection method did not affect viability in these DLBCL cells. Transfection of mimic miR-1244, miR-193b-5p or miR-1231 (100 nM) did not significantly alter the viability compared to cells transfected with the miRNA mimic negative control. However, inhibition of these endogenous miRNA molecules for 48 h significantly decreased by 24.1 ± 5.7%, 28.9 ± 6.5% and 30.9 ± 3.3% cell viability, respectively, suggesting that these miRNAs could be involved in the survival of these tumor cells. In these experiments, the percentage of cell transfection was 89.6%. The effect of miRNA 1244, 193b-5p or 1231 on the response of DLBCL cells to CHOP treatment was also evaluated. Transfection of U-2932 cells with miRNA mimic negative control did not alter its sensibility to CHOP, which dose-dependently decreased its viability with an IC50 of 1.45 μg/ml in NT or transfected cells. Transfection of U-2932 cells with mimic miR-1244 or miR-193b-5p (100 nM) increased cell viability in the presence of vehicle (1% DMSO) and also reverted the inhibitory effect of CHOP (0.3 μg/ml) after 48 h of treatment. However, transfection of U-2932 cells with mimic miR-1231 (100 nM) did not alter the inhibitory effect of CHOP (0.3, 1 and 3 μg/ml) on cell viability at any of the concentrations studied. GEP studies showed that these miRNAs reduced the expression of genes associated to chemosensitivity (MCTP1) in the case of miR-1244 and miR-193-5p or tumoral suppressor genes (NRN1) in the case of miR-1231. Conclusions: We validatedin vitroour previously reported miRNAs about the role of miR-1244, miR-193b-5p and miR-1231 associated to treatment failure. The inhibition of endogenous miR-1244, miR-193b-5p or miR-1231 molecules significantly decreased cell viability. Transfection of U-2932 cells with mimic miR-1244 or miR-193b-5p (100 nM) increased cell viability of cells treated with CHOP. GEP studies showed that these miRNAs were linked to chemoresistance and inhibition of tumoral suppressor genes. Future works will have to translate these results to clinical practice in DLBCL. Disclosures Salar: Celgene:Speakers Bureau;Janssen:Speakers Bureau;Roche:Speakers Bureau.

Effect of pre‑freezing and saccharide types in freeze‑drying of siRNA lipoplexes on gene‑silencing effects in the cells by reverse transfection.

Our previous study reported that reverse (Rev)-transfection with small interfering RNA (siRNA)/cationic liposome complexes (siRNA lipoplexes) freeze-dried in trehalose or sucrose solution resulted in high gene-silencing activity in cells. The current study investigated whether pre-freezing or saccharide types present during the freeze-drying of siRNA lipoplexes affected gene-silencing in cells after Rev-transfection. Three types of cationic cholesterol derivatives and three types of dialkyl or trialkyl cationic lipids were used for the preparation of cationic liposomes. Additionally, six types of siRNA lipoplexes were vacuum-dried in trehalose or sucrose solution without a pre-freezing process in multi-well plates. A strong gene-silencing activity after Rev-transfection was observed regardless of the cationic lipid types in the cationic liposomes. It was also investigated whether saccharide types in the freeze-drying of siRNA lipoplexes affected gene-silencing after Rev-transfection. siRNA lipoplexes freeze-dried in monosaccharides (glucose, fructose, galactose or mannose), disaccharides (maltose, lactose, lactulose or cellobiose) and trisaccharide solution (raffinose or melezitose) demonstrated high gene-silencing activity. However, following Rev-transfection with siRNA lipoplexes freeze-dried in monosaccharides or trisaccharides, certain saccharides induced cytotoxicity and/or off-target effects. The results of the current study indicated that disaccharides may be suitable for the preparation of vacuum-dried or freeze-dried siRNA lipoplexes for Rev-transfection.

A solid-phase transfection platform for arrayed CRISPR screens.

Since the publication of this study, it has come to our attention that a citation to the study by Bulkescher et al (2017) was omitted from the Introduction. The following sentence should have been included in the introduction: “A previously reported solid‐phase reverse transfection method for proteins (Bulkescher et al , 2017) was used for the delivery of RNPs for three endogenous genes suggesting the potential of solid‐phase reverse transfection for CRISPR/Cas9‐based gene editing, despite its low efficiency”. We apologise for any inconvenience this omission may have caused.