Publications by authors named "Niek N Sanders"

82 Publications

OMO-1 reduces progression and enhances cisplatin efficacy in a 4T1-based non-c-MET addicted intraductal mouse model for triple-negative breast cancer.

NPJ Breast Cancer 2021 Mar 17;7(1):27. Epub 2021 Mar 17.

Laboratory of Biochemistry, Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.

c-MET is considered a driver of cancer progression, impacting tumor growth and tumor-supporting stroma. Here, we investigated the therapeutic efficacy of OMO-1, a potent and selective c-MET inhibitor, in an immunocompetent intraductal mouse model for triple-negative breast cancer (TNBC). OMO-1 reduced non-c-MET addicted 4T1 tumor progression dose dependently as monotherapeutic and provided additional disease reduction in combination with cisplatin. At the stromal level, OMO-1 significantly reduced neutrophil infiltration in 4T1 tumors, promoted immune activation, and enhanced cisplatin-mediated reduction of tumor-associated macrophages. OMO-1 treatment also reduced 4T1 tumor hypoxia and increased expression of pericyte markers, indicative for vascular maturation. Corroborating this finding, cisplatin delivery to the 4T1 primary tumor was enhanced upon OMO-1 treatment, increasing cisplatin DNA-adduct levels and tumor cell death. Although verification in additional cell lines is warranted, our findings provide initial evidence that TNBC patients may benefit from OMO-1 treatment, even in cases of non-c-MET addicted tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41523-021-00234-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7969607PMC
March 2021

Corticosteroids and mRNA Vaccines: A Word of Caution.

Mol Ther 2021 03 3;29(3):893-894. Epub 2021 Feb 3.

Laboratory of Gene Therapy, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium; Cancer Research Institute (CRIG), Ghent University, 9000 Ghent, Belgium. Electronic address:

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymthe.2021.01.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934628PMC
March 2021

Lipid-Polyglutamate Nanoparticle Vaccine Platform.

ACS Appl Mater Interfaces 2021 Feb 28;13(5):6011-6022. Epub 2021 Jan 28.

Department of Pharmaceutics, Ghent University, 9000 Ghent, Belgium.

Peptide-based subunit vaccines are attractive in view of personalized cancer vaccination with neo-antigens, as well as for the design of the newest generation of vaccines against infectious diseases. Key to mounting robust antigen-specific immunity is delivery of antigen to antigen-presenting (innate immune) cells in lymphoid tissue with concomitant innate immune activation to promote antigen presentation to T cells and to shape the amplitude and nature of the immune response. Nanoparticles that co-deliver both peptide antigen and molecular adjuvants are well suited for this task. However, in the context of peptide-based antigen, an unmet need exists for a generic strategy that allows for co-encapsulation of peptide and molecular adjuvants due to the stark variation in physicochemical properties based on the amino acid sequence of the peptide. These properties also strongly differ from those of many molecular adjuvants. Here, we devise a lipid nanoparticle (LNP) platform that addresses these issues. Key in our concept is poly(l-glutamic acid) (PGA), which serves as a hydrophilic backbone for conjugation of, respectively, peptide antigen (Ag) and an imidazoquinoline (IMDQ) TLR7/8 agonist as a molecular adjuvant. Making use of the PGA's polyanionic nature, we condensate PGA-Ag and PGA-IMDQ into LNP by electrostatic interaction with an ionizable lipid. We show in vitro and in vivo in mouse models that LNP encapsulation favors uptake by innate immune cells in lymphoid tissue and promotes the induction of Ag-specific T cells responses both after subcutaneous and intravenous administration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsami.0c20607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116839PMC
February 2021

Corticosteroids and cellulose purification improve, respectively, the in vivo translation and vaccination efficacy of sa-mRNAs.

Mol Ther 2021 04 21;29(4):1370-1381. Epub 2021 Jan 21.

Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium; Cancer Research Institute (CRIG), Ghent University, 9000 Ghent, Belgium. Electronic address:

Synthetic mRNAs are an appealing platform with multiple biomedical applications ranging from protein replacement therapy to vaccination. In comparison with conventional mRNA, synthetic self-amplifying mRNAs (sa-mRNAs) are gaining interest because of their higher and longer-lasting expression. However, sa-mRNAs also elicit an innate immune response, which may complicate their clinical application. Approaches to reduce the innate immunity of sa-mRNAs have not been studied in detail. Here we investigated, in vivo, the effect of several innate immune inhibitors and a novel cellulose-based mRNA purification approach on the type I interferon (IFN) response and the translation and vaccination efficacy of our formerly developed sa-mRNA vaccine against Zika virus. Among the investigated inhibitors, we found that corticosteroids and especially topical application of clobetasol at the sa-mRNA injection site was the most efficient in suppressing the type I IFN response and increasing the translation of sa-mRNA. However, clobetasol prevented formation of antibodies against sa-mRNA-encoded antigens and should therefore be avoided in a vaccination context. Residual dsRNA by-products of the in vitro transcription reaction are known inducers of immediate type I IFN responses. We additionally demonstrate a drastic reduction of these dsRNA by-products upon cellulose-based purification, reducing the innate immune response and improving sa-mRNA vaccination efficacy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymthe.2021.01.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058483PMC
April 2021

Sterilizing Immunity against SARS-CoV-2 Infection in Mice by a Single-Shot and Lipid Amphiphile Imidazoquinoline TLR7/8 Agonist-Adjuvanted Recombinant Spike Protein Vaccine*.

Angew Chem Int Ed Engl 2021 04 11;60(17):9467-9473. Epub 2021 Mar 11.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

The search for vaccines that protect from severe morbidity and mortality because of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Here we describe an amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile. It is water-soluble and exhibits massive translocation to lymph nodes upon local administration through binding to albumin, affording localized innate immune activation and reduction in systemic inflammation. The adjuvanticity of IMDQ-PEG-CHOL was validated in a licensed vaccine setting (quadrivalent influenza vaccine) and an experimental trimeric recombinant SARS-CoV-2 spike protein vaccine, showing robust IgG2a and IgG1 antibody titers in mice that could neutralize viral infection in vitro and in vivo in a mouse model.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.202015362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8014308PMC
April 2021

Imidazoquinoline-Conjugated Degradable Coacervate Conjugate for Local Cancer Immunotherapy.

ACS Biomater Sci Eng 2020 09 11;6(9):4993-5000. Epub 2020 Aug 11.

Department of Pharmaceutics, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, 44 Wenhuaxi Road, Jinan, Shandong Province 250012, P. R. China.

Strategies that can reduce the harmful side effects of potent immunomodulatory drugs are in high demand to facilitate clinical translation of the newest generation of immunotherapy. Indeed, uncontrolled triggering of the immune system can lead to life-threatening cascade reactions, such as e.g. cytokine storm. In particular, drug formulations that combine simplicity and degradability are of formidable relevance. Imidazoquinolines are an excellent example of such small molecule immunomodulatory drugs that exhibit in unformulated form a highly undesirable pharmacokinetic profile. Imidazoquinolines are potent inducers of type I interferons that are of great interest in the context of anticancer and antiviral therapy through triggering of Toll like receptors 7 and 8. In this work we aimed to alter the pharmacokinetic profile of imidazoquinolines using a simple, yet efficient, strategy that holds high potential for clinical translation. Hereto, we conjugated an imidazoquinoline to the backbone of poly(aspartate) and further formulated this into a degradable coacervate through complex coacervation with a nontoxic degradable polycation. The intrinsic TLR activity of the imidazoquinoline was well preserved and our formulation strategy offered spatial control over its biological activity in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsbiomaterials.0c00485DOI Listing
September 2020

The Opposing Effect of Type I IFN on the T Cell Response by Non-modified mRNA-Lipoplex Vaccines Is Determined by the Route of Administration.

Mol Ther Nucleic Acids 2020 Dec 11;22:373-381. Epub 2020 Sep 11.

VIB-UGent Center for Medical Biotechnology, VIB, Ghent, Belgium.

mRNA-lipoplex vaccines are currently being explored in phase II clinical trials for the treatment of patients with advanced solid tumors. Mechanistically, these mRNA-lipoplex vaccines are characterized by the induction of type I interferon (IFN) centered innate responses. Earlier studies have identified type I IFNs as major regulators of the T cell response instigated by mRNA-lipoplex vaccines. However, stimulatory or, in contrast, profound inhibitory effects of type I IFNs were described depending on the study. In this mouse study, we demonstrated that the opposing roles of type I IFN signaling on the magnitude of the vaccine-evoked T cell responses is dependent on the route of mRNA-lipoplex administration and is regulated at the level of the T cells rather than indirectly through modulation of dendritic cell function. This study helps to understand the double-edged sword character of type I IFN induction upon mRNA-based vaccine treatment and may contribute to a more rational design of mRNA vaccination regimens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.omtn.2020.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533292PMC
December 2020

In Vivo Validation of a Reversible Small Molecule-Based Switch for Synthetic Self-Amplifying mRNA Regulation.

Mol Ther 2021 03 11;29(3):1164-1173. Epub 2020 Nov 11.

Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium; Cancer Research Institute Ghent (CRIG), Ghent University, 9000 Ghent, Belgium. Electronic address:

Synthetic mRNA therapeutics have the potential to revolutionize healthcare, as they enable patients to produce therapeutic proteins inside their own bodies. However, convenient methods that allow external control over the timing and magnitude of protein production after in vivo delivery of synthetic mRNA are lacking. In this study, we validate the in vivo utility of a synthetic self-amplifying mRNA (RNA replicon) whose expression can be turned off using a genetic switch that responds to oral administration of trimethoprim (TMP), a US Food and Drug Administration (FDA)-approved small-molecule drug. After intramuscular electroporation, the engineered RNA replicon exhibited dose-dependent and reversible expression of its encoded protein upon TMP administration. The TMP serum level needed for maximal downregulation of protein translation was approximately 45-fold below that used in humans for therapeutic purposes. To demonstrate the therapeutic potential of the technology, we injected mice with a TMP-responsive RNA replicon encoding erythropoietin (EPO) and successfully controlled the timing and magnitude of EPO production as well as changes in hematocrit. This work demonstrates the feasibility of controlling mRNA kinetics in vivo, thereby broadly expanding the clinical versatility of mRNA therapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymthe.2020.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934578PMC
March 2021

Immune cells as tumor drug delivery vehicles.

J Control Release 2020 11 28;327:70-87. Epub 2020 Jul 28.

Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium; Cancer Research Institute Ghent (CRIG), 9000 Ghent, Belgium. Electronic address:

This review article describes the use of immune cells as potential candidates to deliver anti-cancer drugs deep within the tumor microenvironment. First, the rationale of using drug carriers to target tumors and potentially decrease drug-related side effects is discussed. We further explain some of the current limitations when using nanoparticles for this purpose. Next, a comprehensive step-by-step description of the migration cascade of immune cells is provided as well as arguments on why immune cells can be used to address some of the limitations associated with nanoparticle-mediated drug delivery. We then describe the benefits and drawbacks of using red blood cells, platelets, granulocytes, monocytes, macrophages, myeloid-derived suppressor cells, T cells and NK cells for tumor-targeted drug delivery. An additional section discusses the versatility of nanoparticles to load anti-cancer drugs into immune cells. Lastly, we propose increasing the circulatory half-life and development of conditional release strategies as the two main future pillars to improve the efficacy of immune cell-mediated drug delivery to tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jconrel.2020.07.043DOI Listing
November 2020

Potent and Prolonged Innate Immune Activation by Enzyme-Responsive Imidazoquinoline TLR7/8 Agonist Prodrug Vesicles.

J Am Chem Soc 2020 07 30;142(28):12133-12139. Epub 2020 Jun 30.

Institute for Experimental Molecular Imaging, Uniklinik RWTH Aachen and Helmholtz Institute for Biomedical Engineering, Faculty of Medicine, RWTH Aachen University, 52074 Aachen, Germany.

Synthetic immune-stimulatory drugs such as agonists of the Toll-like receptors (TLR) 7/8 are potent activators of antigen-presenting cells (APCs), however, they also induce severe side effects due to leakage from the site of injection into systemic circulation. Here, we report on the design and synthesis of an amphiphilic polymer-prodrug conjugate of an imidazoquinoline TLR7/8 agonist that in aqueous medium forms vesicular structures of 200 nm. The conjugate contains an endosomal enzyme-responsive linker enabling degradation of the vesicles and release of the TLR7/8 agonist in native form after endocytosis, which results in high TLR agonist activity. In a mouse model, locally administered vesicles provoke significantly more potent and long-lasting immune stimulation in terms of interferon expression at the injection site and in draining lymphoid tissue compared to a nonamphiphilic control and the native TLR agonist. Moreover, the vesicles induce robust activation of dendritic cells in the draining lymph node .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jacs.0c01928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116109PMC
July 2020

Comparative Profiling of Metastatic 4T1- vs. Non-metastatic Py230-Based Mammary Tumors in an Intraductal Model for Triple-Negative Breast Cancer.

Front Immunol 2019 17;10:2928. Epub 2019 Dec 17.

Laboratory of Biochemistry, Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.

The transition of ductal carcinoma (DCIS) to invasive carcinoma (IC) in breast cancer can be faithfully reproduced by the intraductal mouse model. Envisaging to use this model for therapeutic testing, we aimed to in-depth characterize the tumor immunity associated with the differential progression of two types of intraductal tumors. More specifically, we focused on triple-negative breast cancer (TNBC) and intraductally inoculated luciferase-expressing metastatic 4T1 and locally invasive Py230 cells in lactating mammary glands of syngeneic BALB/c and C57BL/6 female mice, respectively. Although the aggressive 4T1 cells rapidly formed solid tumors, Py230 tumors eventually grew to a similar size through enhanced proliferation. Yet, ductal tumor cell breakthrough and metastasis occurred earlier in the 4T1- compared to the Py230-based intraductal model and was associated with high expression of matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) as well as an increased influx of immune cells (mainly macrophages, neutrophils and T-cells). Moreover, activated cytotoxic T-cells, B-cells and programmed death-1 (PD-1)-positive cells were more prominent in the 4T1-based intraductal model in line with enhanced pro-inflammatory cytokine and gene expression profiles. Py230-based tumors showed a more immunosuppressed anti-inflammatory profile with a high amount of regulatory T-cells, which may account for the decreased T-cell activation but increased proliferation compared to the 4T1-based tumors. Taken together, our results highlight the differential immunological aspects of aggressive metastatic and non-aggressive intraductal progression of 4T1- vs. Py230-based tumors, providing a base for future studies to explore therapy using these intraductal TNBC models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.02928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927949PMC
November 2020

Mononuclear but Not Polymorphonuclear Phagocyte Depletion Increases Circulation Times and Improves Mammary Tumor-Homing Efficiency of Donor Bone Marrow-Derived Monocytes.

Cancers (Basel) 2019 Nov 8;11(11). Epub 2019 Nov 8.

Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium.

Tumor associated macrophages are an essential part of the tumor microenvironment. Consequently, bone marrow-derived monocytes (BMDMs) are continuously recruited to tumors and are therefore seen as ideal delivery vehicles with tumor-targeting properties. By using immune cell depleting agents and macroscopic in vivo fluorescence imaging, we demonstrated that removal of endogenous monocytes and macrophages (but not neutrophils) leads to an increased tumor accumulation of exogenously administered BMDMs. By means of intravital microscopy (IVM), we confirmed our macroscopic findings on a cellular level and visualized in real time the migration of the donor BMDMs in the tumors of living animals. Moreover, IVM also revealed that clodronate-mediated depletion drastically increases the circulation time of the exogenously administered BMDMs. In summary, these new insights illustrate that impairment of the mononuclear phagocyte system increases the circulation time and tumor accumulation of donor BMDMs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers11111752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6896201PMC
November 2019

Expression Kinetics and Innate Immune Response after Electroporation and LNP-Mediated Delivery of a Self-Amplifying mRNA in the Skin.

Mol Ther Nucleic Acids 2019 Sep 7;17:867-878. Epub 2019 Aug 7.

Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium; Cancer Research Institute (CRIG), Ghent University, Ghent, Belgium. Electronic address:

In this work, we studied the expression kinetics and innate immune response of a self-amplifying mRNA (sa-RNA) after electroporation and lipid-nanoparticle (LNP)-mediated delivery in the skin of mice. Intradermal electroporation of the sa-RNA resulted in a plateau-shaped expression, with the plateau between day 3 and day 10. The overall protein expression of sa-RNA was significantly higher than that obtained after electroporation of plasmid DNA (pDNA) or non-replication mRNAs. Moreover, using IFN-β reporter mice, we elucidated that intradermal electroporation of sa-RNA induced a short-lived moderate innate immune response, which did not affect the expression of the sa-RNA. A completely different expression profile and innate immune response were observed when LNPs were used. The expression peaked 24 h after intradermal injection of sa-RNA-LNPs and subsequently showed a sharp drop. This drop might be explained by a translational blockage caused by the strong innate immune response that we observed in IFN-β reporter mice shortly (4 h) after intradermal injection of sa-RNA-LNPs. A final interesting observation was the capacity of sa-RNA-LNPs to transfect the draining lymph nodes after intradermal injection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.omtn.2019.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6722285PMC
September 2019

Immunogenicity and Protection Efficacy of a Naked Self-Replicating mRNA-Based Zika Virus Vaccine.

Vaccines (Basel) 2019 Aug 23;7(3). Epub 2019 Aug 23.

Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium.

To combat emerging infectious diseases like Zika virus (ZIKV), synthetic messenger RNAs (mRNAs) encoding viral antigens are very attractive as they allow a rapid, generic, and flexible production of vaccines. In this work, we engineered a self-replicating mRNA (sr-mRNA) vaccine encoding the pre-membrane and envelope (prM-E) glycoproteins of ZIKV. Intradermal electroporation of as few as 1 µg of this mRNA-based ZIKV vaccine induced potent humoral and cellular immune responses in BALB/c and especially IFNAR1 C57BL/6 mice, resulting in a complete protection of the latter mice against ZIKV infection. In wild-type C57BL/6 mice, the vaccine resulted in very low seroconversion rates and antibody titers. The potency of the vaccine was inversely related to the dose of mRNA used in wild-type BALB/c or C57BL/6 mice, as robust type I interferon (IFN) response was determined in a reporter mice model (IFN-β). We further investigated the inability of the sr-prM-E-mRNA ZIKV vaccine to raise antibodies in wild-type C57BL/6 mice and found indications that type I IFNs elicited by this naked sr-mRNA vaccine might directly impede the induction of a robust humoral response. Therefore, we assume that the efficacy of sr-mRNA vaccines after intradermal electroporation might be increased by strategies that temper their inherent innate immunogenicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/vaccines7030096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789535PMC
August 2019

Potent Lymphatic Translocation and Spatial Control Over Innate Immune Activation by Polymer-Lipid Amphiphile Conjugates of Small-Molecule TLR7/8 Agonists.

Angew Chem Int Ed Engl 2019 10 12;58(43):15390-15395. Epub 2019 Sep 12.

Department of Pharmaceutics, Ghent University, Ghent, Belgium.

Uncontrolled systemic inflammatory immune triggering has hampered the clinical translation of several classes of small-molecule immunomodulators, such as imidazoquinoline TLR7/8 agonists for vaccine design and cancer immunotherapy. By taking advantage of the inherent serum-protein-binding property of lipid motifs and their tendency to accumulate in lymphoid tissue, we designed amphiphilic lipid-polymer conjugates that suppress systemic inflammation but provoke potent lymph-node immune activation. This work provides a rational basis for the design of lipid-polymer amphiphiles for optimized lymphoid targeting.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201905687DOI Listing
October 2019

Improving the Repeatability and Efficacy of Intradermal Electroporated Self-Replicating mRNA.

Mol Ther Nucleic Acids 2019 Sep 28;17:388-395. Epub 2019 Jun 28.

Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium; Cancer Research Institute (CRIG), Ghent University, Merelbeke, Belgium. Electronic address:

Local administration of naked self-replicating mRNA (sr-mRNA) in the skin or muscle using electroporation is effective but hampered by low repeatability. In this manuscript, we demonstrated that intradermal electroporation of sr-mRNA in combination with a protein-based RNase inhibitor increased the expression efficiency, success rate, and repeatability of the data. The RNase inhibitor should be added just before administration because storage of the inhibitor together with the sr-mRNA at -80°C resulted in a partial loss of the beneficial effect. Furthermore, the location of intradermal electroporation also had a major effect on the expression of the sr-mRNA, with the highest and longest expression observed at the tail base of the mice. In contrast with previous work, we did not observe a beneficial effect of calcium ions on the efficacy of naked sr-mRNA after intradermal injection. Finally, another important finding was that the traditional representation of in vivo bioluminescence data as means in logarithmic graphs can mask highly variable data. A more truthful representation can be obtained by showing the individual data points or by displaying median values in combination with interquartile ranges. In conclusion, intradermal sr-mRNA electroporation can be improved by adding an RNase inhibitor and injecting at the tail base.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.omtn.2019.06.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626868PMC
September 2019

Small-molecule-based regulation of RNA-delivered circuits in mammalian cells.

Nat Chem Biol 2018 11 16;14(11):1043-1050. Epub 2018 Oct 16.

Synthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA, USA.

Synthetic mRNA is an attractive vehicle for gene therapies because of its transient nature and improved safety profile over DNA. However, unlike DNA, broadly applicable methods to control expression from mRNA are lacking. Here we describe a platform for small-molecule-based regulation of expression from modified RNA (modRNA) and self-replicating RNA (replicon) delivered to mammalian cells. Specifically, we engineer small-molecule-responsive RNA binding proteins to control expression of proteins from RNA-encoded genetic circuits. Coupled with specific modRNA dosages or engineered elements from a replicon, including a subgenomic promoter library, we demonstrate the capability to externally regulate the timing and level of protein expression. These control mechanisms facilitate the construction of ON, OFF, and two-output switches, with potential therapeutic applications such as inducible cancer immunotherapies. These circuits, along with other synthetic networks that can be developed using these tools, will expand the utility of synthetic mRNA as a therapeutic modality.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41589-018-0146-9DOI Listing
November 2018

Lymph-Node-Targeted Immune Activation by Engineered Block Copolymer Amphiphiles-TLR7/8 Agonist Conjugates.

J Am Chem Soc 2018 10 17;140(43):14300-14307. Epub 2018 Oct 17.

Department of Pharmaceutics and Cancer Research Institute Ghent (CRIG) , Ghent University , Ghent 9000 , Belgium.

Small molecule immuno-modulators such as agonists of Toll-like receptors (TLRs) are attractive compounds to stimulate innate immune cells toward potent antiviral and antitumor responses. However, small molecules rapidly enter the systemic circulation and cause "wasted inflammation". Hence, synthetic strategies to confine their radius of action to lymphoid tissue are of great relevance, to both enhance their efficacy and concomitantly limit toxicity. Here, we demonstrate that covalent conjugation of a small molecule TLR7/8 agonist immunomodulatory to a micelle-forming amphiphilic block copolymer greatly alters the pharmacokinetic profile, resulting in highly efficient lymphatic delivery. Moreover, we designed amphiphilic block copolymers in such a way to form thermodynamically stable micelles through π-π stacking between aromatic moieties, and we engineered the block copolymers to undergo an irreversible amphiphilic to hydrophilic transition in response to the acidic endosomal pH.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/jacs.8b08595DOI Listing
October 2018

Nanoparticle-Conjugate TLR7/8 Agonist Localized Immunotherapy Provokes Safe Antitumoral Responses.

Adv Mater 2018 Nov 1;30(45):e1803397. Epub 2018 Oct 1.

Department of Pharmaceutics, Cancer Research Institute Ghent (CRIG), Ghent University, 9000, Ghent, Belgium.

Localized therapeutic modalities that subvert the tumor microenvironment from immune-suppressive to pro-immunogenic can elicit systemic antitumor immune responses that induce regression of directly treated as well as nontreated distal tumors. A key toward generating robust antitumor T cell responses is the activation of dendritic cells (DCs) in the tumor microenvironment. Treatment with agonists triggering various pattern recognition receptors is very efficient to activate DCs, yet suffers from the induction of serious immune-related adverse effects, which is closely linked to their unfavorable PK/PD profile causing systemic immune activation and cytokine release. Here, it is reported that nanoparticle conjugation of a highly potent TLR7/8 agonist restricts immune activation to the tumor bed and its sentinel lymph nodes without hampering therapeutic antitumor efficacy. On a mechanistic level, it is confirmed that localized treatment with a nanoparticle-conjugated TLR7/8 agonist leads to potent activation of DCs in the sentinel lymph nodes and promotes proliferation of tumor antigen-specific CD8 T cells. Furthermore, therapeutic improvement upon combination with anti-PDL1 checkpoint inhibition and Flt3L, a growth factor that expands and mobilizes DCs from the bone marrow, is demonstrated. The findings provide a rational base for localized tumor engineering by nanomedicine strategies that provide spatial control over immune-activation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/adma.201803397DOI Listing
November 2018

Anti-inflammatory signaling by mammary tumor cells mediates prometastatic macrophage polarization in an innovative intraductal mouse model for triple-negative breast cancer.

J Exp Clin Cancer Res 2018 Aug 15;37(1):191. Epub 2018 Aug 15.

Laboratory of Biochemistry, Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.

Background: Murine breast cancer models relying on intraductal tumor cell inoculations are attractive because they allow the study of breast cancer from early ductal carcinoma in situ to metastasis. Using a fully immunocompetent 4T1-based intraductal model for triple-negative breast cancer (TNBC) we aimed to investigate the immunological responses that guide such intraductal tumor progression, focusing on the prominent role of macrophages.

Methods: Intraductal inoculations were performed in lactating female mice with luciferase-expressing 4T1 mammary tumor cells either with or without additional RAW264.7 macrophages, mimicking basal versus increased macrophage-tumor cell interactions in the ductal environment. Imaging of 4T1-derived luminescence was used to monitor primary tumor growth and metastases. Tumor proliferation, hypoxia, disruption of the ductal architecture and tumor immune populations were determined immunohistochemically. M1- (pro-inflammatory) and M2-related (anti-inflammatory) cytokine levels were determined by Luminex assays and ELISA to investigate the activation state of the macrophage inoculum. Levels of the metastatic proteins matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor (VEGF) as well as of the immune-related disease biomarkers chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) were measured by ELISA to evaluate disease progression at the protein level.

Results: Mice intraductally co-injected with macrophages showed severe splenomegaly with faster ductal breakthrough of tumor cells and increased metastases in axillary lymph nodes and lungs. These mice showed higher M1-related cytokines in the early disease stages (at 1 to 3 weeks post-inoculation) due to the pro-inflammatory nature of RAW264.7 macrophages with increased Ly6G-positive neutrophils and decreased anti-inflammatory macrophages in the tumor microenvironment. However, upon metastasis (at 5 weeks post-inoculation), a prominent increase in M2-related cytokine levels was detected and established a tumor microenvironment with similar immune populations and cytokine responses as in mice which received only 4T1 tumor cells. The observed tumor-associated immune responses and the increased metastasis were associated with significantly induced local and systemic levels of MMP-9, VEGF, CHI3L1 and LCN2.

Conclusions: The current experimental study with an innovative immunocompetent intraductal model for TNBC pinpoints towards a metastasis-supporting M1 to M2 macrophage polarization in the mammary ducts mediated by 4T1-derived signaling. We propose to explore this process as immunotherapeutic target.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13046-018-0860-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6094904PMC
August 2018

Off-Target and Tumor-Specific Accumulation of Monocytes, Macrophages and Myeloid-Derived Suppressor Cells after Systemic Injection.

Neoplasia 2018 08 18;20(8):848-856. Epub 2018 Jul 18.

Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium; Cancer Research Institute Ghent (CRIG), Ghent, Belgium. Electronic address:

Solid tumors frequently coexist with a degree of local chronic inflammation. Recruited myeloid cells can therefore be considered as interesting vehicles for tumor-targeted delivery of therapeutic agents. Using in vivo imaging, the short-term accumulation of systemically injected monocytes, macrophages and myeloid-derived suppressor cells (MDSCs) was compared in mice bearing fat pad mammary carcinomas. Monocytes and macrophages demonstrated almost identical in vivo and ex vivo distribution patterns with maximal tumor-associated accumulation seen 48 hours after injection that remained stable over the 4-day follow-up period. However, a substantial accumulation of both cell types was also seen in the liver, spleen and lungs albeit decreasing over time in all three locations. The MDSCs exhibited a similar distribution pattern as the monocytes and macrophages, but demonstrated a better relative on-target fraction over time. Overall, our findings highlight off-target cell accumulation as a major obstacle in the use of myeloid cells as vehicles for therapeutic tumor-targeted agents and indicate that their short-term on-target accumulation is mainly of nonspecific nature.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neo.2018.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6076377PMC
August 2018

Comparison of the Expression Kinetics and Immunostimulatory Activity of Replicating mRNA, Nonreplicating mRNA, and pDNA after Intradermal Electroporation in Pigs.

Mol Pharm 2018 02 17;15(2):377-384. Epub 2018 Jan 17.

Faculty of Veterinary Medicine, Department of Nutrition, Genetics, and Ethology, Laboratory for Gene Therapy, Ghent University , Heidestraat 19, 9820 Merelbeke, Belgium.

Synthetic mRNA is becoming increasingly popular as an alternative to pDNA-based gene therapy. Currently, multiple synthetic mRNA platforms have been developed. In this study we investigated the expression kinetics and the changes in mRNA encoding cytokine and chemokine levels following intradermal electroporation in pigs of pDNA, self-replicating mRNA, and modified and unmodified mRNA. The self-replicating mRNA tended to induce the highest protein expression, followed by pDNA, modified mRNA, and unmodified mRNA. Interestingly, the self-replicating mRNA was able to maintain its high expression levels during at least 12 days. In contrast, the expression of pDNA and the nonreplicating mRNAs dropped after respectively one and two days. Six days after intradermal electroporation a dose-dependent expression was observed for all vectors. Again, also at lower doses, the self-replicating mRNA tended to show the highest expression. All the mRNA vectors, including the modified mRNA, induced elevated levels of mRNA encoding cytokines and chemokines in the porcine skin after intradermal electroporation, while no such response was noticed after intradermal electroporation of the pDNA vector.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.molpharmaceut.7b00722DOI Listing
February 2018

Evaluation of a xenogeneic vascular endothelial growth factor-2 vaccine in two preclinical metastatic tumor models in mice.

Cancer Immunol Immunother 2017 Dec 3;66(12):1545-1555. Epub 2017 Aug 3.

Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, 9820, Merelbeke, Belgium.

In this study, a xenogeneic DNA vaccine encoding for human vascular endothelial growth factor receptor-2 (hVEGFR-2) was evaluated in two murine tumor models, the B16-F10 melanoma and the EO771 breast carcinoma model. The vaccine was administered by intradermal injection followed by electroporation. The immunogenicity and the biological efficacy of the vaccine were tested in (1) a prophylactic setting, (2) a therapeutic setting, and (3) a therapeutic setting combined with surgical removal of the primary tumor. The tumor growth, survival, and development of an immune response were followed. The cellular immune response was measured by a bioluminescence-based cytotoxicity assay with vascular endothelial growth factor-2 (VEGFR-2)-expressing target cells. Humoral immune responses were quantified by enzyme-linked immunosorbent assay (ELISA). Ex vivo bioluminescence imaging and immunohistological observation of organs were used to detect (micro)metastases. A cellular and humoral immune response was present in prophylactically and therapeutically vaccinated mice, in both tumor models. Nevertheless, survival in prophylactically vaccinated mice was only moderately increased, and no beneficial effect on survival in therapeutically vaccinated mice could be demonstrated. An influx of CD3+ cells and a slight decrease in VEGFR-2 were noticed in the tumors of vaccinated mice. Unexpectedly, the vaccine caused an increased quantity of early micrometastases in the liver. Lung metastases were not increased by the vaccine. These early liver micrometastases did however not grow into macroscopic metastases in either control or vaccinated mice when allowed to develop further after surgical removal of the primary tumor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00262-017-2046-3DOI Listing
December 2017

Immunological, anti-angiogenic and clinical effects of intratumoral interleukin 12 electrogene therapy combined with metronomic cyclophosphamide in dogs with spontaneous cancer: A pilot study.

Cancer Lett 2017 08 28;400:205-218. Epub 2016 Sep 28.

Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, B-9820 Merelbeke, Belgium; Cancer Research Institute Ghent (CRIG), Ghent University, Belgium. Electronic address:

The immunological, anti-angiogenic and clinical effects of metronomic cyclophosphamide and 3 consecutive intratumoral interleukin (IL)-12 gene therapy (electrogene therapy (EGT)) treatments were evaluated in 6 dogs with spontaneous cancer. In all dogs, a decrease in peripheral leukocytes 2 days after IL-12 EGT coincided with erythema and swelling of the tumor. In the tumor, a transient increase in IL-12 levels was measured, whereas a continuous increase in interferon γ (IFNγ) and thrombospondin 1 (TSP-1) were determined in contrast to a continuous decrease in vascular endothelial growth factor (VEGF). In the serum, a transient increase in IL-12 and IL-10 levels were noted in contrast to a transient decrease in VEGF and TSP-1. The treatment resulted in a significant anti-angiogenic effect. Although all primary tumors continued to progress in time, this progression was slower than before treatment according to the contrast-enhanced ultrasound data. Besides the encouraging immunostimulatory and anti-angiogenic effects observed in all dogs we also noticed in 4 out of 6 dogs clinically relevant improvements in quality of life and weight. These results hold great promise for combinatorial strategies of IL-12 EGT and metronomic chemotherapy with conventional antitumor (immuno)therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.canlet.2016.09.015DOI Listing
August 2017

Comparison of the Adipose and Luminal Mammary Gland Compartment as Orthotopic Inoculation Sites in a 4T1-Based Immunocompetent Preclinical Model for Triple-Negative Breast Cancer.

J Mammary Gland Biol Neoplasia 2016 12 6;21(3-4):113-122. Epub 2016 Oct 6.

Laboratory of Biochemistry, Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.

Breast tumorigenesis is classically studied in mice by inoculating tumor cells in the fat pad, the adipose compartment of the mammary gland. Alternatively, the mammary ducts, which constitute the luminal mammary gland compartment, also provide a suitable inoculation site to induce breast cancer in murine models. The microenvironments in these compartments influence tumor cell progression, yet this effect has not been investigated in an immunocompetent context. Here, we compared both mammary gland compartments as distinct inoculation sites, taking into account the immunological aspect by inoculating 4T1 tumor cells in immunocompetent mice. Following tumor cell inoculation in the adipose compartment of non-pretreated/naive, hormonally pretreated/naive and non-pretreated/lactating mice, the primary tumors developed similarly. However, a slower onset of primary tumor growth was found after inoculations in the luminal compartment of non-pretreated/lactating mice. Despite this difference in tumor development rate, metastasis to the liver and lungs was equally observed and was accompanied by lymphatic spreading of tumor cells and progressive splenomegaly with both inoculation types. Chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) served as innovative biomarkers for disease progression showing increased levels in primary tumors and sera of the non-pretreated/lactating inoculation groups. A slower increase in circulating CHI3L1 but not LCN2 levels, was observed after inoculations in the luminal compartment which corroborated the slower tumor development at this inoculation site. Our results highlight the critical impact of different mammary gland compartments on tumor development in syngeneic murine models and support the use of novel tumor progression biomarkers in an immune-competent environment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10911-016-9362-7DOI Listing
December 2016

Coadministration of a Plasmid Encoding HIV-1 Gag Enhances the Efficacy of Cancer DNA Vaccines.

Mol Ther 2016 09 20;24(9):1686-96. Epub 2016 Jun 20.

Advanced Drug Delivery and Biomaterials, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium.

DNA vaccination holds great promise for the prevention and treatment of cancer and infectious diseases. However, the clinical ability of DNA vaccines is still controversial due to the limited immune response initially observed in humans. We hypothesized that electroporation of a plasmid encoding the HIV-1 Gag viral capsid protein would enhance cancer DNA vaccine potency. DNA electroporation used to deliver plasmids in vivo, induced type I interferons, thereby supporting the activation of innate immunity. The coadministration of ovalbumin (OVA) and HIV-1 Gag encoding plasmids modulated the adaptive immune response. This strategy favored antigen-specific Th1 immunity, delayed B16F10-OVA tumor growth and improved mouse survival in both prophylactic and therapeutic vaccination approaches. Similarly, a prophylactic DNA immunization against the melanoma-associated antigen gp100 was enhanced by the codelivery of the HIV-1 Gag plasmid. The adjuvant effect was not driven by the formation of HIV-1 Gag virus-like particles. This work highlights the ability of both electroporation and the HIV-1 Gag plasmid to stimulate innate immunity for enhancing cancer DNA vaccine immunogenicity and demonstrates interesting tracks for the design of new translational genetic adjuvants to overcome the current limitations of DNA vaccines in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/mt.2016.122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5113100PMC
September 2016

Immunogenicity and safety of xenogeneic vascular endothelial growth factor receptor-2 DNA vaccination in mice and dogs.

Oncotarget 2016 Mar;7(10):10905-16

Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium.

Vascular endothelial growth factor receptor-2 (VEGFR-2) is an attractive target in oncology due to its crucial role in angiogenesis. In this study a DNA vaccine coding for human VEGFR-2 was evaluated in healthy mice and dogs, administered by intradermal injection and electroporation. In mice, three doses and vaccination schedules were evaluated. Cellular immune responses were measured by intracellular IFN-gamma staining and a cytotoxicity assay and antibodies by ELISA. Safety was assessed by measuring regulatory T cells and myeloid derived suppressor cells and a wound healing assay. The vaccine was subsequently evaluated in dogs, which were vaccinated three times with 100µg. Cellular immune responses were measured by intracellular IFN-gamma staining and antibodies by a flow cytometric assay. In mice, maximal cellular responses were observed after two vaccinations with 5µg. Humoral responses continued to increase with higher dose and number of vaccinations. No abnormalities in the measured safety parameters were observed. The vaccine was also capable of eliciting a cellular and humoral immune response in dogs. No adverse effects were observed, but tolerability of the electroporation was poor. This study will facilitate the evaluation of the vaccine in tumor bearing animals, ranging from rodent models to dogs with spontaneous tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.7265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4905448PMC
March 2016

Topical imiquimod yields systemic effects due to unintended oral uptake.

Sci Rep 2016 Jan 28;6:20134. Epub 2016 Jan 28.

Inflammation Research Center, VIB, Ghent, Belgium.

Repetitive application of topical imiquimod is used as an experimental model for the induction of psoriasiform skin lesions in mice. The model is characterized by several inflammatory processes, including cytokine production both locally and systemically, cellular infiltration, and splenomegaly. To investigate the production of type I interferons in response to imiquimod-containing Aldara cream, IFNβ-luciferase reporter mice were imaged in vivo and ex vivo. Type I interferons were found to be produced in the skin, but also in the intestinal system caused by unintended ingestion of imiquimod by the mice. Through the use of Elizabethan collars to prevent ingestion, these effects, including psoriasiform lesions were nearly completely prevented. Our findings reveal that topical treatment with Aldara induces a psoriasiform skin inflammation, but that its mode of action depends on ingestion of the chemical, which leads to systemic responses and affects local inflammation. Therefore, potential ingestion of topical treatments during experimental procedures should be taken into account during assessment of cutaneous inflammatory parameters in skin disease models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep20134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4730190PMC
January 2016

Can dendritic cells improve whole cancer cell vaccines based on immunogenically killed cancer cells?

Oncoimmunology 2015 Dec 1;4(12):e1048413. Epub 2015 Jun 1.

Laboratory of Gene Therapy, Department of Nutrition, Genetics and Ethology; Faculty of Veterinary Medicine; Ghent University ; Merelbeke, Belgium.

Immunogenic cell death (ICD) offers interesting opportunities in cancer cell (CC) vaccine manufacture, as it increases the immunogenicity of the dead CC. Furthermore, fusion of CCs with dendritic cells (DCs) is considered a superior method for generating whole CC vaccines. Therefore, in this work, we determined in naive mice whether immunogenically killed CCs per se (CC vaccine) elicit an antitumoral immune response different from the response observed when immunogenically killed CCs are associated with DCs through fusion (fusion vaccine) or through co-incubation (co-incubation vaccine). After tumor inoculation, the type of immune response in the prophylactically vaccinated mice differed between the groups. In more detail, fusion vaccines elicited a humoral anticancer response, whereas the co-incubation and CC vaccine mainly induced a cellular response. Despite these differences, all three approaches offered a prophylactic protection against tumor development in the murine mammary carcinoma model. In summary, it can be concluded that whole CC vaccines based on immunogenically killed CCs may not necessarily require association with DCs to elicit a protective anticancer immune response. If this finding can be endorsed in other cancer models, the manufacture of CC vaccines would greatly benefit from this new insight, as production of DC-based vaccines is laborious, time-consuming and expensive.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/2162402X.2015.1048413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4635695PMC
December 2015

N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice.

J Control Release 2015 Nov 3;217:337-44. Epub 2015 Sep 3.

Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, 500 Technology Square, Cambridge, MA, USA. Electronic address:

Messenger RNA as a therapeutic modality is becoming increasingly popular in the field of gene therapy. The realization that nucleobase modifications can greatly enhance the properties of mRNA by reducing the immunogenicity and increasing the stability of the RNA molecule (the Kariko paradigm) has been pivotal for this revolution. Here we find that mRNAs containing the N(1)-methylpseudouridine (m1Ψ) modification alone and/or in combination with 5-methylcytidine (m5C) outperformed the current state-of-the-art pseudouridine (Ψ) and/or m5C/Ψ-modified mRNA platform by providing up to ~44-fold (when comparing double modified mRNAs) or ~13-fold (when comparing single modified mRNAs) higher reporter gene expression upon transfection into cell lines or mice, respectively. We show that (m5C/)m1Ψ-modified mRNA resulted in reduced intracellular innate immunogenicity and improved cellular viability compared to (m5C/)Ψ-modified mRNA upon in vitro transfection. The enhanced capability of (m5C/)m1Ψ-modified mRNA to express proteins may at least partially be due to the increased ability of the mRNA to evade activation of endosomal Toll-like receptor 3 (TLR3) and downstream innate immune signaling. We believe that the (m5C/)m1Ψ-mRNA platform presented here may serve as a new standard in the field of modified mRNA-based therapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jconrel.2015.08.051DOI Listing
November 2015