Expression kinetics and innate immune response after electroporation and LNP-mediated delivery of a self-amplifying mRNA in the skin

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-beta 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-beta 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

Improving the repeatability and efficacy of intradermal electroporated self-replicating mRNA

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 degrees 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

Mononuclear but not polymorphonuclear phagocyte depletion increases circulation times and improves mammary tumor-homing efficiency of donor bone marrow-derived monocytes

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

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

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

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

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

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

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

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

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

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

© 2020 The American Society of Gene and Cell Therapy 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. Synthetic mRNAs are gaining attention as a safe alternative to viral and DNA-based gene delivery for therapeutic purposes. Recently, self-amplifying mRNAs whose translation can be controlled by the small-molecule drug trimethoprim were described but their utility had not yet been evaluated in mice. Mc Cafferty et al. demonstrate effective modulation of protein expression from intramuscularly electroporated self-amplifying mRNA by oral administration of trimethoprim