Biology subjects

Mager, I.

Publications and source records attributed to Mager, I..

8 recordsLinked to original sources

bioRxiv2026

Mapping, stacking and characterization of a novel resistance haplotype against GpaVvrn-virulent Globodera pallida derived from wild potato relative Solanum brevicaule

Potato production is threatened by pests and pathogens. Among the most harmful are the potato cyst nematodes (PCN), including Globodera pallida. The major control measures are potato cultivars harbouring resistance genes. However, virulent nematode populations are breaking through the most broadly applied resistance genes, and therefore novel resistance genes are required to keep control of G. pallida. In this study we map a resistance locus in wild potato relative Solanum brevicaule to the bottom of chromosome 11 (42.83 - 42.85 Mb) using a k-mer based bulked segregant analysis and name the resistant haplotype GpaXIbrc. The presence of the GpaXIbrc marker in the potato genome significantly increases resistance against a GpaVvrn-virulent G. pallida population (AMPOP01). Furthermore, the GpaXIbrc haplotype was stacked with three other resistance haplotypes: GpaXItar, GpaVvrn, and GpaIXvrn. Stacking of the novel GpaXIbrc haplotype to these other resistance haplotypes resulted in a significant increase in resistance. The largest effect was seen in combinations with 'major' resistance haplotypes GpaVvrn + GpaIXvrn and GpaXItar + GpaXIbrc. To determine whether we are dealing with a novel allele, the resistance mechanism underlying the GpaXIbrc haplotype was described after performing an in vitro bioassay. The resistance mechanism was different from GpaXIspl as described in literature, which is also localized at the bottom of chromosome 11. Therefore, we conclude that we found a novel resistance allele originating from S. brevicaule.

genetics↗

bioRxiv2026

Quantitative Assessment of shRNA Loading and Delivery Efficiency of Engineered Extracellular Vesicles

RNA interference (RNAi) therapeutics enable selective silencing of disease-associated genes. Yet, their clinical application remains largely confined to the liver due to extrahepatic delivery constraints of current platforms such as GalNAc conjugates and lipid nanoparticles. Extracellular vesicles (EVs) offer an attractive alternative delivery strategy owing to their biocompatibility, ability to traverse biological barriers, and amenability to engineering. However, EV-mediated RNA delivery is limited by inefficient endogenous RNA loading and poor cytosolic release following uptake. Here, we establish a modular EV-based platform that addresses both challenges by integrating enhanced endogenous shRNA loading with fusogen-mediated cytosolic delivery. Using Argonaute 2 (AGO2)-assisted loading, we substantially increase shRNA copy numbers per vesicle (up to 3.7 copies/EV) and enable quantitative, molecule-resolved assessment of delivery potency. Engineered EVs achieve robust and reproducible shRNA-mediated gene silencing with picomolar IC50 values across multiple cell types and induce significant target knockdown in the mouse brain following intracerebral administration. Together, these findings demonstrate that coordinated engineering of shRNA loading and cytosolic release can overcome key limitations of EV-mediated small RNA delivery.

bioengineering↗

bioRxiv2024

Controlling reactogenicity while preserving immunogenicity from a self-amplifying RNA vaccine by modulating nucleocytoplasmic transport

Self-amplifying RNA (saRNA)-based vaccines have emerged as a potent and durable RNA vaccine platform. However, RNA vaccine platforms trigger undesirable side effects at protective doses, underscoring the need for improved tolerability. To address this, we leveraged the Cardiovirus leader protein, which is well-characterized to dampen host innate signaling by modulating nucleocytoplasmic transport (NCT). Co-administration of a leader-protein-encoding mRNA (which we have named "RNAx") delivered alongside vaccine cargo saRNA reduced interferon production while enhancing Influenza hemagglutinin (HA) expression in human primary cells and murine models. RNAx potently decreased serum biomarkers of reactogenicity after immunizations with an HA-expressing saRNA-LNP vaccine while maintaining the magnitude of the antibody and cellular response. RNAx also consistently enhanced binding antibody titers after a single injection and in some conditions enhanced binding antibody and neutralization titers post-boost. These findings support RNAx as a promising platform approach for improving tolerability of saRNA-LNP vaccines while preserving or enhancing immunogenicity.

immunology↗

bioRxiv2023

Multimodal engineering of extracellular vesicles for efficient intracellular protein delivery

Extracellular vesicles (EVs) are promising tools to transfer macromolecular therapeutic molecules to recipient cells, however, efficient functional intracellular protein delivery by EVs remains challenging. Here, we have developed novel and versatile systems that leverage selected molecular tools to engineer EVs for robust cytosolic protein delivery both in vitro and in vivo. These systems, termed VSV-G plus EV-sorting Domain-Intein-Cargo (VEDIC) and VSV-G-Foldon-Intein-Cargo (VFIC), exploit an engineered mini-intein (intein) protein with self-cleavage activity to link cargo to an EV-sorting domain and release it from the EV membrane inside the EV lumen. In addition, we utilize the fusogenic protein VSV-G to facilitate endosomal escape and cargo release from the endosomal system to the cytosol of recipient cells. Importantly, we demonstrate that the combination of the self-cleavage intein, fusogenic protein and EV-sorting domain are indispensable for efficient functional intracellular delivery of cargo proteins by engineered EVs. As such, nearly 100% recombination and close to 80% genome editing efficiency in reporter cells were observed by EV-transferred Cre recombinase and Cas9/sgRNA RNPs, respectively. Moreover, EV-mediated Cre delivery by VEDIC or VFIC engineered EVs resulted in significant in vivo recombination in Cre-LoxP R26-LSL-tdTomato reporter mice following both local and systemic injections. Finally, we applied these systems for improved treatment of LPS-induced systemic inflammation by delivering a super-repressor of NF-B activity. Altogether, this study describes a platform by which EVs can be utilized as a vehicle for the efficient intracellular delivery of macromolecular therapeutics for treatments of disease. Graphic summary: Development of VEDIC and VFIC systems for high-efficiency intracellular protein delivery in vitro and in vivo.Intein in tripartite fusion protein (EV-sorting Domain-Intein-Cargo) performs C-terminal cleavage during the process of EV-biogenesis, resulting in enriched free cargo proteins inside of vesicles. Together with fusogenic protein, VSV-G, these engineered EVs achieve high-efficiency intracellular delivery of cargo protein (Cre and super repressor of NF-B) or protein complex (Cas9/sgRNA RNPs) both in reporter cells and in mice models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/535834v4_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@d0da37org.highwire.dtl.DTLVardef@1a6514corg.highwire.dtl.DTLVardef@235c9aorg.highwire.dtl.DTLVardef@194bbfa_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Extracellular vesicle-mediated promotion of myogenic differentiation is dependent on dose, collection media composition, and isolation method

Extracellular vesicles (EVs) have been implicated in the regulation of myogenic differentiation. We observed that treatment of C2C12 murine myoblasts with either GW4869 (to inhibit exosome biogenesis) or heparin (to inhibit EV uptake) reduced myogenic differentiation. Conversely, conditioned media collected from differentiated C2C12 myotubes enhanced myogenic differentiation. Ultrafiltration-size exclusion liquid chromatography (UF-SEC) was used to isolate pure EV preparations and extracellular protein from C2C12 myoblast- and myotube-conditioned media in parallel. UF-SEC purified EVs promoted myogenic differentiation at low doses ([≤]2x108 particles/ml), had no effect at 2x1010 particles/ml, and inhibited myo<genic differentiation at the highest dose tested (2x1011 particles/ml). Similar effects were observed with both myoblast- and myotube-derived EVs. Given that muscle-enriched miRNAs (myomiRs) are largely absent in myoblast cultures, these findings are indicative of a myomiR-independent mechanism underlying the observed pro-myogenic effects. Indeed, individual myomiRs were found to be scarce in EVs (e.g. the most abundant myomiR, miR-133a-3p, was present at 1 copy per 195 EVs). UF-SEC-purified extracellular protein had no effect on myogenic differentiation when collected in serum-free DMEM. However, a potent pro-myogenic effect was observed when Opti-MEM was used as EV harvest media. Opti-MEM contains insulin, which was sufficient to recapitulate the pro-myogenic effect. Similarly, when EVs were isolated by polymer-based precipitation, a pro-myogenic effect was observed, but only when Opti-MEM was used as a collection media. These findings highlight Opti-MEM as a potential confounding factor, and provide further evidence that polymer-based precipitation techniques should be avoided in EV research.

cell biology↗

bioRxiv2020

Engineering of extracellular vesicles for display of protein biotherapeutics

Extracellular vesicles (EVs) have recently emerged as a highly promising cell-free bio-therapeutics. While a range of engineering strategies have been developed to functionalize the EV surface, current approaches fail to address the limitations associated with endogenous surface display, pertaining to the heterogeneous display of commonly used EV-loading moieties among different EV subpopulations. Here we present a novel engineering platform to display multiple protein therapeutics simultaneously on the EV surface. As proof-of-concept, we screened multiple endogenous display strategies for decorating the EV surface with cytokine binding domains derived from tumor necrosis factor receptor 1 (TNFR1) and interleukin 6 signal transducer (IL6ST), which can act as decoys for the pro-inflammatory cytokines TNF and IL6, respectively. Combining synthetic biology and systematic screening of loading moieties, resulted in a three-component system which increased the display and decoy activity of TNFR1 and IL6ST, respectively. Further, this system allowed for combinatorial functionalization of two different receptors on the same EV surface. These cytokine decoy EVs significantly ameliorated disease phenotypes in three different inflammatory mouse models for systemic inflammation, neuroinflammation, and intestinal inflammation. Importantly, significantly improved in vitro and in vivo efficacy of these engineered EVs was observed when compared directly to clinically approved biologics targeting the IL6 and TNF pathways.

bioengineering↗

bioRxiv2020

Engineered extracellular vesicle decoy receptor-mediated modulation of the IL6 trans-signalling pathway in muscle

The cytokine interleukin 6 (IL6) is a key mediator of inflammation that contributes to skeletal muscle pathophysiology. IL6 activates target cells by two different mechanisms, the classical and transsignalling pathways. While classical signalling is associated with the anti-inflammatory activities of the cytokine, the IL6 trans-signalling pathway mediates chronic inflammation and is therefore a target for therapeutic intervention. Extracellular vesicles (EVs) are natural, lipid-bound nanoparticles, with potential as targeted delivery vehicles for therapeutic macromolecules. Here, we engineered EVs to express IL6 signal transducer (IL6ST) decoy receptors to selectively inhibit the IL6 trans-signalling pathway. The potency of the IL6ST decoy receptor EVs was optimized by inclusion of a GCN4 dimerization domain and a peptide sequence derived from syntenin-1 which targets the decoy receptor to EVs. The resulting engineered EVs were able to efficiently inhibit activation of the IL6 transsignalling pathway in reporter cells, while having no effect on the IL6 classical signalling. IL6ST decoy receptor EVs, were also capable of blocking the IL6 trans-signalling pathway in C2C12 myoblasts and myotubes, thereby inhibiting the phosphorylation of STAT3 and partially reversing the anti-differentiation effects observed when treating cells with IL6/IL6R complexes. Treatment of a Duchenne muscular dystrophy mouse model with IL6ST decoy receptor EVs resulted in a reduction in STAT3 phosphorylation in the quadriceps and gastrocnemius muscles of these mice, thereby demonstrating in vivo activity of the decoy receptor EVs as a potential therapy. Taken together, this study reveals the IL6 trans-signalling pathway as a promising therapeutic target in DMD, and demonstrates the therapeutic potential of IL6ST decoy receptor EVs.

molecular biology↗

bioRxiv2020

GAPDH controls extracellular vesicle biogenesis and enhances therapeutic potential of EVs in silencing the Huntingtin gene in mice via siRNA delivery

Extracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication and pathophysiology. Their capacity to transfer biomolecules between cells has sparked efforts to bioengineer EVs as drug delivery vehicles. However, a better understanding of EV biogenesis mechanisms and function is required to unleash their considerable therapeutic potential. Here we demonstrate a novel role for GAPDH, a glycolytic enzyme, in EV assembly and secretion, and we exploit these findings to develop a GAPDH-based methodology to load therapeutic siRNAs onto EVs for targeted drug delivery to the brain. In a series of experiments, we observe high levels of GAPDH binding to the outer surface of EVs via a phosphatidylserine binding motif, designated as G58, and discover that the tetrameric nature of GAPDH promotes extensive EV aggregation. Studies in a Drosophila EV biogenesis model demonstrate that GAPDH is absolutely required for normal generation of intraluminal vesicles in endosomal compartments and promotes vesicle clustering both inside and outside the cell. Fusing a GAPDH-derived G58 peptide to dsRNA-binding motifs permits highly efficient loading of RNA-based drugs such as siRNA onto the surface of EVs. Such vesicles efficiently deliver siRNA to target cells in vitro and into the brain of a Huntingtons disease mouse model after systemic injection, resulting in silencing of the huntingtin gene in multiple anatomical regions of the brain and modulation of phenotypic features of disease. Taken together, our study demonstrates a novel role for GAPDH in EV biogenesis, and that the presence of free GAPDH binding sites on EVs can be effectively exploited to substantially enhance the therapeutic potential of EV-mediated drug delivery to the brain.

neuroscience↗