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The nEXt-RNA project

The nEXt-RNA projectResearch collaborative

Photo: nEXt-RNA project

PhD projects

The 11 nEXt-RNA doctoral candidates (DCs) will work on collaborative research projects led by exRNA experts working on different host-pathogen systems and species; spanning virology, microbiology, nematology, plant and mammalian biology and covering various life science disciplines of molecular biology, cell biology, pathology, immunology, bioinformatics.

The DC projects are further connected within topic-related work packages (WPs) and through collaborations with associated partners both from academic and non-academic, business-related sectors forming a unique and highly synergistic interdisciplinary and intersectoral research network to educate DCs through secondments, network-wide training activities and mentorship programmes.

The individual DC project description below provide further information on specific research objectives, expected outcome, share methods and planned secondments.

  • -> Project DC1
    Tracking ex-mRNAs from the fungal pathogen Ustilago maydis to its crop plant maize
  • -> Project DC2 
    Exploring the role of extracellular RNA in plant viral and viroid infections
  • -> Project DC3 
    Mechanisms of cross-species RNAi in host-bacteria interactions
  • -> Project DC4 
    Quantitative biochemical model for cross-species RNAi in intestinal infection
  • -> Project DC5 
    Exploring fungal EV attachment and uptake into plant cells
  • -> Project DC6
    Comparing exRNA uptake, specificity, and localization when transported by different carriers
  • -> Project DC7
    Extracellular vesicle characteristics and molecular mechanisms that drive RNA cargo delivery into human cells
  • -> Project DC8
    Engineering Citrus-Derived Nanovesicles for RNA Delivery in human cell models
  • -> Project DC9
    Develop microalgae EV (M-EV)-based therapeutics against the fungal pathogen Botrytis cinerea
  • -> Project DC10
    Learning from nature: exploring and applying delivery of ex-mRNA into plant and mammalian hosts
  • -> Project DC11
    Engineer RBPs for improved small RNA delivery and action in target cells

Work packages

WP1: Mechanisms of cross-kingdom exRNA transmission
WP2: Mechanisms of exRNA uptake and delivery
WP3: Next exRNA application and innovations

Project DC1

WP1

Supervisor:
Prof. Dr. Michael Feldbrügge, University of Düsseldorf (UDUS)
Email: feldbrue@hhu.de

Title:
Tracking ex-mRNAs from the fungal pathogen Ustilago maydis to its crop plant maize

Photo: M. Feldbrügge

Co-supervisor:
Prof. Dr. Eva Tutucci, University of Amsterdam

Project objectives:

  • Develop novel tools to track and visualize RNA transmission

  • Identify ex-mRNA functional interaction partners relevant for the transmission

  • Explore functional roles and modes-of action of exRNAs in target cells

Expected outcome:

  • Understand the biological function and the underlying mechanisms of extracellular mRNA delivery during host-microbe interaction

Methods:

  • RNA live imaging
  • Fluorescence in situ hybridisation (FISH)

Secondments:

Project DC2

WP1

Supervisor
Prof. Dr. Kriton Kalantidis, University of Crete (UoC)
Email: kriton"AT"imbb.forth.gr

Title:
Exploring the role of extracellular RNA in plant viral and viroid infections

Photo: K. Kalantidis

Co-supervisor:
Prof. Dr. Esther Nolte t´Hoen (University of Utrecht)

Project objectives:

  • Identify the content of extracellular space of virus- and viroid-infected plants, both inside and outside of EVs

  • Investigate the fate of exRNAs, including their cellular fate

  • Investigate the function of exRNAs in virus- and viroid-infected plants

Expected outcome:

  • Discover exRNAs of the apoplastic space with importance for viroid biology
  • Discover cellular and apoplastic proteins important for viroid infections or in the defence against viroids

Methods:

  • Advanced RNAi reporters

  • Tissue culture

  • Confocal and electron microscopy

Secondments:

Project DC3

WP1

Supervisor:
Prof. Dr. Lionel Navarro, University of Paris (ENS)
Email: lionel.navarro@bio.ens.psl.eu

Title: 
Mechanisms of cross-species RNAi in host-bacteria interactions 

Photo: L. Navarro

 
Co-supervisor:
Dr. Stefania Raimondo (University of Palermo)

Project objectives:

  • Determine plant and human AGOs for internalization into pathogenic bacteria and their contribution to AGS
  • Establish reporter assays to monitor ex-sRNA activity in bacterial cells

Expected outcome:

  • Define factors required for uptake and function of AGOs in recipient bacterial cells
  • Build resources to monitor spatio-temporal dynamics of exRNAs

Methods:

  • Molecular Biology
  • CRISPR-endonuclease-based switch-on siRNA reporter system
  • Quantitative LC MS/MS, Turbo-LOV proximity labelling

Planned secondments:

Project DC4

WP1

Supervisor:
Prof. Dr. Amy Buck, University of Edinburgh (UEDIN)
Email: a.buck"AT"ed.ac.uk

Title:
Quantitative biochemical model for cross-species RNAi in intestinal infection 

Photo: A. Buck

Co-supervisor:
Dr. Cei Abreu-Goodger (University of Edinburgh)
Dr. Matthias Hackl (TAMIRNA)

Project objectives:

  •  Determine absolute copy numbers and target recognition rules of parasite RNAs inside murine intestinal cell populations
  • Identify host proteins associated with parasite RNA-induced silencing complexes
  • Build reporter assays for cross-species RNAi via exWAGO to compare specificity and efficacy in different species

Expected outcome:

  • Quantitative data on exRNAs delivered naturally by exWAGO to mammalian cells
  • New host targets associated with gastrointestinal nematode infection and strategies to block their de-regulation during infection.
  • New tools to examine and compare cross-species RNAi via exWAGO in mammals, plants and bacteria

Methods

  • Cell and intestinal organoid culture
  • FACS
  • Bind-n-seq, immunoprecipitation, computational analyses of small RNA/targets
  • Reporter design and gastrointestinal nematode infection model

Planned secondments

Project DC5

 WP2

Supervisor:
Prof. Dr. Arne Weiberg, University of Hamburg (UHAM)
arne.weiberg@uni-hamburg.de

Title: 
Exploring fungal EV attachment and uptake into plant cells

Photo:  A. Weiberg, Ruf et al. 2022 (CC BY 4.0)

Co-supervisor:
Prof. Dr. Kriton Kalantidis (University of Crete)

Project objectives:

  • Use proximity labelling to characterize fungal EV surface proteins and plant cell surface factors interacting with fungal EVs
  • Characterize EV surface proteins for their role in EV attachment and uptake into plant cells

Expected outcome:

  • Identify fungal and plant proteins involved in EV uptake
  • Gain knowledge on fungal EVs to deliver exRNA to various cell types
  • Correlate EV uptake and fusion for RNA delivery in plants

Methods:

  • EV proximity labelling
  • EV-FUSIM technologies

Secondments:

Project DC6

 WP2

Supervisor:
Prof. Dr. Amy Buck, University of Edinburgh (UEDIN)
Email: a.buck@ed.ac.uk

Title: 
Comparing exRNA uptake, specificity, and localization when transported by different carriers    

Photo: A. Buck, White et al. 2023 (CC-BY)

Co-supervisor:
Prof. Dr. Esther Nolte-t’Hoen (University of Utrecht)

Project objectives:

  • Compare cellular specificity of exRNA uptake by mammalian cells and organoids when delivered via EVs, RNA-binding proteins or naked (non-complexed).
  • Determine differences in sub-cellular localization of imported exRNAs and their associated protein co-factors based on parasite carrier
  • Compare functional differences of exRNA imported by different carriers using reporter assays

Expected outcome:

  • Cross-system comparison on natural exRNA uptake mechanisms and new or refined strategies for therapeutic exRNA delivery
  • New knowledge on sub-cellular localization and protein co-factors of exRNAs inside cells and correlation with functional activity

Methods:

  • ExRNA/EV purification (Ultracentrifugation and Size exclusion chromatography)
  • Fluorescent labelling of exRNAs/carriers, Spectral and Imaging Flow, confocal microscopy
  • Cell and intestinal organoid culture and gastrointestinal nematode infection model.

Secondments:

  • Research stay at Utrecht University to track exRNA import with reporter / fusion assays and compare subcellular localization to other EV forms.

Project DC7

 WP2

Supervisor: 
Prof. Dr. Esther Nolte t´Hoen, Utrecht University (UU)
Email: E.N.M.Nolte@uu.nl

Title:
Extracellular vesicle characteristics and molecular mechanisms that drive RNA cargo delivery into human cells

Photo: E. Nolte van´t Hoen

Co-supervisor:
Prof. Dr. Amy Buck (University of Edinburgh)

Project objectives:

  • Compare EVs from various species/pathogens for their capacity to interact and/or fuse with human cells to deliver RNA cargo
  • Identify cell binding/internalization routes that support EV binding/fusion and RNA cargo delivery
  • Assess the relationship between EV-fusion and subcellular exRNA delivery site and exRNA function

Expected outcome:

  • Cross-species discovery of EV types that promote endosomal escape in mammalian target cells (and therefore promising candidates for EV therapeutics)
  • Understanding of (common) molecular features of EVs that promote EV fusion and cargo delivery
  • c) Understanding if and how subcellular compartments support EV fusion and functional exRNA delivery

Methods:

  • EV isolation methods
  • Diverse techniques to fluorescently label EVs
  • EV characterization methods
  • Genetic engineering of cells to produce EVs with molecular tags or reporter constructs
  • EV-FUSIM technology for live cell microscopic imaging of EV binding, uptake and fusion in time and space
  • Flow cytometry to quantify binding/uptake of EVs to diverse cell types
  • Setting up novel technology for parallel live cell imaging of ExRNA and EV fusion

Secondments:

  • Research stay at Aarhus University to learn (live) tracing of (EV-associated) exRNAs

Project DC8

WP3

Supervisor:
Dr. Stefania Raimondo, University of Palermo (UNIPA)
Email: stefania.raimondo@unipa.it

Title:
Engineering Citrus-Derived Nanovesicles for RNA Delivery in human cell models

Photo: BioRender, Tinnirello et al. (CC BY)

Co-supervisor:
Alice Conigliaro (NAVHETEC)

Project objectives:

  • develop citrus-derived nanovesicles (CDNVs) as a novel and sustainable platform for RNA delivery
  • investigate both the endogenous RNA cargo of CDNVs and strategies for efficient exogenous RNA loading

Expected outcome:

  • identification of optimal methods for RNA encapsulation, the evaluation of CDNV biodistribution, stability, and targeting in advanced human models
  • demonstration of functional RNA delivery in vitro

Methods:

  • 2D and 3D human cell models
  • Molecular biology
  • RNA sequencing
  • Imaging approaches

Secondments:

  • Research stay at Engreen Technologies to use microalgae and vesicle-based RNA delivery technologies with
  • Research stay at Microsynth to advance RNA synthesis and sequencing platform

Project DC9

WP3

Supervisor: 
Prof. Dr. Arne Weiberg, University of Hamburg (UHAM)
Email: arne.weiberg@uni-hamburg.de

Title:
Develop microalgae EV (M-EV)-based therapeutics against the fungal pathogen Botrytis cinerea

Photo: EnGreen Technol

Co-supervisor:
Dr. Antonio Emidio Fortunato (ENGREEN)

Project objectives:

  • Establish RNAi and M-EV fusion reporters in Botrytis
  • Design siRNA probes to control Botrytis via M-EV approaches
  • Run greenhouse trail to assess efficacies of M-EV approaches

Expected outcome:

  •  Develop novel M-EV-based applications to control Botrytis in different crops

Methods:

  • RNAi reporter systems in pathogens
  • M-EV production and plant applications

Secondments:

Project DC10

WP3

Supervisor:
Prof. Dr. Michael Feldbrügge, University of Düsseldorf (UDUS)
Email: feldbrue@hhu.de

Title:
Learning from nature: exploring and applying delivery of ex-mRNA into plant and mammalian hosts

Photo: M. Feldbrügge, Serendip Innovations

https://serendipinnovations.com/
https://www.mikrobiologie.hhu.de/en

Co-supervisor:
Dr. Vianney Poignavent (SERENDIP)

Project objectives:

  • Identify new components of EVs involved in loading and delivery of mRNAs
  • Study the function of most interesting candidates in detail
  • Delivery of mRNAs using orthogonal systems such as virus-like particles for plants or tailor-made EVs for mammalian cells

Expected outcome:

  • Apply novel components for tailor-made delivery of mRNAs to target cells

Methods:

  • Proximity labelling techniques
  • Optogenetics
  • Generation and application of Virus-like particles
  • EV-FUSIM technology

Secondments:

  • Research stay at Serendip Innovations to develop virus-like particle technology
  • Research stay at Utrecht University to apply EV-FUSIM technology

Project DC11

WP3

Supervisor: 
Prof. Dr. Lionel Navarro, University of Paris (ENS)
Email: lionel.navarro@bio.ens.psl.eu

Title:
Engineer RBPs for improved small RNA delivery and action in target cells

Photo: L. Navarro, Serendip Innovations

Co-supervisor:
Dr. Vianney Poignavent (SERENDIP)

Project objectives:

  • Optimize exogenous loading of exAGO/RBP-sRNA into EVs
  • Establish VLPs containing exAGO/RBP-sRNA and coated with antibody fragment recognizing proteins at the surface of bacteria or human cells
  • Assess the silencing efficacy and antibacterial activity of EV/VLP-based delivery of exAGO/RBP-sRNAs

Expected outcome:

  • Unveiling the best EV-RBP-sRNA composition(s) to trigger gene silencing in bacterial and mammalian cells.

  • Unveiling the best molecular biology approach to load RBP-sRNA in VLPs

  • Strategy to enhance the affinity of VLPs for bacterial and human cells

Methods:

  • RT-qPCR, Golden Gate cloning, Low molecular weight Northern analyses, sRNA-seq
  • Plant manipulation (Agrobacterium-transient assay in N. benthamiana)
  • Protein expression and purification from bacteria, western blot analyses

Secondments:

  • Research stay at Serendip Innovations to engineer exAGO/exWAGO/RBPs-sRNA complex into VLPs and enhance their cell targeting capacity