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A perspective on risks associated with dsRNA-based product - Neena Mitter - Centre for Horticultural Science, Australia


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10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.

Published in: Environment
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A perspective on risks associated with dsRNA-based product - Neena Mitter - Centre for Horticultural Science, Australia

  1. 1. A perspective on risks associated with dsRNA-based Products Prof Neena Mitter Director, Centre for Horticultural Science, QAAFI, The University of Queensland, Australia
  2. 2. Global agriculture  10 billion people by 2050  2 trillion calories will be required per day  Increased productivity- Reduce crop losses  Concerns with current crop protection measures  INNOVATION IS KEY DRIVER PESTICIDES:  RESISTANCE  RESIDUE  RUN-OFF  LACK OF SPECIFICITY  NEW CHEMICALS
  3. 3. RNA interference for crop protection In transgenic or GM plants pathogen specific dsRNA is integrated into the genome of the plant to afford protection • Community acceptance • Concerns regarding environmental impact • Regulation of use • Cost and time involved • Lack of transformation protocols
  4. 4. Is there another way? Can we deliver dsRNA as a spray instead of making a GM plant?
  5. 5. dsRNA spray to control virus infection Pepper Mild Mottle Virus dsRNA + Pepper Mild Mottle Virus Tenllado et al. (2003)
  6. 6. dsRNA-based products act on the RNAi pathway  Active against virus, insect and fungal pathogens Christiaens et al., 2016 Koch et al., 2016 Wang et al., 2016 Gan et al., 2010
  7. 7. dsRNA-based products  Pre- and post-harvest topical application of dsRNA-based products represents a paradigm shift  Increased pathogen specificity  Deployment of new sequences to address resistance  Finite amount of dsRNA
  8. 8.  Unstable  Degradation on plant surface  Not protected from UV and sunlight  Can get easily washed off after spray  Short window of protection Issues with spray of naked dsRNA
  9. 9. How can we convert naked dsRNA experiments into a commercially viable system for farmers? Stabilise? Stick to the leaf? Protect from rain? Non-toxic? Degradable? Environmentally friendly? Easy to adopt?
  10. 10. Formulation  A formulated dsRNA product ‒ dsRNA carrier ‒ Surfactants ‒ Stabilizers ‒ Buffers ‒ Anti-foam materials ‒ Sticking agents  Associated risks are on a case-by-case basis
  11. 11. Nanomaterials as carriers of dsRNA  Clay  Silica  Carbon Nanotubes  Polymers  Lipid based formulations  Nanogels
  12. 12. Clay particles as carriers for non-GM, delivery of RNA interference BioClay- Sustainable crop protection Neena Mitter Gordon Xu Max Lu
  13. 13. Case study : BioClay dsRNA loaded on to layered double hydroxide nanoparticles (LDH) • Increases dsRNA stability and period of protection from days to weeks
  14. 14. BioClay- It works Mitter et al, Nature Plants 2017
  15. 15. BioClay version 2  Specifically designed for targeting insect pests  pH switch to release dsRNA under alkaline conditions  Protection from nucleases
  16. 16. DNA nanostructures coordinate gene silencing in mature plants –  DNA origami or nanoscale folding of DNA  DNA nanostructures could be loaded with siRNA, to accomplish gene silencing in plant  Prior work shows that DNA based nanostructures do not exhibit toxicity in mammalian systems  DNA nanostructures do not induce a stress response in plants Zhang, H….. Landry, MP PNAS (2019)
  17. 17. Nanoparticles to deliver genes into plant chloroplasts. Kwak, SY… Strano, MS Nature Nanotechnology, 2019 Chloroplast Transformation Lipid exchange envelope penetration – LEEP nanobionic-plants-that-glow-1213
  18. 18. Nanomaterials as carriers of dsRNA  Releasing active ingredients in a slow/targeted manner  Protecting the active ingredient against premature degradation  The context within which a material is being evaluated  The importance of measuring a specific parameter within that context  The feasibility of measuring the parameter within a specific context. Oberdörster et al (2005) APVMA report 2015,
  19. 19. Putative risks of dsRNA-based products Human health Environment Trade
  20. 20.  Human exposure pathways: ‒ Ingestion ‒ Inhalation ‒ Dermal  Environmental exposure: ‒ Mammals ‒ Fish ‒ Fungi ‒ Soil biota etc.  Trade impacts: ‒ Non-GM designation ‒ Withholding periods Putative risks of dsRNA-based products
  21. 21. Risks to human health – oral exposure  Multiple and redundant biological barriers to absorption and/or biodistribution  dsRNAs and siRNAs consumed in significant quantities via a diet of fruit and vegetables without detectable effect  miRNA studies indicate insufficient uptake for biologically relevant effects (e.g. Sherman et al. 2015; Snow et al., 2013)
  22. 22. Petrick et al., 2015 – dsRNA and siRNA 28-day mouse feeding trials ‒Treatments included 218 nt dsRNA and 4 x 21 nt siRNAs with 100% homology to mouse vATPase transcript ‒No evidence of differential gene expression relative to controls ‒No evidence of impacts on body weight, food consumption, clinical observations, clinical chemistry, hematology, gross pathology, or histopathology endpoints Risks to human health – oral exposure
  23. 23. Food Standards Australia New Zealand on dsRNA Consumption Investigation of the safety of consuming dsRNAs generated via GM crops (2013) 1. Gene silencing or RNAi is a universal mechanism that is naturally present in eukaryotic organisms 2. dsRNAs are a normal constituent of the human diet 3. A number of barriers exist to the systemic and cellular uptake of exogenous nucleic acids, including small RNAs, by humans 4. There is no scientific basis for presuming that dsRNAs produced by GM plants would pose a greater risk than dsRNAs naturally present in food al-on-the-regulation-of-GM-crops-and-foods-developed-using-gene-silencing.aspx
  24. 24.  Dermal and inhalation exposure ‒ Little evidence of transmembrane movement without conjugation or encapsulation  Low likelihood of allergenicity as no new protein produced Risks to human health – inhalation and dermal exposure
  25. 25. Risks to the environment – Off targets Species closely related to the target pathogen most likely to be affected ‒Similar uptake mechanism ‒Target transcript homology Key question: what level of dsRNA homology to a non-target sequence is sufficient to induce biological effects? s
  26. 26.  Non-formulated dsRNA rapidly degrades in the environment, presenting few risks related to persistence Risks to the environment - persistence  No basis for concentration of topically-applied dsRNA in higher organisms in the food chain
  27. 27. Risks to trade – GMO declaration Natural virus infection Topically applied dsRNA • No promotors or terminator regulatory sequences • No transgene insertion • Not heritable / cannot cross pollinate • No protein expression
  28. 28. Risks to trade – GMO declaration  In Australia, the Office of the Gene Technology Regulator has proposed that topically-applied dsRNA be exempt from GMO regulations (Schedule 1A – Techniques that are not gene technology) Presentation at the CSIRO Gene Editing of Crops Workshop 28-30 November 2017
  29. 29. Naked dsRNA: ‒Short or nil withholding periods (set to allow chemical residues in edible commodities for domestic markets) ‒Short or nil export intervals (to satisfy the standards imposed by overseas trading partners) Trade – withholding periods
  30. 30. DsRNA applications for the ‘Agriculture of Tomorrow” Design of regulation and public opinion are crucial FUNGI Hailing Jin, Nature Plants , 2016 VIRUSES INSECTS PROTECTED CROPPING ANIMAL HEALTH BIOSECURITYPACKHOUSES Endless possibilities……