Methods and compositions for the modification of plants
Inventors
VAN ROOIJEN, Maria Helena Christine • TAM, Hok Hei • AMADO, Maier Steve Avendaño • Martin, Barry Andrew • Martinez, Ignacio • KOWALSKI, Piotr Stanislaw • NUKOLOVA, NATALIYA VLADIMIROVNA • Niu, Yajie
Assignees
Flagship Pioneering Inc • Flagship Pioneering Innovations VI Inc
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Publication Number
US-12416009-B2
Publication Date
2025-09-16
Expiration Date
Abstract
Disclosed herein are plant-modifying compositions including a plurality of plant messenger packs (PMPs) (e.g., including a plant extracellular vesicle (EV), or segment, portion, or extract thereof), wherein the PMPs include a plant-modifying agent, and wherein the plant-modifying agent comprising the heterologous RNA. The compositions herein are useful in methods for modifying plants such as to increase plant fitness, wherein the increase in plant fitness comprising an increase in development, growth, yield, resistance to abiotic stressors, or resistance to biotic stressors.
Core Innovation
The invention relates to non-viral delivery of heterologous RNA to an intact plant using lipid-reconstituted plant messenger packs (LPMPs) derived from plant extracellular vesicles (EVs). The LPMP composition includes a plurality of LPMPs, at least one heterologous RNA, an exogenous cationic lipid, and at least one plant protein extracellular vesicle marker.
The exogenous cationic lipid increases RNA loading efficiency compared with an LPMP not comprising the at least one heterologous cationic lipid. Heterologous RNA payloads described include siRNA, tracrRNA, shRNA, miRNA, dsiRNA, dsRNA, antisense RNA, antisense polynucleotides, and guide RNA provided as part of a ribonucleoprotein complex.
The compositions are delivered at an effective concentration to increase plant fitness, including plant development, growth, yield, resistance to abiotic and/or biotic stressors, altered flowering time, and improved harvested-product quality. The document also describes gene modulation in plants, including knockdown and activation phenotypes, and CRISPR systems with Cas9 or Cpf1, CRISPRi, and CRISPRa approaches.
Claims Coverage
The independent claim set is directed to non-viral delivery of heterologous RNA to an intact plant using LPMPs, with an exogenous cationic lipid that improves RNA loading efficiency and delivery at an effective concentration to increase plant fitness. Dependent claims refine the RNA type, cationic lipid identity, RNP delivery, and formulation constraints, for a total of eight inventive features.
Non-viral delivery of lipid-reconstituted plant messenger packs to an intact plant
A method of administering at least one heterologous RNA to an intact plant comprising non-viral delivery to the intact plant of a composition comprising a plurality of lipid-reconstituted plant messenger packs, each comprising the at least one heterologous RNA, an exogenous cationic lipid, and at least one plant protein extracellular vesicle marker.
Exogenous cationic lipid increases RNA loading efficiency in LPMPs
The exogenous cationic lipid increases the RNA loading efficiency as compared to the loading efficiency of an LPMP not comprising the at least one heterologous cationic lipid.
Effective concentration to increase plant fitness
The composition is delivered at an effective concentration to increase plant fitness.
DC-Cholesterol or DOTAP cationic lipid embodiments
The exogenous cationic lipid is DC-Cholesterol or DOTAP.
siRNA or tracrRNA as the heterologous RNA
The at least one heterologous RNA is either siRNA or tracrRNA.
Guide RNA delivered as an RNP complex
The gRNA is part of a ribonucleoprotein complex, and each LPMP comprises the RNP.
Concentration effective to modify a plant trait and increase fitness
The LPMPs are used at a concentration effective to modify a plant trait and increase the fitness of the plant.
DOTAP by-weight range constraint in LPMPs
DOTAP comprises between 25% and 40% of the total lipids in the LPMP by weight.
Overall, the claims focus on administering heterologous RNA to an intact plant via non-viral LPMP delivery where an exogenous cationic lipid improves RNA loading efficiency, combined with delivery at an effective concentration to increase plant fitness. Dependent elements specify particular cationic lipid identities, heterologous RNA types, guide RNA as an RNP complex, and DOTAP content by weight.
Stated Advantages
Increased RNA loading efficiency compared to an LPMP not comprising the exogenous cationic lipid.
Increased plant fitness.
Enhanced development, growth, and yield.
Resistance to abiotic stress and biotic stress.
Altered flowering time.
Improved harvested-product quality, including taste, appearance, and shelf life.
Reduced allergen production that triggers immune response in animal.
Improved plant outcomes by delivering a composition at an effective concentration to increase plant fitness.
Documented Applications
Administering at least one heterologous RNA to an intact plant using non-viral delivery of a composition comprising lipid-reconstituted plant messenger packs.
Plant trait enhancement defined as increased plant fitness outcomes, including enhanced development, growth, yield, resistance to abiotic and biotic stress, altered flowering time, and improved harvested-product quality including taste, appearance, and shelf life.
Reducing allergen production in plant to reduce immune response in animal.
Using an appendix table of plant extracellular vesicle marker proteins across species to support identification and selection of EV markers used in LPMP formulations.
Gene modulation outcomes in plants, including inhibitory RNA effects and CRISPR-based gene editing, CRISPRi, and CRISPRa approaches.
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