Polynucleotide nanoparticles for the modulation of gene expression and uses thereof
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Assignees
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Publication Number
US-10731157-B2
Publication Date
2020-08-04
Expiration Date
Abstract
The present invention is directed to novel self-forming polynucleotide nanoparticles, and the use of such nanoparticles and compositions comprising the same for gene modulation in a variety of organisms.
Core Innovation
The invention provides isolated polynucleotide nanoparticles built from contiguous polynucleotides comprising two or more MV-RNA sequences joined by at least one linkage nucleotide. The contiguous polynucleotide self-forms the nanoparticle into a defined diameter range while exhibiting a specified distribution of stems, with approximately twice the ratio of stems near the surface than at the core and a size on the order of about 40-100 nm.
A further disclosed nanoparticle format is an isolated polynucleotide nanoparticle of approximately 100 nm diameter with at least four times the ratio of stems on the outer surface than at the center. This nanoparticle comprises stacked sets of three MV-RNA, where each stacked MV-RNA set is separated by a linking element. The disclosed designs also include engineered dsRNA tail/open-close sequence changes and variant constructs including aptamers removed.
The disclosure further includes targeting and structural control features for nanoparticle embodiments, including DNA templates for WCR nanoparticle designs with dsRBD uptake signals and plant-application nanoparticle templates targeting Palmer amaranth genes such as PDS, EPSPS, and HPPD. Circularization via cyclase ribozymes is described, alongside follow-on nanoparticle targeting demonstrations and MV-RNA polynucleotide nanoparticle designs and variants.
Claims Coverage
The independent claims are directed to two principal isolated polynucleotide nanoparticle architectures, comprising specified MV-RNA connectivity and stem distribution features. Across the claim set, the inventive features refine the linkage/connectivity structure and optionally add surface-displayed targeting sequences and expression or production contexts.
Self-forming contiguous MV-RNA nanoparticle with surface-biased stem ratio
A contiguous polynucleotide comprising two or more MV-RNA sequences, each MV-RNA sequence joined by at least one linkage nucleotide, self-forming into the nanoparticle having a diameter of approximately 40-100 nm with approximately twice the ratio of stems near the surface of the nanoparticle than at the core of the nanoparticle.
Stacked three MV-RNA sets in an outer-surface-biased ~100 nm nanoparticle
An isolated polynucleotide nanoparticle approximately 100 nm diameter with at least four times the ratio of stems on the outer surface than at the center, comprising stacked sets of three MV-RNA, wherein each stacked MV-RNA set is separated by a linking element.
Surface-displayed aptamer or cellular uptake sequences
The isolated polynucleotide nanoparticle includes aptamer or cellular uptake sequences on one or more surface loops.
Endonuclease-cleavable linkage element
The at least one linkage nucleotide or linking element comprises a stem-loop structure, or a dinucleotide, or is a mononucleotide cleavable by an endonuclease.
Across the independent claims, the coverage centers on isolated polynucleotide nanoparticles formed from MV-RNA connectivity and characterized by a defined surface-to-core stem ratio. Dependent claims further refine these architectures by specifying surface-loop display of aptamer or cellular uptake sequences and endonuclease-cleavable linkage configurations.
Stated Advantages
High trigger molarity.
Nuclease resistance.
Programmable nanoparticle diameter.
Scalable in vitro and in vivo production.
Multi-gene modulation.
Cis/trans-kingdom use.
Documented Applications
Multi-gene prostate cancer targeting.
Western corn rootworm targeting using tri-linked MV-RNA and uptake aptamers.
Palmer amaranth bioherbicide targeting using clathrin-pit and optional removal of aptamers.
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