Co-transcriptional assembly of modified RNA nanoparticles
Inventors
Shapiro, Bruce A. • Afonin, Kirill A. • Kireeva, Maria L. • Kashlev, Mikhail • Jaeger, Luc • Grabow, Wade W.
Assignees
University of California San Diego UCSD • US Department of Health and Human Services
Publication Number
US-9719084-B2
Publication Date
2017-08-01
Expiration Date
2033-09-06
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Abstract
A method is provided for generating RNA nanoparticles having modified nucleotides and/or having increased nuclease resistance where the RNA nanoparticles are formed cotranscriptionally by T7 RNA polymerase in the presence of manganese ions.
Core Innovation
The invention provides a method for generating RNA nanoparticles (NP) comprising modified nucleotides and/or increased nuclease resistance. These RNA nanoparticles are formed cotranscriptionally by wild-type T7 RNA polymerase in a manganese ion containing buffer, allowing incorporation of modified nucleotides into RNA transcripts during in vitro transcription without the need for subsequent purification steps.
The problem addressed arises from limitations in current RNA nanoparticle production methods. These limitations include the cost and size constraints of chemically synthesizing long functional RNA strands, the complexity and optimization required for separate synthesis and assembly protocols, and the low retention time of RNA nanoparticles in the bloodstream due to susceptibility to nuclease degradation. Prior methods utilizing mutant T7 RNA polymerase for incorporating chemical modifications resulted in significantly lower yields, limiting large-scale production of chemically modified RNA nanoparticles.
Claims Coverage
The patent discloses ten inventive features extracted from the independent claims relating to a method for producing modified RNA nanoparticles with specific conditions and compositions.
Co-transcriptional assembly of modified RNA nanoparticles
A method involving mixing one or more double-stranded DNA templates encoding multiple distinct RNA transcripts, wild-type T7 RNA polymerase, a modified nucleotide, and a manganese ion-containing buffer, and incubating the mixture to transcribe the templates and allow co-transcriptional assembly of modified RNA nanoparticles without separate purification of RNA transcripts.
Use of nuclease resistant RNA nanoparticles
Modified RNA nanoparticles produced by the method are nuclease resistant, exhibiting increased serum half-life relative to unmodified RNA nanoparticles.
Manganese ion concentration specificity
The method employs manganese ions in the buffer at a concentration range from 0.25 mM to 0.75 mM, preferably about 0.5 mM, to enable efficient incorporation of modified nucleotides.
Number of distinct RNA transcripts in nanoparticles
The modified RNA nanoparticles produced comprise between 12 and 22 distinct RNA transcripts.
Broad range of modified nucleotides used
The method uses modified nucleotides comprising a broad group of chemically modified nucleosides including but not limited to 5-methylcytidine, 5-methyluridine, various methyladenosines, methylguanosines, and numerous other listed modifications.
Selective modified nucleotides for incorporation
Selective modified nucleotides include 2′-fluoro-dUMP, 2′-fluoro-dCMP, 2′-fluoro-dGMP, 2′-fluoro-dAMP, and 8-N3AMP, with a specific embodiment using 2′-fluoro-dUTP.
Increased serum half-life of nuclease resistant RNA nanoparticles
Nuclease resistant RNA nanoparticles formed have increased serum half-life compared to corresponding wild-type RNA nanoparticles.
Inclusion of RNase H treatment
The method can further comprise an RNase H treatment step to remove undesired extensions or sequences from RNA transcripts during or after transcription.
The inventive features encompass a method for producing modified RNA nanoparticles via co-transcriptional assembly using wild-type T7 RNA polymerase in manganese-containing buffers, enabling high-yield incorporation of various modified nucleotides, producing nuclease resistant nanoparticles of specified sizes, and optionally including RNase H treatment for processing. These features address previous limitations in RNA nanoparticle synthesis and stability.
Stated Advantages
The invention enables high-yield production of chemically modified RNA nanoparticles co-transcriptionally without the need for separate purification of individual RNA strands.
Manganese ions in the transcription buffer increase incorporation efficiency of modified nucleotides by wild-type T7 RNA polymerase without inhibiting transcription of unmodified RNA.
The resulting chemically modified RNA nanoparticles exhibit increased resistance to nucleases present in human blood serum, thus improving stability and retention time.
Chemically modified RNA nanoparticles functionalized with siRNA retain the ability to be processed by human recombinant Dicer, preserving functionality in RNA interference applications.
Functional RNA nanoparticles produced by this method can be directly used in cell culture assays from unpurified transcription mixtures, facilitating rapid functional screening.
Documented Applications
Use as drug delivery vehicles capable of carrying therapeutic agents such as siRNAs, ribozymes, aptamers, and oligonucleotides.
Applications in imaging, nanocircuits, cell growth surfaces, medical implants, medical testing, and gene therapy.
Use as biosensors for pathogen detection and bioterrorism defense.
Utilization as skeletons or scaffolds for tissue growth and biological meshes.
Incorporation of various therapeutic, diagnostic, and delivery agents including chemotherapeutic agents, antibodies, fluorescent dyes, and targeting moieties.
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