Exosome packaging of nucleic acids
Inventors
Gibbings, Derrick • Taylor, James Andrew
Assignees
Publication Number
US-10851372-B2
Publication Date
2020-12-01
Expiration Date
2036-09-30
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Abstract
A method for preparing exosomes or exosome-like vesicles packaged with a nucleic acid of interest is provided. In certain embodiments, the method may comprise: introducing into an exosome-producing cell a nucleic acid construct comprising the nucleic acid sequence of interest incorporated in a pre-miR-451 structural mimic, and allowing the cell to produce exosomes. Nucleic acid constructs, compositions, and uses thereof are also provided.
Core Innovation
The invention provides a method for producing exosomes or exosome-like vesicles packaged with a nucleic acid of interest, specifically by introducing into an exosome-producing cell a nucleic acid construct that incorporates the nucleic acid sequence of interest within a pre-miR-451 structural mimic and allowing the cell to produce exosomes. This method enables selective and robust packaging of gene silencing nucleic acids such as siRNAs, miRNAs, shRNAs, or precursors thereof into exosomes.
The problem addressed is the significant challenge in delivering RNA interference (RNAi) therapeutics to target tissues beyond the liver, due to difficulties in packaging RNAi molecules into delivery vehicles like exosomes. Prior methods including electroporation have reliability issues, such as RNA precipitation and inconsistent exosome function. There was no widely applicable and robust mechanism for packaging nucleic acids of interest efficiently into exosomes. The invention solves this by exploiting the unique pre-miR-451 structural motif, which is naturally enriched in exosomes, to incorporate desired nucleic acid sequences, enabling robust enrichment of gene silencing nucleic acids in exosomes.
Claims Coverage
The patent claims primarily relate to methods and compositions for producing exosomes enriched with nucleic acids of interest using nucleic acid constructs incorporating a pre-miR-451 structural mimic, detailing structural features, cell types, and optional treatments to enhance production.
Method for producing exosomes with nucleic acids incorporated within a pre-miR-451 structural mimic
A method comprising introducing into or expressing in an exosome-producing cell a nucleic acid construct comprising a gene silencing nucleic acid or nucleic acid of interest incorporated within a pre-miR-451 structural mimic, treating the cell with a lysosomal or autophagy inhibitor, producing exosomes or exosome-like vesicles, and collecting or enriching the exosomes.
Pre-miR-451 structural mimic features
The pre-miR-451 structural mimic used in the nucleic acid construct has a stem-loop secondary structure characterized by a blunt end, a 5′ overhang, 3′ overhang, or 5′ and 3′ loose ends, with an overall length of about 25-54 nucleotides and a loop length of about 4-8 nucleotides, and may include at least one base pair mismatch in the stem.
Structural variations positioning base pair mismatches near Drosha cleavage site
Incorporation of at least one base pair mismatch within the first three base pairs adjacent to a Drosha cleavage site of the pre-miR-451 structural mimic in the nucleic acid construct for packaging.
Single-stranded pre-miR-451 structural mimic resembling mature miR-451
Use of a single-stranded structure in the nucleic acid construct including a 3′ portion, which is a loop-derived sequence, effectively mimicking mature miR-451, with lengths about 22-35 nucleotides or specifically about 23-24 nucleotides.
Applicable exosome-producing cell types
The method applies to exosome-producing cells including embryonic stem cell clones H1 or H9, mesenchymal stem cells, cells with low Ago2 expression or activity, primary human mesenchymal stem cells, mouse macrophages, human breast cancer cell lines, mouse or human neuronal cell lines, mouse astrocyte cell lines, mouse microglia cell lines, mouse motor neuron cell lines, HeLa cells, mouse embryonic fibroblasts, and mouse dendritic cells.
Use of culture media
Culturing exosome-producing cells in serum-free media or exosome-depleted serum media during exosome production to reduce contamination with exogenous exosomes.
The claims cover methods for packaging gene silencing nucleic acids or nucleic acids of interest into exosomes using nucleic acid constructs bearing pre-miR-451 structural mimics with defined structural features, across various cell types, optionally utilizing lysosomal or autophagy inhibitors and specialized culture conditions, to produce and enrich exosomes or exosome-like vesicles containing these nucleic acids.
Stated Advantages
Allows robust and specific packaging of gene silencing nucleic acids into exosomes for delivery.
Enables use of various cell types including embryonic stem cells and mesenchymal stem cells for efficient exosome production.
Provides a broadly applicable approach overcoming prior limitations of RNA loading into exosomes, such as precipitation and inconsistent encapsulation.
Permits structural variations in the nucleic acid construct while retaining packaging efficiency.
Potentially enhances production yield of exosomes when combined with lysosomal or autophagy inhibitors.
Enables delivery of gene silencing nucleic acids to difficult tissues beyond the liver, including the brain.
Documented Applications
Packaging and delivery of gene silencing nucleic acids such as siRNAs, miRNAs, shRNAs, or CRISPR guide RNAs via exosomes for therapeutic gene silencing.
Use of exosome-loaded nucleic acids for silencing specific genes in vivo, demonstrated by SOD1 silencing in mouse brain.
Production of exosomes enriched with nucleic acids of interest for use as nucleic acid delivery vehicles to target cells and tissues in research and therapeutics.
Modifying exosome-producing cells or exosomes to improve manufacturing, targeting, or immunogenicity.
Use in drug discovery and target validation for delivering silencing RNAs to cells producing biotherapeutics like antibodies or vaccines.
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