Engineered Cells for Toxic RNA Production in Exosomes
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for delivering toxic RNAi active sequences to cancer cells are inefficient due to the alteration of extracellular vesicles' physical properties during electroporation, leading to reduced efficacy in targeting and killing cancer cells, and cells producing these sequences often die from DISE, limiting the yield of therapeutic agents.
Innovation Solution
Engineering cells deficient in Dicer or Ago2 to express and package toxic RNAs, such as shRNAs and siRNAs, into extracellular vesicles like exosomes, which are resistant to the toxic RNAs, allowing for high-yield production and delivery of these sequences to cancer cells without inducing cell death in the producing cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If electroporation is used to package nucleic acid into exosomes, then nucleic acid delivery is achieved, but the physical properties of exosomes are altered reducing delivery efficiency
Solution Approach 1:
The patent uses an intermediary system consisting of engineered producer cells that naturally package toxic RNA into exosomes through their endogenous secretion pathways. These cells express the toxic RNA sequence along with exosome biogenesis machinery, allowing natural co-packaging without electroporation. The exosomes are then harvested from the culture medium, providing a gentle delivery method that preserves exosome integrity and function.
2Reliability
If cells are engineered to express toxic RNA sequences for therapy, then cancer cells can be targeted, but the producing cells die from DISE limiting agent yield
Solution Approach 1:
The patent segments the system into two distinct functional components: (1) Producer cells that are genetically engineered to express toxic RNA sequences but are protected from DISE through specific genetic modifications, and (2) Target cancer cells that receive the toxic RNA via exosome delivery. This segmentation allows the producer cells to serve as factories for generating therapeutic agents without suffering the intended therapeutic effect, thereby enabling high-yield production.
Solution Approach 2:
The patent converts the harmful effect of toxic RNA (which kills cells via DISE) into a beneficial production system by engineering producer cells with genetic modifications that protect them from DISE while maintaining their ability to package and secrete toxic RNA into exosomes. The harm is redirected exclusively to target cancer cells, while the producer cells benefit by becoming sustainable factories for therapeutic agent production.
3Reliability
If natural delivery systems are used to introduce toxic RNA to cancer cells, then delivery efficiency improves, but the production of toxic RNA in producing cells is limited by cell death
Solution Approach 1:
The patent applies preliminary action by pre-engineering the producer cells with genetic modifications that confer resistance to DISE before they begin producing toxic RNA. These genetic modifications are established in advance, allowing the cells to be cultured and produce large quantities of toxic RNA-loaded exosomes over extended periods without dying from the toxic RNA they produce. This preliminary protection enables sustained high-level production.
Data Source
AI summary
Disclosed are components and methods for producing toxic RNAs and/or extracellular vesicles comprising the toxic RNAs in cells that are resistant to the toxic RNAs, which toxic RNAs may include toxic shRNA/siRNA and/or toxic pre-miRNA/miRNA. The disclosed components may include engineered cells that can be utilized to express the toxic RNAs, in which the engineered cells do not express one or more genes that are required for processing the toxic RNAs for RNA interference (RNAi) and/or one or more genes that are required for executing RNAi. The toxic RNAs and/or extracellular vesicles comprising the toxic RNAs may be utilized in methods for treating diseases and disorders through RNAi.