CCMV Capsid Self-Assembly for RNA Delivery
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Solution Overview
Problem
Current methods for gene delivery to mammalian cells lack effective and efficient use of plant-derived virus-like particles, which have not been explored for direct gene delivery and expression, and existing systems like the baculovector system require recombinant plasmid engineering and cannot be reconstituted in vitro.
Innovation Solution
The use of in vitro reconstituted cowpea chlorotic mottle virus (CCMV) capsids to spontaneously self-assemble around heterologous RNA molecules, forming stable nucleocapsids that protect RNA from degradation and release it in the cytoplasm of mammalian cells, allowing for high-level expression of therapeutic or imaging polypeptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If animal virus-like particles are used for gene delivery, then gene delivery efficiency is improved, but device complexity and manufacturing difficulty increase due to requiring recombinant plasmid engineering and cell culture
Solution Approach 1:
The invention extracts the essential capsid protein function from complex animal virus systems and isolates it into simplified plant viral capsids that can self-assemble. The CCMV capsid proteins are purified from plant expression systems and spontaneously self-assemble around RNA genes without requiring complex animal cell culture or recombinant plasmid engineering, thus extracting the core delivery function while removing manufacturing complexity
Solution Approach 2:
The plant viral capsid proteins exhibit spontaneous self-assembly capability around heterologous RNA molecules in vitro. The capsid proteins automatically organize themselves into complete viral-like particles containing the RNA gene payload without requiring external assembly machinery or complex processing steps, enabling the system to service itself during particle formation
2Ease of manufacture
If plant viral capsids are used for gene delivery, then manufacturing simplicity is improved through in vitro reconstitution, but reliability of gene delivery to mammalian cells is uncertain since plant-derived VLPs have not been previously used for direct gene delivery
Solution Approach 1:
The invention demonstrates that plant viral capsids, originally evolved for plant virus function, can universally package and deliver heterologous RNA genes to mammalian cells. The CCMV capsid system shows multi-functionality by serving both as a protective nanoparticle and as a delivery vehicle that can transduce mammalian cell types including hepatoma, kidney, and breast cancer cells, proving universal applicability across different host systems
Solution Approach 2:
The capsid proteins are pre-expressed and purified from plant systems before being used for RNA packaging. This preliminary production and purification step allows the capsid proteins to be prepared in advance in a controlled manner, ensuring consistent quality and enabling reliable subsequent assembly with RNA genes before delivery to mammalian cells
3Productivity
If baculovector system is used for gene expression, then expression capability is improved, but ease of manufacture deteriorates because the system cannot be reconstituted in vitro and requires recombinant plasmid engineering
Solution Approach 1:
The invention replaces the complex mechanical and biological machinery required by the baculovector system (recombinant plasmid engineering, animal cell culture, viral assembly machinery) with a simplified in vitro reconstitution approach. The plant viral capsid proteins spontaneously self-assemble around RNA genes in buffered solutions without requiring living cell systems or complex engineering machinery, substituting biological complexity with simpler biophysical self-assembly
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables efficient and stable delivery and expression of RNA genes in mammalian cells, overcoming the limitations of existing technologies by providing a robust and monodisperse vector for gene delivery that can be reconstituted from purified components, with the potential for targeted delivery and high-level protein synthesis.
Implementation Method 1
cowpea chlorotic mottle virus (CCMV) capsid protein (CP) is exploited for its unique ability to spontaneously self-assemble around heterologous RNA molecules of widely varying length and sequence
Implementation Method 2
The resulting nucleocapsids—one molecule of RNA surrounded by a rigid single-protein-thick shell—are perfectly monodisperse, stable against aggregation, protect their RNA content against RNases
Implementation Method 3
release their RNA content in the cytoplasm of target mammalian cells
Data Source
AI summary
The invention provides compositions of matter comprising a cowpea chlorotic mottle virus capsid protein (CCMV CP) and a ribonucleic acid, as well as methods for using such compositions. In such compositions, the cowpea chlorotic mottle virus capsid protein envelops the ribonucleic acid so as to for a capsid that can inhibit the degradation of the ribonucleic acid (e.g. by RNAses). A method of delivering a ribonucleic acid into the cytoplasm of a mammalian cell is also provided. Typically, the method comprises the steps of combining the mammalian cell with a composition of matter described herein under conditions selected to allow the cowpea chlorotic mottle virus capsid to contact the mammalian cell and deliver the ribonucleic acid into the cytoplasm of a mammalian cell.