saRNA Microvesicle Delivery Using Envelope Proteins Without Capsids
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Solution Overview
Problem
Current mRNA delivery systems face challenges in efficiently and safely delivering self-amplifying RNA (saRNA) due to issues with viral vectors like VRP, which can generate wild-type viruses and trigger immune responses, while non-viral vectors have low transfection efficiency and high costs.
Innovation Solution
A novel saRNA delivery system using a two-plasmid transfection system to package saRNA into cellular microvesicles mediated by viral envelope proteins, avoiding capsid proteins and nucleoproteins, enabling efficient and targeted delivery of saRNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If viral vectors (VRP) are used for saRNA delivery, then transduction efficiency is improved, but safety deteriorates due to potential generation of wild-type viruses and immune responses
Solution Approach 1:
The patent extracts and removes the problematic capsid protein component from the viral vector system. By using only envelope proteins (E proteins) without capsid proteins, the system eliminates the ability to generate wild-type viruses while maintaining delivery efficiency. This is achieved by transfecting cells with saRNA and E protein expression plasmids, allowing E proteins to self-assemble into microvesicles that package and deliver saRNA without forming complete infectious viral particles.
Solution Approach 2:
The patent segments the viral vector into separate functional components: envelope proteins for delivery and saRNA for therapeutic effect. By separating the capsid protein function (which enables viral replication) from the envelope protein function (which enables cell entry and delivery), the system achieves safe delivery. The E proteins are expressed from separate plasmids and self-assemble with saRNA into non-infectious microvesicles that cannot produce progeny viruses.
2Reliability
If non-viral vectors are used for saRNA delivery, then safety is improved, but transfection efficiency deteriorates
Solution Approach 1:
The patent introduces viral envelope proteins as intermediary structures that bridge the gap between non-viral safety and viral efficiency. The E proteins, when expressed in cells, self-assemble into microvesicles that naturally interact with cell membranes and facilitate efficient saRNA delivery. This intermediary approach maintains the safety of non-viral methods (no capsid proteins, no viral replication) while achieving the high transfection efficiency characteristic of viral vectors.
3Ease of manufacture
If capsid proteins are included in the delivery system, then viral assembly is improved, but immunogenicity and RNA delivery efficiency deteriorate
Solution Approach 1:
The patent removes capsid proteins from the delivery system entirely, retaining only envelope proteins. The E proteins alone are sufficient to form functional delivery vehicles (microvesicles) that can package and deliver saRNA. This extraction eliminates the immunogenicity and harmful effects associated with capsid proteins while maintaining the essential viral assembly capability needed for efficient delivery.
Data Source
Figure 1A~1B
Figure 2~3E
Figure 4A~4B
AI summary
Provided is a method for constructing an saRNA microvesicle delivery system, comprising the following steps: (1) designing an saRNA expression vector 1 and a membrane protein expression vector 2; and (2) co-transfecting the expression vector 1 and the expression vector 2 into a host cell; and (3) culturing the host cell, and isolating microvesicles containing an saRNA, the saRNA containing a target gene sequence, a replicon in the saRNA expression vector being an alphavirus replicon, and the microvesicles not containing capsid protein. The system can efficiently deliver the saRNA to cells or in vivo so as to realize the expression of a target protein. On this basis, different elements in the delivery system can be further transformed so as to develop a high-efficiency low-immunogenicity modular platform aiming at different diseases and different targets. The delivery system may supplement the toolbox of existing viral and lipid nanoparticle delivery vectors and has good application prospects.