Trans-pseudotyping Recombinant AAV Production
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Solution Overview
Problem
Current methods for producing adeno-associated virus (AAV) particles require the continuous provision of AAV genome in trans, which is inefficient and limits the production of AAVs that do not efficiently infect production cell lines such as HEK293 cells.
Innovation Solution
The process of trans-pseudotyping, where first recombinant AAV particles are produced in first host cells with a tropism towards production cell lines, and then second recombinant AAV particles are produced in second host cells with a different tropism towards therapeutic target tissues, using co-infection with adenovirus expressing rep and cap genes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If AAV particles with specific capsid tropism are used to infect target cells, then therapeutic specificity is improved, but production efficiency in standard cell lines deteriorates
Solution Approach 1:
The production process is divided into two distinct stages: first, production of AAV particles with production-efficient capsid (e.g., AAV2) in standard HEK293 cells to generate high viral titers; second, use of these particles to infect target cells where a different capsid (e.g., AAV9) is provided in trans to achieve therapeutic specificity. This segmentation allows optimization of production and therapeutic function separately.
Solution Approach 2:
AAV particles produced with one capsid type serve as an intermediary vehicle to deliver the genome to target cells, where the capsid type is then replaced through co-infection or transduction with particles expressing the desired therapeutic capsid. This intermediary approach enables decoupling of production efficiency from therapeutic specificity.
2Adaptability or versatility
If AAV genome is provided in trans continuously, then production flexibility is improved, but production efficiency deteriorates
Solution Approach 1:
The AAV genome is provided in trans before the production step, allowing the production cell line to be pre-prepared with all necessary viral components except the capsid. This preliminary setup enables efficient production without the need for continuous provision of the genome during the production process.
Solution Approach 2:
The production system is designed to use the provided AAV genome and helper functions to autonomously produce viral particles with the desired capsid type, eliminating the need for continuous external provision of the genome and reducing production bottlenecks.
3Object-affected harmful factors
If rep and cap genes are removed from AAV genome, then vector safety is improved, but production complexity deteriorates
Solution Approach 1:
The rep and cap genes are extracted from the AAV genome to create a replication-defective, non-integrating vector that is safer for therapeutic use. The extracted cap gene is then provided separately in trans during production, allowing the genome to remain simple and safe while production complexity is managed through the separate provision of necessary components.
Solution Approach 2:
The AAV genome is designed to be universal and replication-defective, relying on helper functions provided in trans for production. This multi-functional design allows the same genome to be used for different capsid types and production methods, simplifying the vector design while managing production complexity through standardized helper systems.
Data Source
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AI summary
The present invention relates to a process for producing recombinant adeno-associated virus (AAV) particles, described herein as trans-pseudotyping. The process involves the production of recombinant AAV particles in first host cells and then in second host cells, wherein first and second AAV cap genes are expressed in the first and second host cells, respectively, thus producing first and second recombinant AAV particles which are encapsidated by first and second AAV capsid polypeptides. The first and second recombinant AAV particles have different cell tropisms, preferably towards production cell lines (for high efficiency production of AAVs) and for cells associated with a therapeutic indication (for treatment of such an indication), respectively.