Intron-Modified Cap Cassette for AAV Vector Purity
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Solution Overview
Problem
Current AAV vector production methods face challenges in generating high titers of AAV vectors while minimizing contamination with wild-type AAV virus and reducing immune responses in mammalian subjects, as existing methods often result in contamination with replication-competent AAV and capsid gene expression, leading to immunogenicity issues.
Innovation Solution
The development of an intron-modified cap expression cassette that includes a nucleic acid molecule encoding AAV capsid protein operably linked to a promoter, with at least one heterologous intron sequence, preventing cap gene packaging and reducing immune responses by generating AAV vectors with reduced or undetectable cap gene contamination.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional AAV vector production methods are used, then high titers of AAV vectors can be generated, but contamination with replication-competent AAV and capsid gene expression occurs
Solution Approach 1:
The packaging plasmid is divided into two separate plasmids: one containing the rep gene and another containing the cap gene. This segmentation prevents the formation of replication-competent AAV while maintaining the ability to produce capsid proteins for vector packaging, thereby resolving the contradiction between high productivity and contamination reduction
Solution Approach 2:
The cap gene is extracted from the conventional packaging plasmid and placed in a separate plasmid. This extraction eliminates the risk of capsid gene contamination in the final vector preparation while still allowing capsid protein expression necessary for AAV vector production, thus achieving both high titer and low contamination
2Productivity
If conventional AAV vector production methods are used, then AAV vectors can be produced efficiently, but immune responses occur due to capsid gene expression
Solution Approach 1:
The cap gene is removed from the packaging plasmid and placed in a separate construct. This ensures that while capsid proteins are expressed for vector production, the complete capsid gene sequence is not present in the final AAV vector preparation, thereby reducing immunogenicity while maintaining production efficiency
Solution Approach 2:
The structure of the packaging system is changed by separating rep and cap genes into different plasmids. This parameter change in the packaging architecture allows efficient vector production while eliminating the harmful effect of capsid gene contamination that triggers immune responses
Data Source
AI summary
In one aspect, the present invention provides an intron-modified cap expression cassette useful for generating adeno-associated virus (AAV) vector particles. In another aspect, the present invention provides a method of reducing the immune response in a mammalian subject undergoing treatment with an AAV vector.