Insect Cell Expression Cassette for rAAV VP/Rep Ratio Control
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Solution Overview
Problem
Current methods for producing recombinant adeno-associated virus (rAAV) in insect cells face challenges in achieving stable and large-scale production with appropriate stoichiometric ratios of VP1, VP2, and VP3 capsid proteins, as well as Rep78 and Rep52 proteins, leading to low production efficiency and flexibility in regulating serotype-specific sequences.
Innovation Solution
An expression cassette is designed with overlapping open reading frames and intron alternative splicing to regulate the expression of VP and Rep proteins in insect cells, using a promoter, an artificially constructed sequence, and introns with splicing activity to control the stoichiometric ratios and expression levels of these proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional mammalian cell production systems are used, then the stoichiometric ratio of VP1, VP2, and VP3 proteins can be obtained, but the yield per cell is low and contamination risk is high
Solution Approach 1:
The cap gene is divided into multiple open reading frames (ORFs) with specific start codons (AUG for VP1, ACG for VP2) to enable differential expression control. The gene is segmented into VP1, VP2, and VP3 coding regions that can be independently regulated through splicing mechanisms, allowing precise stoichiometric control while maintaining high productivity in insect cells.
Solution Approach 2:
The invention changes the start codon parameters from the natural AUG for both VP1 and VP2 to a combination of AUG (VP1) and ACG (VP2). This parameter change in the translation initiation codon allows differential translation efficiency, achieving the required 1:1:10 stoichiometric ratio of VP1:VP2:VP3 proteins while using insect cell systems with higher productivity.
2Manufacturing precision
If suboptimal start codon ACG is used for VP1 to express all three VP proteins without splicing, then the stoichiometric ratio can be achieved, but the method is not applicable to all AAV serotypes
Solution Approach 1:
The invention creates a universal expression system that works across multiple AAV serotypes by using a standardized cap gene structure with specific start codon assignments (AUG for VP1, ACG for VP2) and defined splicing sites. This multi-functional design allows the same expression cassette to produce correct stoichiometric ratios for different serotypes (AAV2, AAV5, AAV8, etc.) without requiring serotype-specific optimization, thereby achieving both precision and versatility.
Solution Approach 2:
The invention introduces dynamic splicing regulation that can adapt to different serotype requirements. The splicing acceptor sequences and intron-exon boundaries are designed to be flexible, allowing the expression system to dynamically adjust mRNA processing based on the specific cap gene sequence being expressed, thereby maintaining applicability across diverse AAV serotypes while preserving stoichiometric precision.
3Reliability
If different suboptimal start codon CTG is used to enhance VP1 expression, then the infectivity of AAV5 particles is improved, but the flexibility in regulating relative content of VP1/VP2/VP3 is lost
Solution Approach 1:
The invention applies local quality control by assigning different start codon properties to different protein regions: AUG (optimal) for VP1 to ensure high expression when needed, and ACG (suboptimal) for VP2 to limit its expression to the required stoichiometric level. This localized differentiation of start codon quality at specific positions within the cap gene enables both enhanced VP1 expression for infectivity and controlled VP2 levels for proper capsid assembly, while retaining regulatory flexibility through splicing mechanisms.
4Ease of manufacture
If artificial intron with polh promoter sequence is inserted into VP1 open reading frame, then VP1 and VP2/VP3 can be expressed in the same reading frame, but the relative amounts of VP1/VP2/VP3 cannot be effectively regulated
Solution Approach 1:
The invention uses a splicing mediator mechanism where specific splicing acceptor sequences act as intermediaries between the VP1 and VP2/VP3 coding regions. Instead of directly controlling translation initiation, the splicing mediator (intron-exon boundaries and acceptor sequences) indirectly regulates the relative amounts of VP1 and VP2/VP3 by controlling mRNA processing. This intermediary approach maintains proteins in the same reading frame while achieving precise stoichiometric regulation through post-transcriptional control.
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 stable and large-scale production of rAAV with high packaging efficiency and infectious activity, suitable for various serotypes, by precisely controlling the ratios of VP1, VP2, VP3, and Rep78/Rep52 proteins, enhancing production flexibility and efficiency.
Implementation Method 1
during a post-transcriptional processing process, through an alternative splicing function of the intron, a translation start codon AUG in the artificially constructed sequence is retained or deleted or a translation start codon AUG is formed in the artificially constructed sequence, thereby implementing regulation of translation and expression of different protein encoding genes in the overlapping open reading frames
Data Source
AI summary
The disclosure discloses an expression cassette for expressing a gene including overlapping open reading frames in an insect cell and an application thereof. The expression cassette includes from 5′ to 3′ and operably linked: a promoter capable of driving transcription in the insect cell; an artificially constructed sequence; the overlapping open reading frames missing only a first translation start codon; wherein the artificially constructed sequence includes a native or engineered intron with splicing activity in the insect cell, the intron includes ATG or the intron is located between any two adjacent nucleotides in ATG. A recombinant adeno-associated virus vector including the expression cassette of the disclosure regulates relative expressions of VP1, VP2, and VP3 proteins, and relative expressions of Rep78 and Rep52 proteins by using a designed intron sequence and through an intron splicing function for large-scale production of rAAV.