Engineered AAP Protein Domain Swaps for AAV Vector Yield
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Solution Overview
Problem
Current methods for producing adeno-associated virus (AAV) vectors face challenges in capsid assembly efficiency due to limitations in the assembly activating protein (AAP) function, particularly in serotype specificity and stability, which affects vector yield and transduction efficiency.
Innovation Solution
Engineering the AAP by replacing or deleting specific domains such as the threonine/serine rich region, basic region, and oligomerization domains with heterologous sequences to enhance stability and functionality, allowing for improved capsid assembly and vector production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wildtype AAP is used for capsid assembly, then assembly function is maintained, but vector yield is limited
Solution Approach 1:
The AAP protein is divided into functional domains (N-terminal domain, threonine/serine rich region, basic region, C-terminal domain). By segmenting the protein structure, the invention identifies that the basic region is critical for capsid assembly function while other regions can be modified. This allows engineering of AAP variants with improved stability and yield while preserving the essential assembly function through the conserved basic region.
Solution Approach 2:
The invention applies parameter changes by modifying amino acid sequences in the AAP protein, specifically replacing the threonine/serine rich region and basic region with heterologous sequences. These parameter changes (sequence substitutions) result in AAP variants that exhibit increased stability and up to 200% higher vector yield while maintaining capsid assembly capability.
2Stability of the object's composition
If AAP stability is increased through engineering, then vector production is improved, but serotype specificity may be affected
Solution Approach 1:
The invention applies local quality by making specific modifications only to certain regions of the AAP protein (threonine/serine rich region and basic region) while leaving other critical regions unchanged. The N-terminal domain and C-terminal domain, which are important for serotype specificity and capsid interaction, are preserved. This localized engineering approach increases stability without compromising serotype specificity.
3Productivity
If basic region is replaced with heterologous sequences, then stability and yield increase, but assembly function may be compromised
Solution Approach 1:
The invention uses heterologous basic regions from other AAV serotypes as intermediaries to replace the native basic region. These heterologous basic regions serve as mediators that can be recognized by the capsid proteins and facilitate assembly. For example, replacing AAV1 AAP basic region with AAV2 basic region maintains assembly function while improving stability and yield, as the heterologous basic region acts as a functional intermediary that preserves the essential assembly interface.
Data Source
AI summary
The present invention relates to compositions and methods comprising an engineered assembly activating protein (EAAP).