Naturally occurring singleton residues in AAV capsid impact vector performance and illustrate structural constraints

Area of Science:

• * Molecular biology
• * Virology
• * Gene therapy

Background:

• * Adeno-associated virus (AAV) vectors are key for in vivo gene transfer.
• * The AAV capsid mediates cellular interactions and influences vector properties.
• * Specific capsid amino acids are critical determinants of AAV vector function.

Purpose of the Study:

• * To develop novel AAV vectors by analyzing capsid structure-function relationships.
• * To identify and modify specific capsid residues impacting vector manufacturing and transduction.
• * To create a diverse portfolio of AAV vectors for enhanced gene therapy applications.

Main Methods:

• * Structure-function analysis of naturally occurring AAV capsid isolates.
• * Identification of 'singleton' residues (variable amino acids in conserved positions).
• * Site-directed mutagenesis to alter singleton residues to conserved amino acids.

Main Results:

• * Singleton residues significantly impact AAV vector manufacturability and transduction.
• * Residues at monomer-monomer interfaces are implicated in particle assembly.
• * Modifying singletons rescued defective AAV isolates, creating a portfolio of six clades and three niches.
• * In vivo studies revealed clade-specific tropism for intravenous and intramuscular delivery.

Conclusions:

• * Understanding AAV capsid structure-function relationships enables rational vector design.
• * Targeted modification of capsid singletons enhances AAV vector performance.
• * The developed AAV vector portfolio offers diverse options for gene therapy applications.