Engineering adeno-associated virus serotype 2-based targeting vectors using a new insertion site-position 453-and...

Area of Science:

• * Molecular biology
• * Virology
• * Gene therapy

Background:

• * Genetic modification of adeno-associated viral (AAV) vectors enables targeted gene transfer.
• * Peptide insertion into AAV capsid proteins is a key strategy for AAV targeting.
• * Common insertion sites (587/588) are on the second highest capsid protrusion.

Purpose of the Study:

• * To explore position 453 as a novel insertion site for enhanced AAV targeting.
• * To investigate the impact of position 453 on receptor interaction and gene transfer efficiency.
• * To compare the efficacy of position 453 insertion with established sites.

Main Methods:

• * In silico identification of position 453 for peptide insertion.
• * Construction and comparison of AAV capsid mutants with RGD-4C ligand at positions 453 and 587.
• * Assessment of ligand accessibility, receptor binding specificity, and transduction efficiency.
• * In vivo biodistribution analysis in mice using quantitative PCR.

Main Results:

• * RGD-4C insertion at position 453 initially showed poor receptor binding.
• * Neighboring residues R585/R588 were identified as interfering with ligand binding.
• * Substitutions R585A/R588A at position 453 significantly improved receptor binding and transduction efficiency compared to position 587.
• * In vivo biodistribution was ligand-dependent when primary receptor binding was avoided.

Conclusions:

• * Position 453 is a highly effective site for developing AAV targeting mutants.
• * Linearly distant residues critically influence the efficiency of inserted peptide ligands in AAV vectors.
• * This study advances AAV vector engineering for precise gene delivery.