Comparative structural, biophysical, and receptor binding study of true type and wild type AAV2

Area of Science:

• * Virology
• * Structural Biology
• * Gene Therapy

Background:

• * Adeno-associated viruses (AAV) are crucial gene transfer vectors for monogenic disorders.
• * A rationally engineered variant, AAV-True Type (AAV-TT), exhibits superior neurotropicity for central nervous system applications compared to wild-type AAV2.
• * AAV-TT's enhanced properties are linked to 14 amino acid differences from AAV2, including key residues R585S and R588T.

Purpose of the Study:

• * To structurally and thermally characterize AAV-TT and AAV2 to understand their divergent properties.
• * To investigate the impact of specific amino acid substitutions on capsid stability and function.
• * To elucidate the mechanisms behind AAV-TT's enhanced neurotropism and reduced antigenicity.

Main Methods:

• * Cryo-electron microscopy (cryo-EM) for high-resolution capsid structure determination.
• * Differential scanning fluorimetry (DSF) to assess thermal stability across various pH conditions.
• * Site-directed mutagenesis to reintroduce specific residues into AAV-TT.

Main Results:

• * Cryo-EM revealed structural perturbations in AAV-TT due to amino acid differences from AAV2.
• * AAV-TT displayed a higher melting temperature (Tm) than AAV2, indicating increased capsid stability, with optimal stability at pH 5.5.
• * Reintroducing arginines at positions 585 and 588 in AAV-TT reduced its Tm, confirming their role in stability.

Conclusions:

• * Specific amino acid differences between AAV-TT and AAV2 account for variations in cell binding, transduction, and antigenicity.
• * AAV-TT's stability and altered residue composition suggest it may bypass glycan receptor-mediated entry and possess lower immunogenicity.
• * These findings provide critical structural and thermal insights for optimizing AAV vectors in gene therapy.