Adeno-Associated Virus is the leading vector for gene therapy. Although it is the vector for all in vivo gene therapies approved for clinical use by the US Food and Drug Administration, its biology is still not yet fully understood. It has been shown that different serotypes of AAV bind to their cellular receptor, AAVR, in different ways. Previously we have reported a 2.4Å structure of AAV2 bound to AAVR that shows ordered structure for only one of the two AAVR domains with which AAV2 interacts. In this study we present a 2.5Å resolution structure of AAV5 bound to AAVR. AAV5 binds to the first polycystic kidney disease (PKD) domain of AAVR that was not ordered in the AAV2 structure. Interactions of AAV5 with AAVR are analyzed in detail, and the implications for AAV2 binding are explored through molecular modeling. Moreover, we find that binding sites for the antibodies ADK5a, ADK5b, and 3C5 on AAV5 overlap with the binding site of AAVR. These insights provide a structural foundation for development of gene therapy agents to better evade immune neutralization without disrupting cellular entry.

Adeno-associated virus (AAV) is a promising gene therapy vector and the biophysical characterization of its interactions with host proteins is a critical foundation for engineering tissue targeting and immune escape. Presented here are protocols for the production of: (a) the outer protein shells (virus-like particles or VLPs) for serotype 2 (AAV-2) and (b) two fragments from the binding ectodomain of AAV's cellular receptor, AAVR. His6PKD1-2 comprises the first two polycystic kidney disease (PKD) domains, the minimal required for efficient binding of AAV, expressed with an N-terminal histidine tag. MBP-PKD1-5 is a fusion of the maltose binding protein with all five of the PKD domains of the AAVR receptor. Presented are the expression and purification of milligram quantities, ample for in vitro analyses. For AAV-2, the protocol offers an alternative to the use of (infectious) wild-type virus or transducing vectors. One of the methods for producing transducing vector is in Sf9 cells, and the production of VLPs is based on this. For AAVR, the protocols enable biochemical and biophysical characterization of virus-binding. The minimal two-domain construct allows more saturated binding to symmetry-equivalent sites on the virus, while the larger construct might be better expected to reflect the native receptor.

Human gene therapy has advanced from twentieth-century conception to twenty-first-century reality. The recombinant Adeno-Associated Virus (rAAV) is a major gene therapy vector. Research continues to improve rAAV safety and efficacy using a variety of AAV capsid modification strategies. Significant factors influencing rAAV transduction efficiency include neutralizing antibodies, attachment factor interactions and receptor binding. Advances in understanding the molecular interactions during rAAV cell entry combined with improved capsid modulation strategies will help guide the design and engineering of safer and more efficient rAAV gene therapy vectors.

With 150 clinical trials for 30 diseases under way, AAV is a leading gene therapy vector. Immunotoxicity at high doses used to overcome inefficient transduction has occasionally proven fatal and highlighted gaps in fundamental virology.

Recombinant forms of adeno-associated virus (rAAV) are vectors of choice in the development of treatments for a number of genetic dispositions. Greater understanding of AAV's molecular virology is needed to underpin needed improvements in efficiency and specificity. Recent advances have included identification of a near universal entry receptor, AAVR, and structures by cryo-electron microscopy (EM) single particle analysis (SPA) that revealed, at high resolution, only the domains of AAVR most tightly bound to AAV. Here, cryogenic electron tomography (cryo-ET) is applied to reveal the neighboring domains of the flexible receptor. For AAV5, where the PKD1 domain is bound strongly, PKD2 is seen in three configurations extending away from the virus. AAV2 binds tightly to the PKD2 domain at a distinct site, and cryo-ET now reveals four configurations of PKD1, all different from that seen in AAV5. The AAV2 receptor complex also shows unmodeled features on the inner surface that appear to be an equilibrium alternate configuration. Other AAV structures start near the 5-fold axis, but now β-strand A is the minor conformer and, for the major conformer, partially ordered N-termini near the 2-fold axis join the canonical capsid jellyroll fold at the βA-βB turn. The addition of cryo-ET is revealing unappreciated complexity that is likely relevant to viral entry and to the development of improved gene therapy vectors. IMPORTANCE: With 150 clinical trials for 30 diseases underway, AAV is a leading gene therapy vector. Immunotoxicity at high doses used to overcome inefficient transduction, has occasionally proven fatal and highlighted gaps in fundamental virology. AAV enters cells, interacting through distinct sites with different domains of the AAVR receptor, according to AAV clade. Single domains are resolved in structures by cryogenic electron microscopy. Here, the adjoining domains are revealed by cryo-electron tomography of AAV2 and AAV5 complexes. They are in flexible configurations interacting minimally with AAV, despite measurable dependence of AAV2 transduction on both domains.