107. α2, 3 and α2,6 N-Linked Sialic Acid Facilitate Efficient Binding and Transduction by Adeno-Associated Virus Type 1 and 6

Abstract

Recombinant adeno-associated viruses (AAV) are promising vectors in the field of gene therapy. Different AAV serotypes display distinct tissue tropism, believed to be associated with distribution of their receptors on target cells. Of the eleven well -characterized AAV serotypes, heparan sulfate proteoglycan and sialic acid have been suggested to be the attachment receptors for AAV type 2 and 4 & 5 respectively. In this report, we identify primary attachment molecules for the two closely related serotypes, AAV1 and AAV6. First we demonstrate use of similar receptor for AAV 1 & 6 through competition experiments after co-infection. Second, similar to removal of heparin sulfate for AAV2, enzymatic or genetic removal of sialic acid significantly reduced AAV1 and AAV6 binding and transduction. Third, contrasting AAV5, which uses only |[alpha]|2,3 sialic acid as the major attachment factor for transduction, lectin staining and lectin competition assay identified both |[alpha]|2,3-linked and |[alpha]|2,6-linked sialic acid for efficient AAV 1 & 6 transduction. Inhibitor of O-linked glycosylation did not block transduction, however, significantly reduction was observed on N-glycan deficient Lec1 cells, indicating that N-linked, but not O-linked sialic acid is required for AAV1 and AAV6 transduction. This was confirmed by resialylation experiment using sialic acid deficient Lec2 cells. Furthermore, mucin which binds to O-linked sialic acid and inhibits AAV4 transduction had no inhibition effect on AAV1 and AAV6 transduction. Finally, proteinase K treatment compared to glycolipid inhibitor treatment supports sialic acid modification of glycoproteins rather than glycolipids with respect to AAV1 and AAV6 transduction. Taken together, the data support a glycoprotein with N-linked |[alpha]|2,3 and /or |[alpha]| 2,6 sialic acid serving as a primary attachment molecule for AAV1 and AAV6 transduction.