540. Identification of a 16 Amino Acid-Long Motif in the N-Terminal Half of the AAV9 Capsid Essential for Persistence in the Blood for an Extended Period of up to 3 Days
Authors: Kei Adachi, Hiroyuki Nakai
Journal: Molecular Therapy (2016)

The distinctively prolonged half-life in the blood circulation is one of the key features of AAV9 vectors and is presumed to play an important role in efficient gene delivery to various target organs following systemic vector administration. We have so far analyzed blood clearance rates of nearly one thousand different AAV strains (serotypes and capsid-engineered mutants) in mice following intravenous vector injection, and have discovered that the wild-type AAV9 and many AAV9-derived capsid mutants are particularly unique in that they can persist in the blood circulation at significant levels when measured at 72 hours post-injection. Although non AAV9-derived capsid mutants exist that show a blood vector concentration at a level equivalent to or higher than that of the wild-type AAV9 at 24 hours post-injection, their blood vector concentrations surprisingly and without exception become substantially lower than the wild-type AAV9 concentration at 72 hours post-injection. In AAV vector development, long half-life should be an attractive feature of new vectors; however, the paucity of knowledge of the capsid amino acid sequences responsible for vector persistence in the blood has made it difficult to design novel AAV capsids with a prolonged half-life. Here we report the identification of a 16 amino acid-long motif in the N-terminal half of the AAV9 capsid essential for the vector persistence in the blood for an extended period of up to 3 days, which is the pharmacokinetic profile unique to AAV9. In our previous study, we performed double alanine scanning of the entire C-terminal half of the AAV9 capsids (amino acid positions 356 to 736), and identified approximately 40 amino acids that showed loss of vector persistence in the blood only at the 72-hour time point when mutated to alanine. Many of these amino acids are not unique to AAV9 or are scattered within the capsid amino acid sequence. In the present study, we performed double alanine scanning of the entire N-terminal half of the AAV9 capsids (amino acid positions 4 to 355), and determined their blood clearance rates in mice by the AAV Barcode-Seq approach. Among these 175 double alanine mutants, 52 mutants did not produce viral particles at levels required for the analysis; therefore, the pharmacokinetic data could be collected only from 123 of the 175 mutants. This analysis revealed 7 double alanine mutants that span a stretch of 16 consecutive amino acids and form a group that is clearly distinctive from the other 116 mutants (note: one mutant within this amino acid stretch could not be assessed due to the inability to produce viral particles). That is, the 116 mutants showed the same pharmacokinetic profile as that of the wild-type AAV9 while the distinctive 7 mutants persisted in the blood for only up to 24 hours post-injection at levels similar or superior to the wild-type AAV9 and showed substantially lower concentrations at 72 hours post-injection. Interestingly, this motif contains one of the most variable regions across different serotypes and the amino acid sequence of the identified motif is unique only to AAV9 and other Clade F AAV serotypes. We are currently investigating whether swapping this motif of AAV9 with other serotypes' could result in loss of persistence at 72 hours post-injection or whether replication of this motif in other non-AAV9 capsids could bestow long-term persistence. In summary, our study suggests that the 16 amino acid-long motif in the N-terminal half of the capsid that is unique to the Clade F AAV serotypes plays an essential role in maintaining the persistence of circulating viral particles in the blood for an extended period of several days.