Stabilization of recombinant adenovirus: Site-directed mutagenesis of key asparagine residues in the hexon protein

The quality and stability of recombinant adenovirus preparations for gene t herapy trials are currently analyzed by anion-exchange HPLC. It was shown t hat the retention time of the adenovirus peak increased over the course of time upon storage in current liquid formulations. The number of isoaspartat e residues at the outer surface of the particles also increased in parallel in these preparations. A recombinant adenovirus AV3760 was constructed wit h a modified hexon protein in which all four accessible Asn residues engage d in Asn-Gly pairs were changed into Leu. (Asn244, Asn255, Asn437) or Ala ( Asn276). The retention time of AV3760, as measured by HPLC, was unchanged a fter 2 months at +20 degreesC as opposed to the control adenovirus. This de monstrates that the shift in retention time is caused by an increase in car boxyl groups on the outer surface of the virion due to deamidation of the a bove Asn residues. The modifications introduced in the hexon protein reduce d the rate of Asn deamidation, without adverse effects on the infectivity o f the virions. By reducing microheterogeneity in recombinant adenovirus, th is work represents a significant advance in the development of a stable pha rmaceutical vector for gene therapy. (C) 2001 Academic Press.