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 therapy trials are currently analyzed by anion-exchange HPLC. It was shown that the retention time of the adenovirus peak increased over the course of time upon storage in current liquid formulations. The number of isoaspartate residues at the outer surface of the particles also increased in parallel in these preparations. A recombinant adenovirus AV3760 was constructed with a modified hexon protein in which all four accessible Asn residues engaged 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 after 2 months at +20 degrees C as opposed to the control adenovirus. This demonstrates that the shift in retention time is caused by an increase in carboxyl groups on the outer surface of the virion due to deamidation of the above Asn residues. The modifications introduced in the hexon protein reduced the rate of Asn deamidation, without adverse effects on the infectivity of the virions. By reducing microheterogeneity in recombinant adenovirus, this work represents a significant advance in the development of a stable pharmaceutical vector for gene therapy.
Blanche
F
F
Gencell, a Division of Aventis Pharma, 13, quai Jules Guesde, 994403 Vitry/Seine Cedex, France. francis.blanche@aventis.com
Cameron
B
B
Somarriba
S
S
Maton
L
L
Barbot
A
A
Guillemin
T
T
D000256:Adenoviridae / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D001216:Asparagine / Q000737:chemistry / Q000235:genetics; D002213:Capsid / Q000737:chemistry / Q000235:genetics*; D036022:Capsid Proteins; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004274:DNA, Recombinant / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics; D026581:Isoaspartic Acid / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D013048:Specimen Handling
D036022:Capsid Proteins; D004274:DNA, Recombinant; D026581:Isoaspartic Acid; C018315:hexon capsid protein, Adenovirus; D001216:Asparagine