Engineering adeno-associated virus for one-step purification via immobilized metal affinity chromatography.
Adeno-associated virus (AAV) is a promising vehicle for gene therapy, which will rely on the generation of high-titer, high-purity recombinant vectors. However, numerous purification protocols can involve challenging optimization or scalability issues, and most AAV serotypes do not bind heparin or sialic acid, used for AAV2/3 or AAV4/5 purification, requiring the development of new chromatography strategies. Immobilized metal affinity chromatography (IMAC) allows for robust protein purification via affinity tags such as the hexahistidine (His(6)) sequence. Through the combination of a diverse AAV2 library and rational peptide insertions, we have located an optimal His(6) tag insertion site within the viral capsid. This mutant and a related AAV8 variant can be purified from clarified cell lysate in a single gravity column step at infectious particle yields exceeding 90%. Furthermore, injection of IMAC-purified vector into the brain demonstrates that it mediates high-efficiency gene delivery in vivo, equivalent to that of wild-type capsid, with minimal immune cell activation. This affinity chromatography method may offer advantages in ease of purification, final vector purity, and process scalability. Moreover, a combined rational design and high-throughput library selection approach can aid in the design of enhanced viral gene delivery vectors.
Koerber
James T
JT
Department of Chemical Engineering and Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720, USA.
Jang
Jae-Hyung
JH
Yu
Julie H
JH
Kane
Ravi S
RS
Schaffer
David V
DV
D000595:Amino Acid Sequence; D000818:Animals; D002213:Capsid / Q000737:chemistry; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D000229:Dependovirus / Q000737:chemistry / Q000235:genetics*; D005260:Female; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009532:Nickel / Q000737:chemistry; D011487:Protein Conformation; D015202:Protein Engineering; D051381:Rats; D011916:Rats, Inbred F344
D036022:Capsid Proteins; C442558:VP3 protein, Dependovirus; D009532:Nickel