Engineering adeno-associated virus for one-step purification via immobilized metal affinity chromatography

James T. Koerber, Jae Hyung Jang, Julie H. Yu, Ravi S. Kane, David V. Schaffer

Research output: Contribution to journalArticlepeer-review

46 Citations (Scopus)


Adeno-associated virus (AAV) is a promising vehicle for gene therapy, which will rely on the generation of Mgh-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 (His6) sequence. Through the combination of a diverse AAV2 library and rational peptide insertions, we have located an optimal His6 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.

Original languageEnglish
Pages (from-to)367-378
Number of pages12
JournalHuman Gene Therapy
Issue number4
Publication statusPublished - 2007 Apr

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Molecular Biology
  • Genetics


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