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 journalArticle

36 Citations (Scopus)

Abstract

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 (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.

Original languageEnglish
Pages (from-to)367-378
Number of pages12
JournalHuman Gene Therapy
Volume18
Issue number4
DOIs
Publication statusPublished - 2007 Apr 1

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His-His-His-His-His-His
Dependovirus
Affinity Chromatography
Capsid
Metals
Peptide Library
Viral Genes
Gravitation
N-Acetylneuraminic Acid
Genetic Therapy
Libraries
Heparin
Chromatography
Injections
Brain
Genes
Proteins

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

Koerber, James T. ; Jang, Jae Hyung ; Yu, Julie H. ; Kane, Ravi S. ; Schaffer, David V. / Engineering adeno-associated virus for one-step purification via immobilized metal affinity chromatography. In: Human Gene Therapy. 2007 ; Vol. 18, No. 4. pp. 367-378.
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Engineering adeno-associated virus for one-step purification via immobilized metal affinity chromatography. / Koerber, James T.; Jang, Jae Hyung; Yu, Julie H.; Kane, Ravi S.; Schaffer, David V.

In: Human Gene Therapy, Vol. 18, No. 4, 01.04.2007, p. 367-378.

Research output: Contribution to journalArticle

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