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 (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 language | English |
|---|---|
| Pages (from-to) | 367-378 |
| Number of pages | 12 |
| Journal | Human Gene Therapy |
| Volume | 18 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - 2007 Apr 1 |
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All Science Journal Classification (ASJC) codes
- Molecular Medicine
- Molecular Biology
- Genetics
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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 journal › Article
TY - JOUR
T1 - Engineering adeno-associated virus for one-step purification via immobilized metal affinity chromatography
AU - Koerber, James T.
AU - Jang, Jae Hyung
AU - Yu, Julie H.
AU - Kane, Ravi S.
AU - Schaffer, David V.
PY - 2007/4/1
Y1 - 2007/4/1
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=34248162591&partnerID=8YFLogxK
U2 - 10.1089/hum.2006.139
DO - 10.1089/hum.2006.139
M3 - Article
C2 - 17437357
AN - SCOPUS:34248162591
VL - 18
SP - 367
EP - 378
JO - Human Gene Therapy
JF - Human Gene Therapy
SN - 1043-0342
IS - 4
ER -