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 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
- 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 (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.
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 (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.
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U2 - 10.1089/hum.2006.139
DO - 10.1089/hum.2006.139
M3 - Article
VL - 18
SP - 367
EP - 378
JO - Human Gene Therapy
JF - Human Gene Therapy
SN - 1043-0342
IS - 4
ER -