PMID- 17437357
OWN - NLM
STAT- MEDLINE
DCOM- 20070619
LR  - 20131121
IS  - 1043-0342 (Print)
IS  - 1043-0342 (Linking)
VI  - 18
IP  - 4
DP  - 2007 Apr
TI  - Engineering adeno-associated virus for one-step purification via immobilized 
      metal affinity chromatography.
PG  - 367-78
AB  - 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.
FAU - Koerber, James T
AU  - Koerber JT
AD  - Department of Chemical Engineering and Helen Wills Neuroscience Institute, 
      University of California, Berkeley, CA 94720, USA.
FAU - Jang, Jae-Hyung
AU  - Jang JH
FAU - Yu, Julie H
AU  - Yu JH
FAU - Kane, Ravi S
AU  - Kane RS
FAU - Schaffer, David V
AU  - Schaffer DV
LA  - eng
GR  - EB003007/EB/NIBIB NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PL  - United States
TA  - Hum Gene Ther
JT  - Human gene therapy
JID - 9008950
RN  - 0 (Capsid Proteins)
RN  - 0 (VP3 protein, Dependovirus)
RN  - 7OV03QG267 (Nickel)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Capsid/chemistry
MH  - Capsid Proteins/chemistry/genetics
MH  - Cells, Cultured
MH  - Chromatography, Affinity/*methods
MH  - Dependovirus/chemistry/*genetics
MH  - Female
MH  - Genetic Vectors/chemistry/*genetics/*isolation & purification
MH  - Humans
MH  - Molecular Sequence Data
MH  - Mutation
MH  - Nickel/chemistry
MH  - Protein Conformation
MH  - *Protein Engineering
MH  - Rats
MH  - Rats, Inbred F344
EDAT- 2007/04/18 09:00
MHDA- 2007/06/20 09:00
CRDT- 2007/04/18 09:00
PHST- 2007/04/18 09:00 [pubmed]
PHST- 2007/06/20 09:00 [medline]
PHST- 2007/04/18 09:00 [entrez]
AID - 10.1089/hum.2006.139 [doi]
PST - ppublish
SO  - Hum Gene Ther. 2007 Apr;18(4):367-78. doi: 10.1089/hum.2006.139.