PMID- 11199265
OWN - NLM
STAT- MEDLINE
DCOM- 20010301
LR  - 20060421
IS  - 1099-498X (Print)
IS  - 1099-498X (Linking)
VI  - 2
IP  - 6
DP  - 2000 Nov-Dec
TI  - Scaleable chromatographic purification process for recombinant adeno-associated 
      virus (rAAV).
PG  - 444-54
AB  - BACKGROUND: Adeno-associated virus (AAV) is a human parvovirus currently being 
      developed as a vector for gene therapy applications. Traditionally AAV has been 
      purified from cell lysates using CsCl gradients; this approach however is not 
      likely to be useful in large-scale manufacturing. Moreover gradient-purified AAV 
      vectors tend to be contaminated with significant levels of cellular and 
      adenoviral proteins and nucleic acid. To address the issue of purification we 
      have developed a process scale method for the rapid and efficient purification of 
      recombinant AAV (rAAV) from crude cellular lysates. METHODS: The preferred method 
      for the purification of rAAVbetagal includes treatment of virally infected cell 
      lysates with both trypsin and nuclease followed by ion exchange chromatography 
      using ceramic hydroxyapatite and DEAE-Sepharose in combination with cellufine 
      sulphate affinity chromatography. RESULTS: Purification of rAAV particles from 
      crude cellular lysates co-infected with adenovirus was achieved using column 
      chromatography exclusively. Column-purified rAAV was shown to be greater than 90% 
      pure, free of any detectable contaminating adenovirus, biologically active, and 
      capable of directing efficient gene transfer to the lungs of both cotton rats and 
      mice. CONCLUSIONS: This study demonstrates the feasibility of using column 
      chromatography alone for the isolation of highly purified rAAV vector. The 
      methods described here are advancements in procedures to purify rAAV and are 
      adaptable for commercial production of clinical-grade rAAV vector.
FAU - O'Riordan, C R
AU  - O'Riordan CR
AD  - Genzyme Corporation, Framingham, MA 01701-9322, USA. coriordan@genzyme.com
FAU - Lachapelle, A L
AU  - Lachapelle AL
FAU - Vincent, K A
AU  - Vincent KA
FAU - Wadsworth, S C
AU  - Wadsworth SC
LA  - eng
PT  - Journal Article
PL  - England
TA  - J Gene Med
JT  - The journal of gene medicine
JID - 9815764
RN  - 0 (DNA, Recombinant)
RN  - 0 (Recombinant Fusion Proteins)
RN  - EC 3.2.1.23 (beta-Galactosidase)
SB  - IM
MH  - Animals
MH  - Cell Line
MH  - Chromatography/*methods
MH  - Chromatography, Ion Exchange
MH  - DNA, Recombinant/isolation & purification
MH  - Dependovirus/genetics/*isolation & purification
MH  - Gene Expression Regulation
MH  - Gene Transfer Techniques
MH  - Genetic Vectors/genetics/isolation & purification
MH  - Humans
MH  - Lung/metabolism/virology
MH  - Mice
MH  - Mice, Inbred BALB C
MH  - Recombinant Fusion Proteins/genetics/metabolism
MH  - Sigmodontinae
MH  - beta-Galactosidase/genetics/metabolism
EDAT- 2001/02/24 12:00
MHDA- 2001/03/07 10:01
CRDT- 2001/02/24 12:00
PHST- 2001/02/24 12:00 [pubmed]
PHST- 2001/03/07 10:01 [medline]
PHST- 2001/02/24 12:00 [entrez]
AID - 10.1002/1521-2254(200011/12)2:6<444::AID-JGM132>3.0.CO;2-1 [doi]
PST - ppublish
SO  - J Gene Med. 2000 Nov-Dec;2(6):444-54. doi: 
      10.1002/1521-2254(200011/12)2:6<444::AID-JGM132>3.0.CO;2-1.