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Biotech / Medical : TGEN - Targeted Genetics Corporation -- Ignore unavailable to you. Want to Upgrade?

To: Mike McFarland who wrote (351)	2/13/2002 9:59:33 PM
From: Mike McFarland	Read Replies (1) \| Respond to of 557

Just some old stuff, making AAV A method for the preparation of highly purified adeno-associated virus using affinity column chromatography, protease digestion and solvent extraction AU: Anderson-R; Macdonald-I; Corbett-T; Whiteway-A; Prentice-HG SO: JOURNAL-OF-VIROLOGICAL-METHODS. MAR 2000; 85 (1-2) : 23-34 FTXT: ScienceDirect (tm) sciencedirect.com PY: 2000 IS: 0166-0934 AB: Recombinant adeno-associated virus (AAV) is becoming the vector of choice for many gene therapy protocols. There has been much recent progress made toward increasing AAV titres but a continuing problem in using AAV has been that it is relatively difficult to concentrate and purify. Traditional methods, such as caesium chloride (CsCl) gradients, have drawbacks, notably extended purification times and the ability to process only limited volumes. Where the target cells of interest require a high multiplicity of infection (MOI), or to complete in vivo experiments, there is a requirement for both the production of high titre and a large volume of virus. This is laborious to obtain using traditional methods. A simple technique is described here for purifying AAV, involving affinity chromatography, protease digestion and solvent extraction that retains both the high yields and titres obtained using CsCl gradients. In addition, this technique displays a fast throughput and may be used to purify AAV from larger volumes than CsCl gradients. The high yield and purity of these virus preparations has allowed us to achieve good levels of expression in the target cell types tested. The purification technique described here will be applicable to any protocol that requires high titre, high purity recombinant AAV (rAAV). (C) 2000 Elsevier Science B.V. All rights reserved. UD: 200013 Adeno-associated virus vectors show variable dependence on divalent cations for thermostability: Implications for purification and handling AU: Turnbull-AE; Skulimowski-A; Smythe-JA; Alexander-IE SO: HUMAN-GENE-THERAPY. MAR 1 2000; 11 (4) : 629-635 PY: 2000 IS: 1043-0342 AB: Recombinant adeno-associated virus (rAAV) shows significant promise as a vector for gene transfer in preclinical models of human disease, and is currently being evaluated in human clinical trials. As a consequence, increasing attention is being turned to the important tasks of optimizing rAAV titer, purity, and stability. We have observed dramatic variation in divalent cation dependence for thermostability of different rAAV vectors. To further investigate this observation, the thermostability of eight different vector constructs ranging in size from 73 to 107% of wild-type genome size (4.68 kilobases) was determined in the presence and absence of divalent cations, Virions containing smaller genomes (i.e., <85% wild type) were relatively divalent cation independent for thermostability. In contrast, virions containing recombinant genomes close to, or exceeding, wild-type size (i.e., >95% wild type) were dependent on divalent cations for thermostability. Genome sequence also appeared to be a factor in the thermostability of the larger rAAV vectors. These observations are of both practical and theoretical significance, Divalent cations should be included in all buffer solutions used during rAAV purification and storage, and unnecessary heat exposure avoided. These data also demonstrate that different recombinants of a particular virus should not be assumed to possess the same thermostability profile. UD: 200013