Volume 6, Issue 5, Pages 678-686 (November 2002) A Versatile and Scalable Two-Step Ion-Exchange Chromatography Process for the Purification of Recombinant Adeno-associated Virus Serotypes-2 and -5  Nicole Brument, Robert Morenweiser, Véronique Blouin, Estelle Toublanc, Isabelle Raimbaud, Yan Chérel, Sébastien Folliot, Florence Gaden, Pierre Boulanger, Gabrielle Kroner-Lux, Philippe Moullier, Fabienne Rolling, Anna Salvetti  Molecular Therapy  Volume 6, Issue 5, Pages 678-686 (November 2002) DOI: 10.1006/mthe.2002.0719 Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 1 FPLC elution profiles during rAAV2 and rAAV5 purification. rAAV2 particles were produced using either adenovirus-infected HeRC32GFP cells (A) or triple-transfected 293 cells (B). rAAV5 particles were produced by triple transfection of 293 cells (C). The left and right sides show the elution profiles obtained during the first (SP Sepharose HP) and the second (Source 15Q) chromatography step, respectively. The small insets on the left represent the complete purification profile during the first chromatography step. The dark line corresponds to the 280-nm profile and the light line the conductivity expressed in mS/cm. The × axis indicates the number of column volumes (CV). The gray area indicates the presence of rAAV particles as determined by measuring the transducing units. Molecular Therapy 2002 6, 678-686DOI: (10.1006/mthe.2002.0719) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 2 Silver staining and western blot analysis of rAAV-containing samples. rAAV2 particles were produced using either adenovirus-infected HeRC32GFP cells (A) or triple-transfected 293 cells (B). rAAV5 particles were produced by triple transfection of 293 cells (C). Samples containing known amounts of rAAV particles (measured by dot blot) and indicated on the top of each lane were resolved by SDS–PAGE and either silver stained or transferred to a membrane and analyzed by western blot. I, input cell lysate; C1, pool of the rAAV-containing fractions collected after the first chromatography step; C2, pool of the rAAV-containing fractions collected after the second chromatography step; I/H, rAAV2 particles purified by iodixanol/heparin; I/H*, rAAV2 particles purified by iodixanol/heparin following treatment of the cell lysate with deoxycholate and Benzonase; 2xCsCl, rAAV5 particles purified on a double CsCl gradient; Ad, purified adenoviral particles; indicated as total particles at the top of the lane. Molecular Therapy 2002 6, 678-686DOI: (10.1006/mthe.2002.0719) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 3 EM analysis of rAAV2 and rAAV5 particles purified either by the two-step ion-exchange chromatography procedure (FPLC; right) or by the conventional methods (left), i.e., iodixanol/heparin and CsCl for rAAV2 and rAAV5 particles, respectively. (A and B) rAAV2 particles produced using adenovirus-infected HeRC32GFP cells; (C and D) rAAV2 particles produced by triple transfection on 293 cells; (E and F) rAAV5 particles produced by triple transfection on 293 cells. Molecular Therapy 2002 6, 678-686DOI: (10.1006/mthe.2002.0719) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 4 Ratio of total to full (or genome-containing) particles in purified rAAV2 preparations. Total particles (TP) in rAAV2 stocks prepared by FPLC or iodixanol/heparin were measured by ELISA [28] and genome-containing particles (FP) by dot blot. The ratio (TP/FP) obtained for each preparation is indicated by a dot. The horizontal line indicates the average value and the vertical line the standard deviation. Molecular Therapy 2002 6, 678-686DOI: (10.1006/mthe.2002.0719) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 5 Transduction of the muscle and the retina by rAAV2 and rAAV5 particles purified using different methods. For muscle injections, a total of 2 ×1010 rAAV2CMVnlsLacZ particles (as measured by dot blot) were injected into the mouse tibialis anterior muscle and the tissues stained with X-gal 3 weeks postinjection (A and B; original magnification × 2.5). Some tissue sections were counterstained with hematoxylin and eosin to detect cellular infiltrates (C and D; original magnification × 10). For subretinal injections, 2.5 × 109 rAAV5GFP particles (as measured by dot blot) were injected into rats as previously described [4] (E and F). Photographs were taken 15 days postinjection. Molecular Therapy 2002 6, 678-686DOI: (10.1006/mthe.2002.0719) Copyright © 2002 American Society for Gene Therapy Terms and Conditions