Volume 11, Issue 2, Pages 320-326 (February 2005) Herpesvirus-based infectious titering of recombinant adeno-associated viral vectors  Imran Mohiuddin, Scott Loiler, Irina Zolotukhin, Barry J. Byrne, Terence R. Flotte, Richard O. Snyder  Molecular Therapy  Volume 11, Issue 2, Pages 320-326 (February 2005) DOI: 10.1016/j.ymthe.2004.08.030 Copyright © 2004 Terms and Conditions

FIG. 1 (A) Schematic diagram of the replication center assay (RCA). Permissive cells are infected with adenovirus, wild-type AAV, and dilutions of the rAAV vector to be titered. (B) Infectious center assay (ICA). rep/cap-containing cells are infected with adenovirus and dilutions of the rAAV vector to be titered. (C) HSV infectious center assay (HSVICA). Permissive cells are infected with a herpes vector that expresses rep and cap and dilutions of the rAAV vector to be titered. Following infection, cells are incubated for 24–48 h and analyzed for (1) transgene-specific protein expression, (2) vector genomes by PCR [38,40] or hybridization following isolation of the low-molecular-weight DNA [47], or (3) infected cells by immobilizing the cells onto a membrane, lysing the cells, denaturing the DNA, and fixing it to the membrane with NaOH and probing with vector-specific sequences. Molecular Therapy 2005 11, 320-326DOI: (10.1016/j.ymthe.2004.08.030) Copyright © 2004 Terms and Conditions

FIG. 2 (A) Permissive cell screening. 2 × 105 cells per well were plated into 16-well glass chamber slides (Lab-Tek, Nalge Nunc International) in 100 μl of growth medium. One day later, the cells were infected in triplicate with 1 μl of either AAV1/CB-lucEYFP-WPRE (3.0 × 1012 vg/ml) or AAV5/CB-lucEYFP-WPRE (1.3 × 1012 vg/ml) vector per well. Six days postinfection, the cells were lysed with 100 μl of 1× CCLR lysis reagent from Promega. 10 μl of cell lysate was added to 90 μl of luciferin reagent (Promega Luciferase Assay System) and analyzed for luciferase activity. Results are ±SEM. BNL CL.2 (ATCC TIB-73) cells are normal mouse liver cells grown in DMEM with 10% FBS. VA cells, a rat pulmonary artery endothelial cell line, were isolated by mechanical methods [48] and grown in medium 199 with Earle’s salts (Sigma), sodium bicarbonate (pH 7.4), 10% fetal bovine serum, 10 mM glutamine, and antibiotic/antimycotic solution at 37°C in 5% CO2. (B) 293 cell transduction assay. 2 × 104 293 cells seeded in 96-well plates were transduced with serial dilutions of AAV1-TR-UF-11, AAV5-TR-UF-11, or AAV2-TR-UF-11 vector and co-infected with HSVr/c at an m.o.i. of 10 or 40 or with adenovirus type 5 at an m.o.i. of 2. After 30 h, GFP-positive cells were counted at different dilutions and the transducing titers (TU/ml) were calculated. Recombinant AAV vectors were produced and purified as described [25]. The CB-lucEYFP-WPRE vector is a rAAV vector with the CB promoter driving expression of a fusion protein of luciferase and enhanced yellow fluorescent protein. This vector is flanked on each end by AAV2 ITRs and also contains a WPRE for higher levels of expression. The TR-UF-11 vector is a rAAV vector with the CB promoter driving expression of GFP and is also flanked by AAV2 ITRs. Molecular Therapy 2005 11, 320-326DOI: (10.1016/j.ymthe.2004.08.030) Copyright © 2004 Terms and Conditions

FIG. 3 ICA vs HSVICA. 96-well plates seeded with 2 × 104 293, HS578T, C12, or COS7 cells were infected with serial dilutions of AAV1-TR-UF-11, AAV5-TR-UF-11, or AAV2-TR-UF-11 vector. These cells were co-infected with the indicated m.o.i. of either Ad or HSVr/c. After 48 h, the cells were transferred to a nylon membrane and lysed, and the nucleic acid was immobilized following treatment with NaOH. Filters were hybridized with a radiolabeled CMV immediate early enhancer probe. Cells productively infected with rAAV generated a spot following autoradiography. HSVr/c has been described and was propagated on V27 cells [22]. Adenovirus type 5 (ATCC VR-5) was propagated on 293 cells [49]. The C12 cell line [21] has been described. The 293, HS578T [50], and COS-7 (ATCC CRL-1651, transformed with SV40 T antigen) cell lines were grown in DMEM with 10% FBS. Molecular Therapy 2005 11, 320-326DOI: (10.1016/j.ymthe.2004.08.030) Copyright © 2004 Terms and Conditions