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Volume 16, Issue 12, Pages 1960-1967 (December 2008)
High-throughput Screening and Biophysical Interrogation of Hepatotropic AAV Samuel L Murphy, Anand Bhagwat, Shyrie Edmonson, Shangzhen Zhou, Katherine A High Molecular Therapy Volume 16, Issue 12, Pages (December 2008) DOI: /mt Copyright © 2008 The American Society of Gene Therapy Terms and Conditions
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Figure 1 Generation of a monoclonal antibody against intact AAV8 particles. AAV2 and AAV8 vector particles were coated in triplicate onto an ELISA plate at a concentration of 2 × 1010 vg/ml. Vector particles were either coated without treatment (black bars) or were denatured by incubation for 30 minutes at 100 °C (stippled bars) before coating. Monoclonal antibodies A20, B1, and were used to detect vector particles. HRP-conjugated secondary antibody and ONPD substrate were used for detection. This assay was repeated twice with similar results. Molecular Therapy , DOI: ( /mt ) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions
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Figure 2 Protein interactions detected for AAV2 and AAV8 with a human protein microarray. (a) Protein interactions for AAV2 and AAV8 were detected by incubating protoarray human protein chips with vector particles at a concentration of 5 × 1012 vp/ml. A representative section of protein interactions detected for AAV2 and AAV8 is shown. Protein chips were exposed to AAV2 or AAV8 vector particles and bound vector particles were detected with A20 and monoclonal antibodies, respectively. Secondary Alexa647-conjugated donkey antimouse antibody was used to detect the bound primary antibodies. The control plate was incubated with A20 and secondary antibody in the absence of vector particle addition. Yellow circles and rectangles are used to identify corresponding areas on each chip. The area shown represents one-twelfth of the total chip for each assay. (b) Pie chart classifying protein interactions shared by AAV2 and AAV8 by molecular function. Analysis was performed using pantherDB software. Molecular Therapy , DOI: ( /mt ) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions
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Figure 3 Inhibition of CDK2 increases vector transduction. Hep3B cells were treated with 25 μmol/l SU9516 or vehicle only (dimethyl sulfoxide) at the time of vector transduction. Cells were transduced with AAV2-GFP or AAV8-GFP at a multiplicity of infection of 1 × 105 vg/cell. After 72 hours, cells were harvested and green fluorescent protein fluorescence for cells treated with SU9516 (green line), cells treated with vehicle only (red line) and untransduced cells (blue line) were measured by flow cytometry. The experiment shown is representative of three independent experiments. Each experimental condition was tested in triplicate. Statistical significance was measured using an unpaired Student's t-test (P < 0.02 for AAV2 with SU9516 versus without SU9516 and P < for AAV8 with SU9516 versus without SU9516). MFI, mean fluorescence intensity. Molecular Therapy , DOI: ( /mt ) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions
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Figure 4 In vitro interaction of vector particles with CDK2/cyclin A. (a) Purified CDK2/cyclinA was coated onto ELISA plates at a concentration of 1 μg/ml. Vector particles were incubated with blocked plates at room temperature for 1 hour and bound vector particles were detected with A20 (AAV2) or (AAV8) monoclonal antibodies and an HRP-conjugated donkey antimouse secondary antibody. Heat-treated vector particles were incubated at 60 °C for 30 minutes before addition to incubation. (b) Inhibition of CDK2 kinase activity in an in vitro assay is shown. Phosphorylation of Ser-Thr12 peptide substrate was measured in the presence of inhibitors SU9516, Cdk2/cyclin inhibitory peptide I, AAV2, or AAV8. Each inhibitor was incubated with purified CDK2/cyclinA, ATP, and peptide substrate for 60 minutes at room temperature. Inhibition of phosphorylation was measured using a FRET-based reporter assay. Inhibition curves are shown for SU9516 (dark blue), CCIPI (red), AAV2 (yellow), heat-treated AAV2 (green), AAV8 (purple), and heat-treated AAV8 (orange). Starting concentrations of inhibitors tested were 250 μmol/l for SU9516, 5 mmol/l for CCIPI, and 600 pmol/l for each AAV. Nine threefold dilutions were made for each inhibitor. The eleventh lane contains no inhibitor for each sample. Each sample shown is the average of four quadruplicate samples. This experiment was repeated twice with similar results. Molecular Therapy , DOI: ( /mt ) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions
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Figure 5 Linkage of capsid thermostability and genome uncoating for AAV2 and AAV8. (a) AAV2 vector particles were heat treated for 30 minutes at the indicated temperature and then coated in sextuplicate wells of an ELISA plate overnight. Monoclonal antibodies A20 (squares) and B1 (triangles) were used together with donkey antimouse HRP secondary antibody to detect vector particles in triplicate wells for each indicated temperature in an ELISA. (b) AAV8 vector particles were used to perform the same assay as in a, except that monoclonal antibodies (squares) and B1 (triangles) were used for detection of vector particles. (c) AAV2 (open circles) and AAV8 (closed circles) vector particles were heated to the indicated temperature for 30 minutes. Each sample was then separately digested with DNAse enzyme for 1 hour at 37 °C. DNAse enzymatic activity was subsequently quenched with EDTA and heat treatment. Vector particles were then diluted and loaded onto a real-time PCR plate for genome quantification using a primer/probe set designed to detect the human Factor IX transgene. Average CT is shown for each vector with increasing temperature used during heat treatment. Experiments shown in a, b, and c were each repeated three times with similar results. HHL5 cells were transduced with AAV2-GFP vector at a multiplicity of infection of 100,000 vg/cell. Shown on the left are green fluorescent protein (GFP) fluorescence (upper left) and B1 antibody staining (lower left) for the same cells. Shown on the right are GFP fluorescence (upper right) and A20 staining (lower right) for the same cells. Cells were harvested 4 hours (dark blue line), 24 hours (green line), or 48 hours (orange line) after transduction, or in the absence of transduction (shaded red areas). Molecular Therapy , DOI: ( /mt ) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions
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Figure 6 Kinetic analysis of vector genome uncoating and vector particle degradation. HHL5 cells were transduced with AAV2-GFP vector at a multiplicity of infection of 100,000 vg/cell. Shown on the left are GFP fluorescence (upper left) and B1 antibody staining (lower left) for the same cells. Shown on the right are GFP fluorescence (upper right) and A20 staining (lower right) for the same cells. Cells were harvested 4 hours (dark blue line), 24 hours (green line), or 48 hours (orange line) after transduction, or in the absence of transduction (shaded red areas). Molecular Therapy , DOI: ( /mt ) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions
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