Volume 9, Issue 4, Pages 625-631 (April 2004) A Quantitative Method for Measuring Gene Expression Magnitude and Volume Delivered by Gene Therapy Vectors  Kenneth N Barton, Xueqing Xia, Hui Yan, Hans Stricker, Gregory Heisey, Fang-Fang Yin, Tavarekere N Nagaraja, Guopei Zhu, Andrew Kolozsvary, Joseph D Fenstermacher, Mei Lu, Jae Ho Kim, Svend O Freytag, Stephen L Brown  Molecular Therapy  Volume 9, Issue 4, Pages 625-631 (April 2004) DOI: 10.1016/j.ymthe.2004.01.011 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Modulation transfer function of 99mTcO4− autoradiography. Change in contrast (MTF) is plotted as a function of radioactive spot separation. The example shown represents the sharpest contrast of 24 spots sampled. The resolution at 5% contrast is indicated. Molecular Therapy 2004 9, 625-631DOI: (10.1016/j.ymthe.2004.01.011) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 99mTcO4− autoradiography calibration curve. Optical density from digitized autoradiographs of 100-μm-thick cryosections was plotted as a function of 99mTc activity. Each point on the curve was generated from the average of 10 pixel values. The limit of detection is 2 standard deviations above background (13 ± 1). The optical densities of the dog prostate autoradiographs fell predominantly within the most sensitive region of the calibration curve (30–120). Molecular Therapy 2004 9, 625-631DOI: (10.1016/j.ymthe.2004.01.011) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 In situ hybridization analysis of Ad5-yCD/mutTKSR39rep-hNIS viral DNA in the dog prostate. The center autoradiograph represents a single 100-μm-thick section of a dog prostate injected in the left and right lobes with 3 × 1011 vp of Ad5-yCD/mutTKSR39rep-hNIS 1 day prior to systemic delivery of 20 mCi of Na99mTcO4. (A and B) In situ hybridization of Ad5-yCD/mutTKSR39rep-hNIS viral DNA corresponding to regions of high TcO4− uptake. (C and D) In situ hybridization of Ad5-yCD/mutTKSR39rep-hNIS viral DNA corresponding to regions of no TcO4− uptake. Blue staining indicates the presence of Ad5-yCD/mutTKSR39rep-hNIS viral DNA in infected cells. Molecular Therapy 2004 9, 625-631DOI: (10.1016/j.ymthe.2004.01.011) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Digitization of hNIS gene expression in the dog prostate. hNIS gene expression from digitized autoradiographs is shown for a series of 100-μm-thick sections of a prostate injected with Ad5-yCD/mutTKSR39rep-hNIS in the left and right lobes. (A) Autoradiographs; (B) isodose curves. The color scale on the right indicates the relative level of gene expression. (C) High-resolution image of gene expression from a single prostate section. The color scale on the right indicates level of gene expression in μCi/g tissue. Molecular Therapy 2004 9, 625-631DOI: (10.1016/j.ymthe.2004.01.011) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Three-dimensional reconstruction of the viral spread and gene expression within the prostate. A series of individual isodose curves (as shown in Fig. 4B) was stacked to generate a three-dimensional image of the prostate (blue) and gene expression volume (red). Six projections (A–E) rotated clockwise in 60° increments are shown. The optical density threshold of gene expression was set to 83 based on the results of in situ hybridization analyses for Ad5-yCD/mutTKSR39rep-hNIS viral DNA. l, left; r, right; b, base; ant, anterior face; ur, urethra; p, PEG 400 vehicle; s, saline vehicle. Molecular Therapy 2004 9, 625-631DOI: (10.1016/j.ymthe.2004.01.011) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Graphical representation of gene expression in the dog prostate. The total number of pixels at each gene expression magnitude is plotted. Blue line, virus prepared in saline; red line, virus prepared in PEG 400. Molecular Therapy 2004 9, 625-631DOI: (10.1016/j.ymthe.2004.01.011) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions