Volume 11, Issue 2, Pages 284-293 (February 2005) Gestational age of recipient determines pattern and level of transgene expression following in utero retroviral gene transfer  Christopher D. Porada, Paul J. Park, Graça Almeida-Porada, Wansheng Liu, Ferhat Ozturk, Hudson A. Glimp, Esmail D. Zanjani  Molecular Therapy  Volume 11, Issue 2, Pages 284-293 (February 2005) DOI: 10.1016/j.ymthe.2004.09.009 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Transduction of liver cells following direct intraperitoneal injection of vector in utero. Paraffin-embedded sections were prepared from the liver of sheep injected in utero with the MSCV-RFP-NeoR retroviral vector and analyzed by immunohistochemistry with an antibody specific for RFP. RFP-positive cells appear brown. (A) A representative section of liver exhibiting numerous transgene-positive hepatocytes, (B) two foci of transduced hepatocytes, and (C) transgene-expressing hepatic endothelium. Molecular Therapy 2005 11, 284-293DOI: (10.1016/j.ymthe.2004.09.009) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Transduction of lung tissue following direct intraperitoneal injection of vector in utero. Paraffin-embedded sections were prepared from the lungs of sheep injected in utero with the MSCV-RFP-NeoR retroviral vector and analyzed by immunohistochemistry with an antibody specific for RFP. RFP-positive cells appear brown. (A) A representative section of lung exhibiting numerous transgene-positive epithelial cells and fibroblasts within the interalveolar septi, (B) a cluster of transduced chondrocytes within the forming hyaline cartilage, (C) the transduction of smooth muscle cells within the lung of one of the in utero-treated sheep, and (D) a transgene-positive alveolar macrophage. Molecular Therapy 2005 11, 284-293DOI: (10.1016/j.ymthe.2004.09.009) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Direct intraperitoneal injection of vector supernatant results in low level of gene transfer to brain. Paraffin-embedded sections were prepared from various regions of the brain of sheep injected in utero with the MSCV-RFP-NeoR retroviral vector and analyzed by immunohistochemistry with antibodies specific to NPT and RFP. Very minimal staining was seen with either of these antibodies in all of the sheep. Shown is a single NPT-positive (brown) cell with morphology suggestive of a neuron that was seen in a brain section obtained from one of the in utero-transduced sheep. Molecular Therapy 2005 11, 284-293DOI: (10.1016/j.ymthe.2004.09.009) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Transduction efficiency of hepatocytes is higher at earlier points in gestation. Paraffin-embedded sections were prepared from the liver of sheep injected in utero with the MSCV-RFP-NeoR retroviral vector at varying gestational ages and analyzed by immunohistochemistry with an antibody specific for RFP to assess the effect recipient age had on efficiency of hepatocyte transduction. (A) Representative sections of liver from sheep injected with vector at 54, 59, 65, or 102 days of gestation. RFP-positive cells appear brown. (B) Transgene expression in hepatocytes assessed by immunohistochemistry. A graphical analysis of the data we obtained by counting 5000 hepatocytes in each section and determining the percentage of these that were transgene-positive is shown. Molecular Therapy 2005 11, 284-293DOI: (10.1016/j.ymthe.2004.09.009) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Analysis of liver transgene expression and transduction at 1 week post-vector injection. To assess whether the differences in the number of transgene-positive hepatocytes were truly due to differences in the efficiency of transduction of this cell type at each age or, rather, the result of differing levels of transgene expression following transfer at each age, we performed immunohistochemistry and in situ hybridization on liver sections obtained from in utero-treated sheep at 1 week postinjection to allow quantitation of the percentage of hepatocytes harboring proviral DNA versus the percentage expressing the transgene. (A) A bar graph showing the percentages of transgene-expressing hepatocytes at 1 week after vector injection into fetal sheep of varying age. (B) A bar graph showing the percentages of hepatocytes that contained proviral DNA following gene transfer at each gestational age. (C) A representative in situ hybridization result in which two provirus-containing nuclei are indicated with black arrows. Molecular Therapy 2005 11, 284-293DOI: (10.1016/j.ymthe.2004.09.009) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Transduction efficiency of lung tissue increases as recipient age increases. Paraffin-embedded sections were prepared from the lungs of sheep injected in utero with the MSCV-RFP-NeoR retroviral vector at varying gestational ages and analyzed by immunohistochemistry with antibodies specific for NPT and RFP to assess the effect recipient age had on efficiency of hepatocyte transduction. The results are from staining with the anti-RFP antibody. RFP-positive cells appear brown. (A) A representative section of lung from a sheep injected with vector at 54 days of gestation, exhibiting essentially no transgene-positive cells. (B) A representative section of lung from a sheep injected with vector at 59 days of gestation, exhibiting small numbers of transgene-positive cells. (C) A representative section of lung from a sheep injected with vector at 65 days of gestation, exhibiting significantly higher numbers of transgene-positive cells compared to sheep injected at 54 or 59 days. (D) A representative section of lung from a sheep injected with vector at 102 days of gestation, which exhibits widespread transduction/transgene expression in multiple cell types throughout the section. Molecular Therapy 2005 11, 284-293DOI: (10.1016/j.ymthe.2004.09.009) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions