THE JOURNAL OF GENE MEDICINE RESEARCH ARTICLE J Gene Med 2005; 7: 926–935. Published online 2 March 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.740 Solid tissues can be manipulated ex vivo and used as vehicles for gene therapy E. Hasson 1 Y. Slovatizky 1 Y. Shimoni 1 H. Falk 2 A. Panet 2 E. Mitrani 1 * 1 Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel 2 Department of Virology, Hadassah Medical School, Hebrew University of Jerusalem, Jerusalem, Israel *Correspondence to: E. Mitrani, Institute of Life Sciences, The Hebrew University of Jerusalem, Givat-Ram, Jerusalem 91904. E-mail: mitrani@vms.huji.ac.il Received: 7 October 2004 Revised: 24 November 2004 Accepted: 1 December 2004 Abstract Background Organ fragments can be cultured for weeks in vitro if they are prepared of microscopic thickness and if the basic organ structure is preserved. Such organ fragments, which we termed micro-organs (MOs), express in culture endogenous tissue-specific gene products. We have exploited this methodology to engineer MOs ex vivo by gene transfer. Methods MOs prepared from spleen, lung, colon and skin were infected using: herpes simplex type-1, adeno virus, vaccinia virus and murine leukemia virus (MuLV), carrying the reporter gene β -galactosidase. Results All four viral vectors infected MOs in culture, with adeno infection giving significantly higher values. After optimization, high levels of expression (>15% positive cells), comparable to those obtained with the adeno construct, were also obtained using the MuLV construct both in vitro and after implantation into syngeneic hosts. After implantation, the engineered tissue was found to remain localized, become vascularized, and to express the transduced gene for several months. Conclusions The system can be used to study interactions between viruses and tissues both ex vivo and in vivo. Furthermore, the approach proposes a novel platform for ex vivo gene therapy. Such engineered structures could be used as autologous biological pumps for continuous secretion in vivo of gene products of clinical importance. Copyright  2005 John Wiley & Sons, Ltd. Keywords ex vivo gene therapy; epithelial–mesenchymal interactions; adeno virus; murine leukemia virus; herpes simplex type-1; vaccinia virus Introduction One main goal of gene therapy is to target cells within complex solid tis- sues [1]. However, little is known as to the interactions between virus and intact organs in vivo [2–4]. Lack of availability of in vivo-like three- dimensional systems suitable for long-term culture and effective transduction in vitro has hampered further progress in this important field. Because of these limitations, most current clinical trial protocols have used an approach where gene transfer to cells is achieved ex vivo. This way efficiencies may be better controlled and gene expression levels can be assessed prior to cell transplantation. For obvious reasons, a large number of studies have used CD34+ bone-marrow (BM)-derived stem cells [5–8] or BM-derived mesenchymal cells [9,10]. In others, primary cells or permanent cell lines, derived from mesoderm and epithelial cell lineages, have been used for gene transfer ex vivo [11–14]. Genetically modified cells of fibroblast or epithe- lial origin are usually transplanted either as a suspension of single cells, or Copyright  2005 John Wiley & Sons, Ltd.