Gene Therapy (2000) 7, 1947–1953  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt VIRAL TRANSFER TECHNOLOGY RESEARCH ARTICLE Subcellular post-transcriptional targeting: delivery of an intracellular protein to the extracellular leaflet of the plasma membrane using a glycosyl-phosphatidyl inositol (GPI) membrane anchor in neurons and polarised epithelial cells O Brown 1 *, RL Cowen 1 *, CM Preston 2 , MG Castro 1 and PR Lowenstein 1 1 Molecular Medicine and Gene Therapy Unit, School of Medicine, University of Manchester, Manchester, UK; and 2 MRC Virology Unit, Institute of Virology, Glasgow, UK The effectiveness of viral vector-mediated gene transfer depends on the expression of therapeutic transgenes in the correct target cell types. So far, however, little attention has been given to targeted subcellular distribution of expressed transgenes. Targeting individual transgenes to particular subcellular compartments will provide various advantages in increasing the safety, efficacy, and specificity of viral vector- mediated gene delivery. Viruses normally hijack the cellular protein synthesis machinery for their own advantages. It is thus unknown whether cells infected with viral vectors will be able to target proteins to the correct subcellular organelles, or whether the subcellular targeting machinery would be selectively disrupted by viral infection. In this article we explored whether a herpes simplex virus type 1-derived vector could be used to deliver a transgene engineered to be targeted to the extracellular membrane of target cells. To Keywords: viral vectors; targeting; herpes simplex vectors; neuronal polarity Introduction The capacity to display a therapeutic protein specifically on the outside of target cells could have many advan- tages in the design of gene therapy approaches. In parti- cular, it could be designed to modify cell–cell interac- tions, the function of plasma membrane proteins or their interactions with other membrane proteins. It could also be used to target a prodrug converting enzyme to the plasma membrane of target cells, therefore enhancing the bystander effect. Marais et al 1 highlighted the benefits of expressing the bacterial enzyme carboxypeptidase G2 (CPG2) as a trans- membrane protein for use in gene-directed enzyme pro- drug therapy (GDEPT). This retargeting was achieved by the fusion of the transmembrane region of the human Correspondence: PR Lowenstein, Molecular Medicine and Gene Therapy Unit, Room 1.302 Stopford Building, School of Medicine, Oxford Road, Manchester, M13 9PT, UK *These authors contributed equally to this work Received 18 May 2000; accepted 17 August 2000 do so we constructed a temperature-sensitive mutant HSV-1 vector, tsK-TT21 expressing a recombinant marker protein, tissue inhibitor of metalloproteinases (TIMP), linked to sequence encoding a signal for the addition of a glycosyl- phosphatidylinositol (GPI)-anchor within the endoplasmic reticulum. Our results demonstrate that HSV1-derived viral vectors can be used to target transgenes as GPI anchored proteins to the outside leaflet of plasma membranes, without disrupting the targeting machinery of host epithelial cells or neurons. This approach could then be used to target specific proteins to the cell membrane to modify cell–cell interac- tions, the function of specific plasma membrane proteins, or their interactions with other membrane proteins, and also to target a prodrug converting enzyme to the plasma mem- brane of target cells, therefore enhancing its cell killing effects. Gene Therapy (2000) 7, 1947–1953. tyrosine kinase receptor c-erb B2 to the C-terminus of CPG2. Stable expression of this chimaeric protein on the outer membrane of the breast carcinoma cell line MDA MB 361 rendered these tumour cells sensitive to the pro- drug 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl- l-glutamic acid (CMDA) which cannot cross the plasma membrane. Cells expressing membrane-bound CPG2 were able to allow the extracellular conversion of CMDA into its non-polar cytotoxic metabolite (which can thus cross the plasma membrane), and thus elicit an important bystander effect following prodrug administration. 1 This strategy would pave the way for new enzyme prodrug combinations where potential prodrugs are unable to cross the plasma membrane of cells and could improve existing GDEPT systems that require cell to cell com- munication to give a bystander effect such as thymidine kinase/ganciclovir. 2–4 Cell surface display of proteins is also being investi- gated as a way of improving existing immunotherapy strategies for cancer gene therapy. The surface expression of an exogenous protein per se may be used to stimulate in specific ways the cell–cell interactions needed to stimu-