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

The effectiveness of viral vector-mediated gene transfer depends on the exp ression of therapeutic transgenes in the correct target cell types. So far, however, little attention has been given to targeted subcellular distribut ion 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. V iruses normally hijack the cellular protein synthesis machinery for their o wn 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 vi rus type I-derived vector could be used to deliver a transgene engineered t o be targeted to the extracellular membrane of target cells. To do so we co nstructed 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 glycosylphosph atidylinositol (GPI)-anchor within the endoplasmic reticulum. Our results d emonstrate that HSV1-derived viral vectors can be used to target transgenes as GPI anchored proteins to the outside leaflet of plasma membranes, witho ut disrupting the targeting machinery of host epithelial cells or neurons. This approach could then be used to target specific proteins to the cell me mbrane to modify cell-cell interactions, the function of specific plasma me mbrane proteins, or their interactions with other membrane proteins, and al so to target a prodrug converting enzyme to the plasma membrane of target c ells, therefore enhancing its cell killing effects.