Developing herpes simplex virus type 1 amplicon vectors for gene therapy: Hepatic gene transfer and steroid-inducible gene expression.

摘要

Herpes simplex virus type 1 (HSV-1) amplicon vector is packaged from a conventional prokaryotic plasmid that contains HSV cis-elements for DNA replication and packaging. We tested the hypotheses that HSV-1 amplicon vector can cause efficient and persistent hepatic gene transfer, and that steroid hormone receptors can regulate the expression of a transgene in the context of HSV-1 amplicon vectors.;A prototypical HSV-1 amplicon vector efficiently transduced reporter gene E. coli lacZ into mouse primary hepatocytes in culture. A mouse intrasplenic hepatocyte transplantation model was used to test ex vivo hepatic gene transfer by this vector. Vector-transduced hepatocytes were injected into the spleen of recipient mice, and lacZ gene expression under the control of HSV IE 4/5 promoter lasted in the mouse liver for up to two weeks. The survival of transplanted hepatocytes and persistence of amplicon vector genome in recipient organs were demonstrated for eleven weeks. These results indicated that HSV-1 amplicon vectors could lead to efficient and persistent hepatic gene transfer, although HSV IE 4/5 promoter used here did not support persistent gene expression in hepatocytes in vivo.;To test steroid regulated gene expression, promoters containing responsive elements to glucocorticoid receptor (GR) or Drosophila ecdysone receptor (EcR) were constructed in HSV-1 amplicon vectors to regulate human growth hormone (hGH) expression. In mammalian cell cultures, GR induced 40- to 50-fold increase in hGH expression in modified HSV-1 amplicon vectors. We demonstrated that a shortened, 0.24 Kb HSV origin of DNA replication (ORIs) was sufficient for viral packaging and eliminated the basal expression associated with conventional 1.0 Kb HSV ORIs. Introduction of EcR into mammalian cells by transfection was sufficient for ecdysteroid inducible gene expression, although transferring EcR system by HSV-1 amplicon vectors produced low inducibility of gene expression. In dual transgene vectors, induction of GR-controlled hGH did not affect expression of IE 4/5 promoter-driven lacZ gene from a single vector, suggesting the possibility of simultaneously delivering EcR gene and EcR-inducible gene by HSV-1 amplicon vectors into mammalian cells for regulated gene expression.