Antisense oligonucleotides (ODNs) that target viral genes specifically invOlved in disease pathogenesis are a promising approach to the development of rational antiviral chemotherapy. Using oligodeoxy- methylphosphonates (d-OMPs) we demonstrated that inhibition of Herpes simplex virus (HSV) IE genes reduces virus growth in vitro and in infected animals (mouse ear model). Newly developed OMPs with alternating 2'-O-methylribonucleoside methylphosphonate and phosphodiester bonds (alt-mr-OMPs) have a higher affinity for their target mRNAs and significantly improved antiviral activity. We recently developed a nucleic acid free vector (KALM) consisting of adenovirus and influenza peptides which are involved in cell penetration and endosomolysis, and showed that it increases OMP antiviral activity. Here we propose to examine whether KALM-mediated delivery alters OMP intracellular localization and increases its intracellular biodistribution and antiviral activity. OMP delivered by a KALM vector which lacks the fusogenic influenza protein component as well as free OMP and OMP complexed to BSA will serve as controls. FITC/[32P]-labeled OMPs will be used to examine uptake, intracellular localization and duration of intracellular bioavailability. Analysis and quantitation of fluorescent images will be done by confocal microscopy. To examine OMP uptake/trafficking, colocalization of the FITC labeled OMP with proteins of coated endocytic vesicles, caveolae and beta-COP will be examined by double immunofluorescent staining with rhodamine-labeled antibodies to vesicle proteins. Inhibition of targeted HSV gene expression will be determined by immunoblotting with respective antibodies and the results compared to antiviral activity. The in vivo uptake and biodistribution of OMPs delivered as KALM complexes and their antiviral activity will be determined in the mouse ear model. KALM-delivered OMPs will be studied for their ability to interfere with HSV gene expression and virus growth in cultured primary neuronal cells in which virus gene expression is differently regulated, at the time of first infection and at re- initiation of virus replication.
Perkins, D; Pereira, E F R; Gober, M et al. (2002) The herpes simplex virus type 2 R1 protein kinase (ICP10 PK) blocks apoptosis in hippocampal neurons, involving activation of the MEK/MAPK survival pathway. J Virol 76:1435-49 |
Aurelian, L; Smith, C C (2000) Herpes simplex virus type 2 growth and latency reactivation by cocultivation are inhibited with antisense oligonucleotides complementary to the translation initiation site of the large subunit of ribonucleotide reductase (RR1). Antisense Nucleic Acid Drug Dev 10:77-85 |
Smith, C C; Kulka, M; Aurelian, L (2000) Modified adenovirus penton base protein (UTARVE) as a non-replicating vector for delivery of antisense oligonucleotides with antiviral and/or antineoplastic activity. Int J Oncol 17:841-50 |