Modified adenovirus penton base protein (UTARVE) as a non-replicating vector for delivery of antisense oligonucleotides with antiviral and/or antineoplastic activity.
Antisense oligonucleotides that selectively inhibit gene expression are a genetic approach for disease treatment and prevention. However, their use as therapeutic agents is complicated by their low rate of transport across cellular membranes and their sequestration within endocytic-like vesicles. We report that the adenovirus type-2 penton base protein modified to include the fusogenic peptide of the influenza virus hemagglutinin protein is a non-replicating vector (designated UTARVE) that improves delivery of antisense oligonucleotides. Approximately 10-18% of the input vector was internalized by A549 and HeLa cells as determined by immunoblotting. It was cleared by proteolysis within 48 h. The vector had endosome disruptive potential as evidenced by erythrocyte lysis activity at low pH and a primarily diffuse cytoplasmic distribution in treated cells. Despite concentration and time-dependent cell detachment, UTARVE was not cytotoxic in the dye release assay. We used R1T1, an antisense oligonucleotide that inhibits expression of the multifunctional herpes simplex virus type-2 (HSV-2) R1 protein, HSV-2 growth and the proliferation of R1 PK transformed cells to examine vector-mediated delivery. Conjugated FITC-labeled R1T1 was rapidly (15-30 min) internalized by all cells treated at low (80 nM) concentration and the oligomer was intracellularly dissociated from the vector. This compares to 65-83% of cells internalizing the unconjugated R1T1 when treated for 24 h. In antiviral assays, the IC50 and time required to inhibit HSV-2 growth were significantly lower for the conjugated (2 nM; 30 min) as compared to unconjugated (100 nM; 24 h) R1T1. The data indicate that the bioavailability and biological activity of R1T1 were significantly increased by its delivery with UTARVE.
Smith
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Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Kulka
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Aurelian
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D000970:Antineoplastic Agents / Q000494:pharmacology*; D000998:Antiviral Agents / Q000494:pharmacology*; D001692:Biological Transport; D002213:Capsid / Q000235:genetics* / Q000494:pharmacology; D036022:Capsid Proteins; D002448:Cell Adhesion / Q000187:drug effects; D002451:Cell Compartmentation; D016373:DNA, Antisense / Q000235:genetics* / Q000494:pharmacology; D004274:DNA, Recombinant / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems / Q000379:methods; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics* / Q000494:pharmacology; D006367:HeLa Cells; D018258:Herpesvirus 2, Human / Q000187:drug effects / Q000254:growth & development; D006801:Humans; D018613:Microscopy, Confocal; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D012264:Ribonucleotide Reductases / Q000235:genetics; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism / Q000821:virology
D000970:Antineoplastic Agents; D000998:Antiviral Agents; D036022:Capsid Proteins; D016373:DNA, Antisense; D004274:DNA, Recombinant; C043322:penton protein, adenovirus; C100671:ICP10 protein, herpes simplex virus type 2; D012264:Ribonucleotide Reductases; D017346:Protein Serine-Threonine Kinases