Evaluation of delivery agents used for introduction of small interfering RNAs into feline corneal cells.
OBJECTIVE
To evaluate agents used for delivery of small interfering RNAs (siRNAs) into feline corneal cells, toxicity of the delivery agents, and functionality of anti-feline herpesvirus 1 (FHV-1)-specific siRNA combinations.


METHODS
Feline primary corneal cells and 19 six-month-old colony-bred cats.


METHODS
siRNA delivery into corneal cells via various delivery agents was evaluated via flow cytometric detection of labeled siRNAs. Cellular toxicity was evaluated with a proliferation assay. Functionality was tested via quantitative reverse transcriptase PCR assay, plaque assay, and flow cytometry. In vivo safety was evaluated with an ocular scoring method following topical application of delivery agents containing siRNAs into eyes. Corneal biopsy specimens were used to assess safety and uptake of siRNAs into corneal cells.


RESULTS
Use of 3 delivery agents resulted in > 95% transfection of primary corneal cells. Use of a peptide for ocular delivery yielded approximately 82% transfection of cells in vitro. In cultured corneal cells, use of the siRNA combinations resulted in approximately 76% to 89% reduction in FHV-1-specific mRNA, 63% to 67% reduction of FHV-1-specific proteins in treated cells, and 97% to 98% reduction in FHV-1 replication. The agents were nonirritating in eyes, caused no substantial clinical ocular signs, and were nontoxic. Histologically, corneal epithelium and stroma were normal in treated cats. However, none of the agents were effective in delivering siRNAs into the corneal cells in vivo.


CONCLUSIONS
The tested anti-FHV-1-specific siRNAs could potentially be used as a treatment for FHV-1 if a successful means of in vivo delivery can be achieved.
Department of Biomedical and Diagnostic Research, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA. beckpen@utk.edu
Rebecca P
RP
Wilkes
Dan A
DA
Ward
Kim M
KM
Newkirk
Joleen K
JK
Adams
Stephen A
SA
Kania
D000818:Animals; D000998:Antiviral Agents / Q000008:administration & dosage; D002371:Cat Diseases / Q000235:genetics / Q000628:therapy; D002415:Cats; D002478:Cells, Cultured; D003315:Cornea / Q000187:drug effects*; D004337:Drug Carriers / Q000008:administration & dosage / Q000009:adverse effects*; D005128:Eye Diseases / Q000235:genetics / Q000628:therapy / Q000662:veterinary; D005260:Female; D006566:Herpesviridae Infections / Q000235:genetics / Q000628:therapy / Q000662:veterinary*; D008297:Male; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000009:adverse effects; D014162:Transfection / Q000662:veterinary; D018140:Varicellovirus / Q000187:drug effects* / Q000235:genetics; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D004337:Drug Carriers; D034741:RNA, Small Interfering; D014764:Viral Proteins