Feline herpesvirus 1 is a DNA virus that causes the most clinically important respiratory tract disease of cats. The clinical manifestations of chronic disease mainly affect the eyes and can lead to blindness due to repeated recrudescence.1 Vaccines only induce partial protection from clinical disease.2 Antiviral medications approved for treatment of a related virus, herpes simplex virus type 1 in humans, are currently the best treatment available for these chronic cases in cats, but these are not uniformly effective.3,4 Therefore, our goal is to develop a successful product that specifically targets several FHV-1 genes, is nontoxic, and reduces FHV-1 replication to mitigate clinical disease.
Recently, the authors have proven that RNA interference using siRNAs can be used to target essential genes of FHV-1, including the viral DNA polymerase5 and glycoprotein D,6 a surface protein that appears to function in viral attachment, cell penetration, or both. Ribonucleic acid interference of these genes results in excellent suppression of virus replication, with reductions up to 98 ± 1% (mean ± SD) in an immortalized cell line (Crandell-Rees feline kidney cells).5
The challenge with use of siRNAs as a treatment is delivery of the molecules to the intended cells. Ideally, an siRNA preparation would be delivered topically to the cornea. This circumvents the necessity to develop targeted systemic delivery of the siRNAs to virus-infected cells, and topical delivery also reduces the potential for off-target effects that may occur with siRNA use. Clinical disease associated with FHV-1 is usually localized, so systemic delivery in most cases is unnecessary. Also, topical delivery to the affected area allows for higher concentrations of siRNAs in the infected cells, which in the case of corneal epithelial cells, are normally devoid of a blood supply. However, the major challenges to topical delivery are the hydrodynamics of blinking and tear flow, which will quickly remove the siRNAs from the corneal surface. A previous study7 evaluating delivery of nucleic acids topically to the cornea in mice resulted in successful uptake of naked nucleic acid only after the corneal surface had been scarified. Although this was plasmid DNA (≥ 2,000 kDa) and not siRNAs (approx 13.5 kDa), the problem with delivery of nucleic acid (RNA or DNA) to cells is its polyanionic charge, which is repelled by the negatively charged cell membrane.7 Another study8 performed in rats resulted in unsuccessful delivery of naked nucleic acids to intact corneas. Therefore, identification of a delivery molecule to enhance cell penetration of these siRNAs is necessary to the potential success of this product as a therapeutic agent.
Successful delivery of nucleic acids to intact corneas has been reported in rats with use of a low dose of cationic lipoplexes.8 Cell-penetrating peptides have also been evaluated for their ability to deliver nucleic acids to cells.9 These peptides are much less toxic than transfection agents,9 and 1 particular peptide, POD, is taken up into corneal cells of mice within 5 minutes.10
Primary feline corneal cells have been used for in vitro testing of antiviral medications.11,12 Primary cell lines are more difficult to transfect with siRNAs than are immortalized cell lines, so in vitro testing with corneal cells is necessary prior to in vivo studies.
The purpose of the study reported here was to determine whether siRNAs could be delivered into feline corneal cells, initially in cell culture, and to evaluate siRNA toxicity and functionality in the corneal cells, in an vitro method of FHV-1 infection that closely mimics the in vivo environment. Our hypothesis was that the delivery agents would facilitate siRNA uptake.
Feline herpesvirus 1
Peptide for ocular delivery
Quantitative reverse transcriptase PCR
Small interfering RNA
2000, Invitrogen, Carlsbad, Calif.
Lipofectamine RNAiMAX, Invitrogen, Carlsbad, Calif.
Lipofectamine LTX, Invitrogen, Carlsbad, Calif.
TransIT-TKO (TKO), Mirus BIO, Madison, Wis.
TransIT-siQUEST, Mirus BIO, Madison, Wis.
siPORT Amine (PORT), Ambion-Applied Biosystems, Austin, Tex.
Kindly provided by Dr. Dorothee Bienzle, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada.
Biopeptide Co LLC, San Diego, Calif.
Life Technologies, Carlsbad, Calif.
Epics XL, Beckman Coulter, Fullerton, Calif.
Stealth RNAi siRNA negative control Lo GC Duplex #1 labeled with Alexafluor 488, Invitrogen, Carlsbad, Calif.
OPTI-MEM I, Invitrogen, Carlsbad, Calif.
DMEM F-12, Lonza, Allendale, Ga.
Atlanta Biologicals, Atlanta, Ga.
CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega, Madison, Wis.
ELx 800, BIO-TEK Instruments Inc, Winooski, Vt.
FHV-1 strain No. VR-636, ATCC, Manassas, Va.
VMRD, Pullman, Wash.
Liberty Research Inc, Waverly, NY.
ProLong Gold antifade reagent with DAPI, Invitrogen, Carlsbad, Calif.
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