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.
Materials and Methods
Delivery agents—Six commercially available transfections agentsa–f were evaluated for delivery of siRNAs into primary feline corneal cells.11,g Additionally, a POD10,h was evaluated.
In vitro siRNA delivery—Initial transfection experiments were performed according to manufacturers' instructions with a 100nM total concentration (2.5 μL of a 20μM solution) of a fluorescently labeled, nontargeting siRNAi in 1 mL/well in 12-well plates containing confluent primary feline corneal cells and incubated overnight at 37°C and 5% CO2.
Transfection efficiency was determined via flow cytometryj as described.6 The commercial agents with the best transfection efficiencies were additionally tested for their ability to rapidly transfect the corneal cells in vitro; toxicity was also evaluated in vitro.
Rapid transfections were performed in 12-well plates containing confluent primary feline corneal cells. One microgram (3 μL of a 20μM solution) of a fluorescently labeled nontargeting siRNAk was combined with 2 μL of transfection agentd,f in cell culture medial using an 80-μL total volume/sample, according to manufacturer's instructions, or approximately 375 POD particles/siRNA duplex in an 80-μL total volume as described.10 Cells were incubated for 5 minutes or 10 minutes at 37°C and 5% CO2. Samples were tested in duplicate via flow cytometry as described,6 and each experiment was performed twice.
In vitro toxicity testing—Transfections were performed with a total volume of 80 μL/well of the preparations described (rapid transfections), with a nonlabeled siRNA negative control,k in 12-well plates containing confluent primary feline corneal epithelial cells. Cells were incubated for 10 minutes at 37°C and 5% CO2. Transfection solutions were then removed, and the cells were washed with cell culture medium.m Cell culture mediumm with 10% fetal bovine serumn was added to the wells (1 mL/well) and the plates were incubated at 37°C in 5% CO2 for 48 hours. Nontransfected cells were used as a control. Cells were then tested for toxic effects by use of a cell proliferation assayo as described.13 A product induced by metabolically active cells was detected by absorbance at 490 nmp and was directly proportional to the number of live cells in the sample. Experiments were performed in triplicate, and each delivery agent was tested at least twice.
siRNA efficiency in primary feline corneal epithelial cells—To determine siRNA efficiency, 100nM total concentration (2.5 μL of a 20μM solution) of combinations of anti–FHV-1–specific siRNAs targeting the FHV-1 DNA polymerase (DNA1 and DNA3) or glycoprotein D (gD1)5/well were transfected with a commercially available reagentf according to manufacturer's instructions in 12-well plates with confluent primary feline corneal cells, 24 hours prior to FHV-1q infection with 3 × 103 plaque forming units/well. Untreated FHV-1 infected cells were used as controls. Sample evaluation was performed 24 hours following infection. Interference efficacy was assessed by measuring amounts of glycoprotein D and DNA polymerase mRNA via real-time qRT-PCR assay in treated and untreated control samples as described.5 The 28S rRNA was used as an RNA standardization control. Reduction in viral protein expression on the surface of infected cells was evaluated via flow cytometry using fluorescein isothiocyanate–labeled polyclonal anti–FHV-1 antibodiesr as described.5 A total of 104 cells/sample was analyzed. Plaque assays were performed to titer infectious virus in the cell culture supernatant of each sample as described.5 Experiments were performed in duplicate and repeated twice.
Two commercially available transfection agentsd,f and POD were also tested with one of the combinations of anti–FHV-1–specific siRNAs (DNA1/gD1) by use of the reduced transfection period of 10 minutes, as described. Cells were infected with 3 × 103 plaque forming units of FHV/well, 8 hours following transfection. Treatments were evaluated approximately 24 hours following viral infection via plaque assay and flow cytometry as described.5
In vivo testing—Twenty 6-month-old colony-bred cats,s including 10 sexually intact males and 10 sexually intact females, were obtained for the study. The study protocol was approved by the Animal Care and Use Committee at the University of Tennessee and was conducted in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care requirements. Cats were randomly assigned to 5 equal groups (2 male and 2 female cats/group). There were 3 treatment groups and 2 control groups. Prior to the start of the study, 1 male cat was removed from the study because of aggressive temperament, leaving 3 cats in 1 control group. Ophthalmologic examinations were performed for each cat by a board-certified veterinary ophthalmologist (DAW) prior to initiation of the study. Three delivery agentsd,f,h were evaluated by use of a single concentration (1 μg) of the fluorescence-labeled negative control siRNAk for each agent, prepared as described. One control group was administered diluted siRNAs only, with no delivery agent. The other control group was administered the diluent (cell culture medial) only, with no siRNAs or delivery agent. The treatment or control preparations were delivered topically to both eyes. The cats were evaluated by an observer (DAW) who was unaware of group assignments at 5 minutes, 1 hour, and 24 hours after agent administration for signs of conjunctival and corneal irritation according to a described scoring system.14
Twenty-four hours following drug administration, cats were administered general anesthesia, and a 6-mm central core corneal biopsy specimen extending to 20% of the stromal depth was collected from the left eye of each cat. The siRNAs were expected to be taken up by the corneal epithelial cells, but the depth of biopsy (which included stroma) would allow determination of the extent of siRNA tissue penetration. Cats recovered from anesthesia and were treated with topical administration of neomycin-polymixin B-gramicidin solution 3 times daily, topical administration of atropine ointment once daily, and oral administration of meloxicam (0.1 mg/kg) once daily as needed for signs of pain, until corneal ulcers healed (approx 96 hours), at which time the cats were adopted to individual owners.
The corneal tissues were formalin fixed, paraffin embedded, sectioned at 5 μm, and mounted with a fluorescence-protecting reagent.t The sections were examined via fluorescence microscopy to evaluate the percentage of cells that took up the siRNA molecules. Sections stained with H&E were also prepared and evaluated via light microscopy by a board-certified veterinary pathologist (KMN) for evidence of any histologic changes as a result of treatment. The pathologist was unaware of treatment group assignments.
Statistical analysis—A randomized design ANOVA was used to compare siRNA combinations among in vitro treatment and control groups to evaluate logarithmic reduction in virus titer results. An ANOVA was also used to compare means from the in vitro transfection experiment with the means from the in vitro toxicity experiment. A least significant difference method was used for paired post hoc tests to compare results among treatment groups in each in vitro experiment. A Fisher exact test was used to evaluate the relationship between treatment and clinical score (conjunctival or corneal irritation). For all tests, a value of P < 0.05 was considered significant.
Results
Use of 3 commercially available transfection agentsd–f resulted in > 95% transfection efficiencies in primary feline corneal cells, following an overnight incubation period, when used at amounts suggested by the manufacturers. The others did not perform as well, with transfection efficiencies of approximately 20%,b 75%,c and 85%.a One of the highly effective agentse was considered too toxic for additional studies on the basis of the appearance of numerous granulated and dead cells in culture. Two of the agentsd,f were further evaluated for their ability to facilitate rapid transfection of the feline corneal cells in vitro. Transfection of the cells with these agents was evaluated at 10 minutes initially, followed by a more stringent exposure time of 5 minutes to account for expected rapid removal of the agents in vivo. Use of 1 agentf resulted in 89 ± 1% transfection (mean ± SD) and use of the otherd resulted in 91 ± 2% transfection of corneal cells within 10 minutes and 76 ± 7% and 74 ± 4% transfection of corneal cells, respectively, within 5 minutes after delivery. The POD was also tested. Transfection of cells following overnight incubation was 82 ± 4%, but because of its previous success in mice in in vivo studies,10 POD was also evaluated at 10 and 5 minutes. Treatment with POD resulted in 27 ± 2% and 13 ± 1% transfection of cells within 10 and 5 minutes, respectively. Means were not significantly different between the 2 transfection agents at either time point, but each agent was significantly better than POD for corneal cell transfection.
In vitro, the 2 transfection agentsd,f and POD, combined with siRNAs, were nontoxic to feline corneal epithelial cells. Mean toxicity did not differ by treatment and there was no significant (P < 0.05) difference between the siRNA treatment groups and the controls.
Two combinations of siRNAs determined to be superior for reduction of virus replication in Crandell-Rees feline kidney cells (DNA1/gD1 and DNA1/DNA3)5 reduced DNA polymerase mRNA expression by 76 ± 5% (mean ± SD) and 82 ± 5%, respectively, and glycoprotein D mRNA expression by 89 ± 11%, respectively, and 87 ± 11% in feline corneal cells, determined via real time relative qRT-PCR assay. Treatment with DNA1/gD1 and DNA1/DNA3 resulted in reductions of 67 ± 4% and 63 ± 2%, respectively, in FHV-1 proteins detected on the surface of infected corneal cells, compared with untreated controls (Figure 1). Reduction in virus replication was 97 ± 1% (mean ± SD) by treatment with DNA1/DNA3 and 98 ± 1% by treatment with DNA1/gD1 in cell culture supernatants of the treated cells versus untreated cells.
Results of flow cytometric evaluation of the effects of treatment with siRNA combinations DNA1/gD1 and DNA1/DNA3 on the expression of FHV-1 proteins on the surface of infected primary corneal cells of cats. A—Fluorescence histogram for nontreated, FHV-1–infected cells. B—Fluorescence histogram for FHV-1–infected primary corneal cells treated with siRNA combination DNA1/gD1. C—Fluorescence histogram for FHV-1–infected primary corneal cells treated with siRNA combination DNA1/DNA3. D— Fluorescence histogram for uninfected cells. FL1 log = Log fluorescence intensity.
Citation: American Journal of Veterinary Research 74, 2; 10.2460/ajvr.74.2.243
Results of flow cytometric evaluation of the effects of treatment with siRNA combinations DNA1/gD1 and DNA1/DNA3 on the expression of FHV-1 proteins on the surface of infected primary corneal cells of cats. A—Fluorescence histogram for nontreated, FHV-1–infected cells. B—Fluorescence histogram for FHV-1–infected primary corneal cells treated with siRNA combination DNA1/gD1. C—Fluorescence histogram for FHV-1–infected primary corneal cells treated with siRNA combination DNA1/DNA3. D— Fluorescence histogram for uninfected cells. FL1 log = Log fluorescence intensity.
Citation: American Journal of Veterinary Research 74, 2; 10.2460/ajvr.74.2.243
Results of flow cytometric evaluation of the effects of treatment with siRNA combinations DNA1/gD1 and DNA1/DNA3 on the expression of FHV-1 proteins on the surface of infected primary corneal cells of cats. A—Fluorescence histogram for nontreated, FHV-1–infected cells. B—Fluorescence histogram for FHV-1–infected primary corneal cells treated with siRNA combination DNA1/gD1. C—Fluorescence histogram for FHV-1–infected primary corneal cells treated with siRNA combination DNA1/DNA3. D— Fluorescence histogram for uninfected cells. FL1 log = Log fluorescence intensity.
Citation: American Journal of Veterinary Research 74, 2; 10.2460/ajvr.74.2.243
One of these siRNA combinations was also tested with a reduced transfection period using the 2 superior transfection agentsd,f and POD. Microscopically, the treated cells had no signs of toxicity and appeared similar to nontreated, control cells. On the basis of plaque assay results from the supernatant of treated wells versus nontreated wells, there were 86 ± 4% (mean ± SD) and 89 ± 1% reductions in viral replication by treatment with the siRNAs delivered by the transfection agentsd,f and 50 ± 15% reduction by treatment with siRNAs delivered by POD. Evaluation of viral replication in the cells was performed via flow cytometry, and there were 59 ± 6% and 64 ± 4% reductions in FHV-positive cells by treatment with siRNAs delivered by transfection agents, compared with a 37 ± 7% reduction by treatment with siRNAs delivered by POD. There was a significant difference between the means of each treatment group, compared with the controls, proving functionality despite a reduced concentration of siRNAs per cell, compared with overnight transfection protocols. There was no significant difference among the means of the groups treated with the transfection agents, but delivery with each of these agents yielded results significantly better than treatment with siRNAs delivered by POD.
In vivo study—No abnormal findings were seen in ophthamologic examinations. When tested in vivo, the delivery agents combined with the siRNAs were nonirritating, inducing no clinical score consistently greater than baseline in any time period evaluated. No significant association between a treatment and the presence of a condition (ie, blepharospasm, ocular discharge, conjunctival hyperemia, chemosis, or corneal ulceration) was noted (P = 0.486). Histologically, corneal epithelium and stroma were normal in treated cats. No fluorescence was detected in any corneal tissue sample via fluorescence microscopy; therefore, there was no evidence of uptake of the siRNAs into the corneal cells.
Discussion
In this study, RNA interference using combinations of siRNAs to target FHV-1 DNA polymerase and glycoprotein D mRNAs was successful in reducing replication of FHV-1 in primary feline corneal epithelial cells. These 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. The DNA1/gD1 combination is nontoxic in an immortalized cell line5 and according to microscopic evaluation in the present study, was also safe in the primary feline corneal cells when delivered with 3 agents.
Despite rapid transfection of siRNAs into corneal cells in vitro, none of the agents tested were effective in delivering siRNAs into the corneal cells in vivo, or at least to a level that was detectable in the tissues by fluorescence microscopy at 24 hours after treatment. Fluorescent siRNAs in cultured corneal cells were observed with each of the delivery agents within 5 minutes after application by microscopy, suggesting that these siRNAs could be detected by this method. Fluorescence was also detectable in cell cultures 24 hours after transfection; however, it is possible that if delivery efficiency was poor, the ability to observe the fluorescently labeled siRNA in the tissues at 24 hours after administration could have been diminished. The time lapse prior to biopsy was established to allow many cats to have the biopsy procedure on the same day and to provide a highly stringent standard to predict therapeutic success. Also, the delay between treatment and biopsy was used to avoid the ocular changes that accompany anesthesia that could have affected the results, although a 24-hour period may have been excessive for this purpose. Sampling at an earlier time point may have provided different results.
The tissues were processed rapidly, and the slides were prepared in dim light and stored in the dark for 2 days prior to microscopic evaluation for fluorescence. A fluorescence anti-fade reagent was used for tissue mounting to preserve any fluorescence present in the tissues. However, if the fluorescence had been altered by the formalin fixation, use of the anti-fade reagent would not have helped with detection. In future studies, chemical detection or a different processing technique will be used to exclude potential detection problems due to formalin fixation.
Tear film turnover in humans is approximately 17.78%/min15 and in horses is 13.21%/min.16 The turnover rate in cats is probably similar, which means at the higher rate, approximately 90% of product instilled into the eye will be removed from the surface of the cornea within 5 minutes after application. Therefore, assuming no problems with detection, it is likely the siRNA solutions were diluted and removed too rapidly to be effective. Although multiple applications of the siRNA solutions may have enhanced delivery, avoidance of multiple applications is preferable. Inadequate compliance with administering multiple daily treatments is likely the cause of treatment failures with current antiviral medications.3 Oral administration of the antiviral medication famciclovir to cats induces adequate concentrations of the drug at the ocular surface17 and is also beneficial for treatment of experimentally induced primary feline herpesvirus infection.18 Although oral or systemic administration of an antiviral treatment bypasses the problems associated with ocular administration, use of siRNAs for inhibition of herpesvirus replication would best be done by topical administration. Cats with ocular disease due to FHV-1 infection often have corneal ulcers, and damage to the epithelial cells may allow for better uptake of the siRNAs; however, future studies are needed to identify a means of increasing contact time between the corneal cells and siRNAs to allow adequate time for optimal siRNA delivery, while also preventing the need for multiple applications. One possibility is use of an ointment, as has been done with an antiviral drug,19 to prolong duration of contact with the cornea.
ABBREVIATIONS
| FHV-1 | Feline herpesvirus 1 |
| POD | Peptide for ocular delivery |
| qRT-PCR | Quantitative reverse transcriptase PCR |
| siRNA | 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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