WO2013162350A2

WO2013162350A2 - Circular antiviral rna

Info

Publication number: WO2013162350A2
Application number: PCT/MY2013/000088
Authority: WO
Inventors: Abdul Rahman Omar; Bimo A. TEJO; Sheau Wei TAN; Oi Kuan CHOONG
Original assignee: Universiti Putra Malaysia
Priority date: 2012-04-25
Filing date: 2013-04-23
Publication date: 2013-10-31
Also published as: WO2013162350A3

Abstract

The present invention relates to triple helix forming oligonucleotides (TFO) for use in the treatment of diseases mediated by single stranded RNA viruses, for example corona viruses. The invention provides a selection of circular TFO sequences that target different regions of the genome of the virulent Feline coronavirus and that efficiently block the virus' replication. Thereby the TFO of the present invention provide new means for therapy against viral diseases. The invention relates to the novel TFOs, pharmaceutical compositions comprising the same, the new TFO design and their uses in the treatment of viral diseases.

Description

Circular Antiviral RNA

Field of the Invention The present invention relates to triple helix forming oligonucleotides (TFO) for use in the treatment of diseases mediated by single stranded RNA viruses, for example corona viruses. The invention provides a selection of circular TFO sequences that target different regions of the genome of the virulent Feline coronavirus and that efficiently block the virus' replication. Thereby the TFO of the present invention provide new means for therapy against viral diseases. The invention relates to the novel TFOs, pharmaceutical compositions comprising the same, the new TFO design and their uses in the treatment of viral diseases.

Background of the Invention Feline Infectious Peritonitis Virus (FIPV) was firstly discovered and observed by Zook et al (1968) through the tissues of experimentally infected feline. Later, the Feline Infectious Peritonitis Virus (FIPV) was identified and grouped under Feline Coronavirus (FCoV) which classified as family Coronaviridae. FCoV is assorted into two biotypes according to the pathogenicity differences (Takano et al., 2008). The biotypes are Feline Enteric Coronavirus (FECV), the ubiquitous enteric biotype of FCoV, and Feline Infectious Peritonitis Virus (FIPV), the virulent biotype of FCoV. According to Vennema et al (1998), FIPV is proved as mutants of FECV which arise within an individual cat with the presence of deletion mutations in open reading frames (ORFs) 3c and 7b of the viral genome. This showed both viruses have a highly similar genome organization.

FIPV strain 79-1 146 is an enveloped virus containing a positive sense single stranded RNA genome positioned in helical symmetry, and is mainly composed of nucleocapsid, transmembrane and viral spike peplomers (Olsen, 1993). The spike peplomers function as receptors for the virus attachment to the cellular surface proteins (Hartmann et al., 2005). The genomic sequence of FIPV 79-1 146 comprises 29355 nucleotide (nt) with the GenBank accession number AY994055. This genome sequence incorporates several functional genes encoded with structural and non-structural proteins of the virus which included 5' and 3' untranslated region (UTR), open reading frames (ORFs) la/lb, gene S, gene nsp, gene E, gene M and gene N. The 3' untranslated region encodes the structural protein of the virus (Hegyi et al., 2002) while the 5' untranslated region is believed to interact with host and perhaps viral proteins to control RNA replication which includes the synthesis of positive and negative strand genomic length RNA (Weiss & Navas-Martin, 2005). The ORF la and lb are functioning as replicase gene which encode the polyproteins called ORF la-encoded protein and C-terminally extended frameshift protein, respectively. Both these ORF la and lb are specially connected by a ribosomal frameshift site where the sequences are overlapped by 46 nucleotides at the region nts 12355-12361 (Hegyi et al., 2002; Dye & Siddell, 2007). These replicative polyproteins are processed extensively by viral proteases to produce the functional subunits of the virus replication or transcription machinery. The gene S, gene E, gene M and gene N are encoded with the spike glycoprotein (S), envelope protein (E), the membrane protein (M) and the nucleocapsid protein (N) respectively (Dye & Siddell, 2007). The gene nsp 3 and nsp 7 are implicated in viral pathogenicity or virulence (Dye & Siddell, 2007). According to Pedersen et al (2009), the deleterious mutations of the 3c gene is the majority cause for a cat to acquire feline infectious peritonitis (FIP) disease.

Feline Infectious Peritonitis (FIP) is a severe fatal immune mediated disease which occurs more frequently in cats from catteries, boarding facilities and multicat households. It was first discovered by Holzworth as an 'important disorder of cats' in 1963. This disease is triggered by Feline Infectious Peritonitis Virus (FIPV) where it acquired the ability to invade and replicate in the blood monocytes following spreads systemically (Dewerchin et al., 2005). FIPV itself, will not encourage a pandemic disease in spite of feline enteric coronavirus (FECV), is a common and highly infectious feline virus. FECV infection is the first step in a chain of events leading to FIP as we know the FIPV arises from the mutation of FECV. Generally, FECV causes asymptomatic infection sometimes mild to often unapparent enteritis in the infected feline. Even though the infected feline is asymptomatic, they are producing and shedding a large amount of virus in faeces as a source of infection. For FIPV, it becomes pathogenic and causing symptomatic disease as the development of immune complex vasculitis accompanied by necrosis and pyogenic granulomatous inflammation, fibrinous serositis with accumulation of fluids in body cavities and hypergammaglobulinemia (Takano et al., 2008; Haagmans et al., 1996).

Before the etiology agent of FIP was discovered, several experiments had been done to remedying the felines infected with FIP in order to come out with a treatment. The first therapy was done by Disque et al (1968) by using prednisolone, penicillin and dihydrostreptomycin. Later, several different drugs such as cyclophosphamide, ozagrel hydrochloride, glucocorticoids were used to suppress the immune system (Hartmann & Ritz, 2008). After the identification of FIPV, several different drugs such as Ribavirin, Human interferon-alpha and Feline interferon- omega were used to test the antiviral effect. Over the past several decades, the felines infected with the disease were treated symptomatica! ly and with antiviral chemotherapy. Although there were a lot of antiviral drugs had been tested on the feline, the results were not applicable due to the toxicity of the drugs being used. Besides that, there are several studies used attenuated virus attempts to develop effective vaccines against FIP (Fehr et al., 1997; Hebben et al., 2003; Haijema et al., 2004) still the results are not encouraging mainly because of antibody-dependent enhancement (ADE). Treatment of cats with FIP remains frustrating and success is limited to a few cases that respond favourably within the first few days of illness. In addition, the available clinical intervention and anti-viral therapy are unable to revert this progressively fatal disease.

Hence, the invention of a new nucleic acid based molecular therapy may able to treat FIP rather than controlling the infection. Molecular therapy is a novel and rapidly evolving therapeutic regimens which applied to correct genetic and acquired diseases through the design and development of oligonucleotide, peptide and protein. Recent review, there are many different type of oligonucleotides are being designed, modified and reshaped according to the practical application such as silence RNA (siRNA), microRNA (miRNA), antisense oligonucleotide, peptide nucleic acid (PNA) chimeric DNA/RNA, DNA/RNA aptamers, triple helix oligonucleotide formation (TFO) and so on. These oligonucleotide therapies are mainly implementing in cancer research, infectious diseases and genetic diseases.

In view of the absence of working therapeutics against corona virus diseases, like FIP, it was an object of the present invention to provide novel agents having an antiviral activity that is sufficient to enable a treatment of viral diseases rather than merely controlling the infection of the virus.

The present invention provides a new antiviral therapeutic especially targeting the single stranded RNA viruses. This antiviral therapeutic is constructed by a circular RNA which is specially designed to inhibit single stranded RNA virus's replication during the viral propagation stage. Succinctly, this antiviral therapeutic is a specific array of oligonucleotide base therapeutic which apply as 'inhibitor' The above problem is solved in a first aspect by a triple helix forming oligonucleotide (TFO), comprising a circular nucleic acid molecule, which is in a preferred embodiment a RNA, preferably a circular single stranded RNA molecule. In one specific preferred embodiment the TFO is used in the treatment of a viral disease. Here, the Triple Helix-Forming Oligonucleotide (TFO) is chosen as a potential molecular therapy agent to treat Feline Infectious Peritonitis (FIP) infection by inhibiting the post- transcription of the FIPV.

By referring to the previous research in FIP disease, all the antiviral drugs and vaccines invented did not give a promising result. The FIP disease still remains unsolved. Here, TFO is chosen as a new remedy to treat the FIP disease. The TFO is mainly targeting the nucleic acid subsequently forming a triple helix by binding to the target region with Hoogsteen hydrogen bond. This action takes place when the viral genome is release into the cell. The triple helix is forming a barricade in the viral genome and indirectly blocking the replication of the viral genome. The viral genome, most probably, will remain dormant or degrade gradually inside the cell.

At the end, the virus cannot propagate inside the host and this is the way to treat the FIP disease through TFO. Since the TFO is a nucleic base molecular therapy, it would not trigger the ADE as the vaccine did in the immune system of feline. And, the toxicity of the TFO can actually be assured or eliminated during the designation of the TFO. TFO is a very sensitive and specific molecular therapy. This is because the designation of the TFO is totally depending on the target region's sequence. Other than that, the TFO is unable to response and gives no effect. In this research, few TFOs were designed and tested on the Crandell-Reef Feline Kidney (CRFK) cells to study the inhibitory of TFOs towards the FIPV.

TFO is a homopyrimidine oligonucleotides which can form a sequence specific triple helix by Hoogsteen bonds to the major groove of a complementary homopyrimidine · homopurine stretch in duplex DNA (Valentin et al., 1992). Apart from that, double helical RNA or DNA-RNA hybrids can also be targeted as a template for triple helix formation once the strand composition on the stabilities of triple helical complexes is determined (Han & Dervan, 1993). This TFO has the special abilities such as the binding characteristics and sequence-specificity of triplex forming molecules, a diverse class of DNA oligonucleotides and their analogs, and the ability to compete successfully with other DNA binders makes TFO become a great potential tools to alter gene expression ( nauert & Glazer, 2001). Thus, the TFOs have been proposed as a potential nucleic acid based antiviral therapy to treat FIP disease due to its unique aspects.

The triple helical nucleic acid structure formation was firstly discovered and observed in 1957 by Felsenfeld, it was not been universalized as pharmacologically active compounds since the function and characteristic of triple helical nucleic acid was unknown. Over the past few years, this TFO has attracted a tremendous attention due to its potential to become a new remedy in medical research. The advantage of the triple helix approach in improving binding affinity and selectivity towards the single stranded target, as opposed to standard Watson±Crick recognition, stems from three aspects. Firstly, the formation within the same complex of both Watson-Crick and Hoogsteen hydrogen bonds enhances the stability in terms of enthalpy. Secondly, Hoogsteen binding is far more sensitive to mismatches than Watson-Crick binding and thus the interaction gains in specificity. Thirdly, the entropic advantage brought about by the joining together in the proper orientation of two out of the three strands of the triple helix is considerable in terms of free energy.

The main attractive characteristic of TFOs is the sequence specificity. TFO only can bind to the duplex when it has the high specificity and affinity towards the duplex. With this characteristic, the TFOs can effectively used to manipulate the genetic in multiple ways. The TFOs have been proof to use to inhibit transcription or blocking the initiation or elongation of transcriptional step by competing successfully with other DNA binders and transcriptional factors to the promoter. According to Carine et al., 1997, TFOs has been used to control gene transcription within the chromatin structure of the cell which containing the HIV provirus as endogenous genes. Apart from that, Shen et al., 2001, conducted an experiment by using TFOs to inhibit expression of bcl- 2 proto-oncogene in vivo by blocking the functions of the 3' UTR which regulate the bcl-2 gene expression and the bcl-2 is significantly down regulated in HeLa cells. TFOs also have been reported to inhibit and/or interfere with gene expression in a sequence specific manner. Kautz et al., 2005, designed a sequence specific single stranded RNA as TFO to interfere with an overlapping SP-l/AP-1 target and subsequently inhibit the MCP-1 gene expression. The TFO according to the present invention comprises a nucleic acid sequence having a purine rich make up. The term "purine-rich" can be understood as referring to a nucleic acid which has an overall composition of at least 50%, 60%, 70%, 80%, 90%, 95%, 98% purine bases. Purine bases in RNA and DNA are guanine or adenine. In a preferred embodiment the TFO according to the invention comprises two purine rich domains which are circular linked by two clamp domains. The two purine rich domains independently comprise at least 50%, 60%, 70%, 80%, 90%, 95%, 98% purine bases. The clamp domain comprises in a preferred embodiment two cysteine bases. The circular TFO of the present invention is in a preferred embodiment produced by preparing a single stranded linear RNA, wherein the 5' and 3' ends were modified into a phosphate (P0₄) and hydroxide (OH) group, respectively. The circulation of the molecule is then performed by standard procedures in the art, preferably using a T4 RNA ligase protocol. Another preferred embodiment of the invention relates to the TFO as described herein, wherein the two purine rich domains form with a single stranded target sequence a purine-purine- pyrimidine triple helix. The target sequence is preferably located in the genome of the targeted virus, and most preferably an essential region for the virus' replication. As described above, the triplex formation blocks the further replication cycle of the virus in the host cell.

Hence, the TFO according to the invention is in a preferred aspect used for use in the treatment of viral diseases. Such viral diseases are in one embodiment mediated by a single stranded RNA virus, like a corona virus, preferably Feline Infectious Peritonitis Virus (FIPV). Further preferred is that the TFO according to the invention comprises a nucleotide sequence - the "target sequence" which is complementary to a part of the genome of the virus, which mediates said viral disease; preferably wherein the TFO forms a purine-purine-pyrimidine triple helix with said part of the genome, and thereby blocks viral replication.

A complementary sequence is complementary to said target sequence in case it binds to said target sequence under stringent conditions as defined in Maniatis, Molecular Cloning: A Laboratory Manual.

It is preferred that the TFO comprises a sequence complementary to parts of the replicase gene or the virulence gene - as target sequences - of the virus mediating said viral disease. It is preferred for any of the herein described embodiments that in case the TFO is intended to inhibit a virus, the preferred target sequence is selected from the any region comprising the 5' untranslated region (UTR), Open Reading Frame la (ORFla), ORFlb, Gene S, Gene nsp3abc, Gene E (encodes envelope protein), Gene M (encodes membrane protein), Gene N (encodes nucleocapsid protein), Gene nsp7ab, or the 3' UTR.

For the present invention a TFO is preferred comprising a sequence having a sequence identity of at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 100% compared to any one of SEQ ID Nos 1 to 5. Another aspect of the invention relates to any of the aforementioned TFO according to the invention for use in the treatment of viral diseases, specifically wherein the viral disease is mediated by a single stranded RNA virus, such as a corona virus, for example Feline Infectious Peritonitis Virus (FIPV). Yet another aspect is the TFO according to the invention for use in manufacturing a medicament.

The invention also relates to the inventive TFO for use in manufacturing a medicament, wherein said medicament is against a viral diseases, preferably wherein said viral disease is mediated by a single stranded RNA virus, such as a corona virus, for example Feline Infectious Peritonitis Virus (FIPV).

Furthermore described is a method of treating a viral infection, comprising the steps of administering to a subject in need of such a treatment a therapeutically effective amount of a TFO according to the invention described in the aforementioned embodiments. The viral infection is preferably a coronavirus, preferably a feline corona virus, such as Feline Infectious Peritonitis Virus (FIPV).

Also preferred is that said subject is an animal or human in need of such a treatment, preferably wherein said animal is a pet, such as a cat.

For treating the diseases as mentioned herein, also a combination of different TFOs according to the invention may be administered to said subject.

In a final aspect, the invention relates to a pharmaceutical composition comprising a TFO as described herein together with pharmaceutically acceptable carriers and/or exipients. Such a pharmaceutical composition is preferably pharmaceutical composition for used in a method as described herein before.

The carrier and/or excipient must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. It is preferred that the pharmaceutical composition comprising a TFO or a pharmaceutically acceptable derivative thereof in association with a pharmaceutically acceptable carrier and/or excipient is for use in therapy, and in particular in the treatment of human or animal subjects suffering from a condition or disease that is susceptible to a treatment said TFO, in particular selected from a viral infection.

The pharmaceutical composition for use according to the present invention may be formulated for oral, buccal, parenteral, topical, rectal or transdermal administration or in a form suitable for administration by inhalation or insulation (either through the mouth or the nose). For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium starch glycollate); or wetting agents (e.g. sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions or they may be presented as a dry product for constitution with water or other suitable vehicles before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g. almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g. methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

For buccal administration the compositions may take the form of tablets or lozenges formulated in a conventional manner. The TFO according to the present invention may be formulated for parenteral administration by injection, e.g. by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g. in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.

The TFO according to the present invention may be formulated for topical administration by insufflations and inhalation. Examples of types of preparation for topical administration include sprays and aerosols for use in an inhaler or insufflator.

Powders for external application may be formed with the aid of any suitable powder base, for example, lactose, talc or starch. Spray compositions may be formulated as aqueous solutions or suspensions or as aerosols delivered from pressurized packs, such as metered dose inhalers, with the use of a suitable propellant. The TFO according to the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, e.g. containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compositions of the invention may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously, transcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds according to the present invention may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the invention within the principles and scope of the broadest interpretations and equivalent configurations thereof.

Description of the Drawings

Figure 1: Strategy for single stranded targets which utilizes two domain oligonucleotide ligands, linked by clamps (c) to form a circular domain, to bind tightly to pyrimidine rich target sequences. | is the Hoogsteen hydrogen bond and | is the Watson and Crick hydrogen bond.

Figure 2: 20% Denaturing PAGE gel. 01 to 07 is the linear TFOs while cirl to cir7 is the circularized TFOs.

Figure 3: EMSA result. Gl to G5 refer to the target gene while Cirl to Cir 5 is the circular

TFOs. Cir 2 shows negative result of triple helix formation. Figure 4: The inhibitory effect of TFOs towards FIPV. 1 to 5 indicate the circular TFOl to

TF05, L3 and L4 indicate the linear TF03 and TF04. 7 is the Unrelated circular TFO. Comb. (TF01-TF05) is the combination treatment for TFOl to TF05.

Figure 5: In vitro antiviral effects of different concentrations of TFOl towards FIPV replication.

Figure 6: The morphology changes of CRFK cell with TFO treatment after 72 hours in I OX magnification. Red arrow shows the cytopathic effect (CPE) virus. Figure 7: In vitro antiviral assays of circular TFOl and TF05 towards influenza virus,

H1N1 replication.

Figure 8: Interaction between TFO and target region. The binding affinity of the TFO with its t arget region is measured with the association constant (K_a).

SEQ ID No 1 - 6: triple helix forming oligonucleotides

SEQ ID No 7 - 11: target regions Detailed Description of the Invention

Examples:

Triple Helix Oligonucleotide Designation

Generally, triplex consists of a duplex, where the base pairs are formed via Watson-Crick hydrogen bond, and a third strand, whose bases form Hoogsteen hydrogen bond with one base of each pair of the duplex. The purines have potential hydrogen bonding donors and acceptors that can form two hydrogen bonds with incoming third bases. By contrast, pyrimidine bases already involved in the duplex can form only one additional hydrogen bond with incoming third bases. Here, the F1PV is a single stranded RNA virus. In order to form a triplex, a double stranded domain is necessary to be designed. The approach applied here was a double-length purine rich oligonucleotide binds a target strand, folding back to form an antiparallel pur-pur-pyr triple helix (Figure 1). Clamps were inserted into both ends of the purine rich domains to form a circular purine rich domain where this domain is highly resistant to exo and endonucleases comparing to linear domain. Five different sequence of TFO were designed specially targeting the replication gene and virulence of FIPV and one unrelated TFO. The sequences of TFOs are shown in Table 1.

Table 1 : Sequence of TFOs and t e r target sequence.

Synthesis of Designed Oligonucleotide

The designed linear RNAs were synthesized by Dharmacon where the 5' and 3' end of the linear RNAs were modified into Phosphate (P04) group and Hydroxide (OH) group, respectively. These modifications are necessary for the circularization of linear RNA.

Circularization of Linear RNAs

The linear RNAs were circularized by using T4 RNA ligase induced circularization method. The T4 RNA ligase 1 (ssRNA ligase) from New England Biolabs was chosen in this process. This is because this T4 RNA ligase can specially apply to ligate the single stranded RNA into a single stranded circular RNA. T4 RNA Ligase 1 catalyzes the ligation of a 5' phosphoryl-terminated nucleic acid donor to a 3' hydroxyl-terminated nucleic acid acceptor through the formation of a 3'→5' phosphodiester bond, with hydrolysis of Adenosine-5 '-triphosphate (ATP) to Adenosine Monophosphate (AMP) and pyrophosphate (PPi). This circular RNAs were prepared according to the manufacturer's protocol.

Denaturing Polyacrylamide Gel Electrophoresis (PAGE)

Denaturing PAGE was carried out after the ligation process to confirm the formation of the circular RNA. 20% of denaturing PAGE was used to separate the samples (Table 2). The gel was pre-run at 20-40Volts for 45 minutes. After pre-run, the samples were loaded into the wells and run at 200V for 45 minutes. The gel was stained with ethidium bromide and viewed with the Bio- Rad GelDoc system. The circular RNA is moved faster than the linear RNA (Figure 2).

Components 20% gel

40% Stock polyacrylamide 5mL

10X TBE buffer ImL 8M Urea 4.80g

DEPC treated water 3.9mL

TEMED * 10pL

Ammonium Persulphate (APS) * 90μί

Total volume lOmL

*added after the urea dissolve.

Table 2: Components of 20% denaturing PAGE

Recovery of Circular RNAs

The circular RNAs were recovered from the ligation process through a conventional method called ethanol precipitation. This method is commonly used technique for concentrating nucleic acid. 0.8M (final concentration) of lithium chloride and 2.5 volume of the absolute ethanol were added into the mixture with ligated RNA and mixed it by gentle shaking. Then the mixture was incubated in -20 ° C for half an hour. After incubation, the mixture was centrifuged at 4 ° C, 13500rpm for 1 hour to precipitate the RNA. The RNA pellet was washed with 70% ethanol to remove any residual salt away from the pellet. The RNA pellet was air dried and resuspended in RNase free water.

Electrophoretic Mobility Shift Assay (EMSA)

EMSA is a common affinity electrophoresis technique which applied in interaction study. This EMSA was performed to study how capable of the TFOs with the special design sequence can hybridized with the target region to form triple helix. 2\iL of TFO was mixed with 4μΕ of target gene in IX binding buffer with ΙΟμί as final volume and the solution was incubated in 37 ° C for 2 hours. After 2 hours, the sample was run with 15% native gel in cool condition. The gels were stained with ethidium bromide and viewed with the Bio-Rad GelDoc system. The band shifted above showed the triple helix formation (Figure 3).

Components 15% gel

40% Stock polyacrylamide 3.75mL

10X TBE buffer ImL

DEPC treated water 5.14mL

TEMED * \0 iL

Ammonium Persulphate (APS) * ΙΟΟμί

Total volume lOmL *added at last

Table 3 Components of 15% native gel

Table 4 Components of 10X binding buffer

Cell and Virus

Crandell-Reef Feline Kidney (CRFK) cells were employed in this in vitro study because this cell line is susceptible to FIPV. CRFK cells were maintained in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum, 100 IU of penicillin, and 100 g of streptomycin per mL. FIPV serotype II strain 79-1 146 is propagated in CRFK cells (Groot-Mijnes et al., 2004). The FIPV is inoculated into the confluence flask of CRFK cells. This virus inoculated CRFK cells is maintained in MEM supplemented with 1% fetal bovine serum, 100 IU of penicillin, and 100μg of streptomycin per mL. The virus can be harvest when 80% of the cells show cytopathic effect (CPE). The virus titer was determined by using infectivity assay (TCID50) and calculated by using Reed-Muench method.

In vitro antiviral effect of TFOs towards FIPV

In vitro TFO formation efficacy studies were performed in a 96-wells plate by transfect the TFOs into CRFK cell. 3xl0⁴ cells were seeded into each well in 96-wells plate. The plate was incubated in 37 °C, 5%C02 for 24 hours to allow the cells attach to the surface of the well. After 24 hours, ΙΟΟηΜ of TFOs and a combination treatment of TFOl to TFO 5 were transfected separately into the plate by using HiPerFect Transfection Reagent (Qiagen), according to manufacturer's protocol, and the plate was incubated for 6 hours in 37 °C, 5% C02. Later, I OOTCID50 of FIPV was inoculated into the plate for 1 hour at 37 °C, following which the virus inoculums was replaced by fresh maintenance media. Samples were harvested after 72 hours. Before harvest, the morphology of the cells for each treatment was captured and the picture was showed in Figure 6. Samples were stored at -80 °C prior further process.

Quantitative Real Time Reverse Transcriptase PCR (qRT-PCR)

Total RNA was extracted using QIAamp Viral RNA Mini Kit (Qiagen), according to manufacturer's protocol. RNA yield and purity was determined by spectrophotometry (OD280/OD260). Purified RNA was used immediately or stored at -80 °C prior further process. A set of published primer (Simons et al., 2004) was used in this quantitative real time PCR reaction. Quantitative real time PCR reactions were performed using a Bio-rad CFX96 real time system (Bio-rad) and the reagent used was SensiMix SYBR No-ROX One-Step Kit (Bioline). The cycling conditions are shown below. The results were analyzed based on copies of viral RNA genome (Figure 4).

Dose-response of TFO in inhibiting FIPV replication

TFOl was employed to study the effective working concentration to inhibit FIPV replication. A 96-wells plate with 3xl04cells/well of CRFK cells was incubated in 37DC, 5% C02 for 24 hours. Then, different concentrations of TFOl (25nM, 50nM, ΙΟΟηΜ and 500nM) were transfected separately into the cells associated with the HiPerFect Transfection Reagent® (Qiagen, Germany). The plate was incubated in 37 DC, 5% C02 for 6 hours to allow the TFOl transfected into CRFK cells. After 6 hours, the media inside the plate was discarded and ΙΟΟμίΛνεΙΙ of 100TCID50 of FIPV was inoculated into the plate and the plate was incubated at 37DC, 5% C02 for 1 hour. Later, the media inside the plate was discarded and ΙΟΟμίΛνεΙΙ of maintenance media was added into the plate. The plate was incubated at 37DC, 5% C02 for 72 hours. Lastly, the samples were collected and the viral RNA was extracted for Quantitative Real Time Reverse Transcription PCR analysis. The results were analyzed based on copies of viral RNA genome (Figure 5). In vitro specificity study of TFOs towards Influenza A virus subtype H1N1 New Jersey 8/76

In vitro specificity study of TFOs towards Influenza A virus subtype HlNl New Jersey 8/76 is an experiment to examine the specificity of the TFOs to inhibit the virus replication other than FIPV. The cell concentration of 4x10⁴ MDCK cells (ATCC^® No: CCL-34™) was seeded into each well in 96-wells plate. The plate was incubated in 37 °C, 5%C02 for 24 hours to allow the cells attach to the surface of the well. After 24 hours, ΙΟΟηΜ of TFOl and TF05 were transfected separately into the plate by using HiPerFect Transfection Reagent (Qiagen), according to manufacturer's protocol, and the plate was incubated for 6 hours in 37 °C, 5% C02. Later, 100TOD₅o of Influenza A virus subtype H1N1 New Jersey 8/76 was inoculated into the plate for 1 hour at 37 °C, following which the virus inoculums was replaced by fresh DMEM media containing Trypsin-TPCK (Sigma^®, USA). Samples were harvested after 48 hours. Samples were stored at -80 °C prior further process for qRT-PCR. The results were analyzed based on copies of viral RNA genome (Figure 7).

Interaction between TFO and target region

Study on the interactions and hybridization of TFO with its target region is crucial since the stronger the binding, the more stable is the triplex structure formation. The strength of the binding can be measured using nano isothermal titration calorimetry (ITC). The samples RNA were prepared in different concentration accordingly by using lx binding buffer (Table 4) as the diluent. The nano ITC contains a small volume of sample cell and syringe. The high concentration of RNA is loaded into the syringe (50μ1) while the low concentration of RNA is injected into the sample cells (300μΤ). Besides the cell, the nano ITC also contains a reference cell. The solution inside the reference cell must be the same as the sample cell. The experiment is run at 37°C with 2μΐνίη}εΰΙϊοη for total 25 times of injection. The data is collected in each 250 seconds of time interval. The data is then analyzed by using the software provided by the manufacturer (Figure 8).

Discussion

The invention describes the development of triple helix-forming oligonucleotide (TFO) as antiviral agent against coronavirus, specifically against a fatal immune-mediated feline infectious peritonitis (FIP) disease in cats. Specific TFO targeted to the selected regions of virulent Feline coronavirus (FeCoV) strain FIPV WSU 79-1146 genomes were designed and tested in FIPV infected Crandell-Reef Feline Kidney (CRFK) cell line. Five different circular TFOs (TFOl to TF05) and one unrelated circular TFO (TF07) were designed and tested for in vitro antiviral effects. Results from this study showed 50 to 100 nM of circular TFO is sufficient to inhibit virus replication. To study the antiviral effects of TFOs, ΙΟΟηΜ of each TFO were used on cells infected with IOOTCID₅₀, separately, and the antiviral efficacy of TFOs was determined by qRT- PCR. Results showed the viral RNA genome copy numbers of cells treated with TFOl, TF02, TF03, TF04, TF05 and TF07 are 3.65 x 10⁹, 3.22 x 10¹⁴, 5.04 x 10⁹, 5.01 x 10⁹, 4.41 x 10⁹ and 3.96 x 10¹⁴, respectively (Figure 4). As expected the mock transfected cells showed high viral RNA genome copy number, 4.03 x 10¹⁴ (Figure 5). The cytophathic effect of the circular TFOs of the present invention was confirmed in a morphology study (Figure 6). Hence, all the circular TFO except for TF02 demonstrated antiviral effect on FIPV. This study describes the potential used of TFO as antiviral agent against coronavirus, FIPV in cats.

References

Zook, B.C., King, N.W., Robinson, R.L., McCombs, H.L. Ultrastructural evidence for the viral etiology of feline infectious peritonitis. Pathol Vet 1968, 5: 91-95.

Vennema, H., Poland, A., Foley, J. & Pedersen, N. C. Feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses. Virology 1998, 243: 150-157. Hegyi, A., Friebe, A., Gorbalenya, A. E. & Ziebuhr, J. Mutational analysis of the active centre of coronavirus 3C-like proteases. Journal of General Virology 2002, 83: 581-593.

Weiss, S.R. & Navas-Martin, S. Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. American Society for Microbiology 2005, Vol. 69, No.4, 635-664.

Dye, C. & Siddell, S.G. Genomic RNA sequence of feline coronavirus strain FCoV CI Je. Journal of Feline Medical Surgery 2007, 9(3):202-213. Pedersen, N.C., Liu, H., Dodd, K.A. & Pesavento, P.A. Significance of coronavirus mutants in feces and diseased tissues of cats suffering from feline infectious peritonitis. Viruses 2009, 1: 166-184. Dewerchin, H.L., Cornelissen, E. & Nauwynck, H.J. 2005. Replication of feline coronaviruses in peripheral blood monocytes. Archives of Virology 150: 2483-2500. Haagmans, B.L., Egberink, H.F. & Horzinek, M.C. 1996. Apoptosis and T-Cell depletion during feline infectious peritonitis. Journal of Virology 70(12): 8977-8983.

Holzworth, J. E. 1963. Some important disorders of cats. Cornell Veterinary 53: 157-60. Takano, T., Kawakami, C, Yamada, S., Satoh, R. & Hohdatsu, T. 2008. Antibody-dependent enhancement occurs upon re-infection with the identical serotype virus in feline infectious peritonitis virus infection. Journal of Veterinary Medical Science 70(12): 1315-1321.

Fehr, D., Holznagel, E., Bolla, S., Hauser, B., Herrewgh, A.A.P.M, Horzinek,M.C. & Lutz, H. 1997. Placebo-controlled evaluation of a modified life virus vaccine against feline infectious peritonitis: safety and efficacy under field conditions. Vaccine Vol 15, No. 10, 1101-1 109.

Hebben,M., Duquesne,V., Cronier, J., Rossi, B. & Aubert,A. 2004. Modified vaccine virus Ankara as a vaccine against feline coronavirus: immunogenicity and efficacy. Journal of Feline Medicine and Surgery. 6, 111-118.

Haijeme,B.J., Volders,H. & Rottier, PJ.M. 2004. Live, attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis. Journal of Virology. Vol.78, No.8, 3863-3871.

Valentin, G.D., Thuong, N.T. & Helene, C. 1992. Specific inhibition of transcription by triple helix-forming oligonucleotides. Proceedings of the National Academy of Science USA, Vol. 89, 504-508. Han,H. & Dervan, P.B. 1993. Sequence-specific recognition of double helical RNA and RNA-DNA by triple helix formation. Proceedings of the National Academy of Science USA, Vol.90, 3806-3810. Knauert, M.P. & Glazer, P.M. 2001. Triplex forming oligonucleotides : Sequence-specific tools for gene targeting. Human Molecular Genetics, Vol. 10, No. 20, 2243-2251. Groot-Mijnes, J. D. F., Van Dun, J. M., Van Der Most, R. G. & De Groot, R. 2004. Natural history of a recurrent feline coronavirus infection and the role of cellular immunity in survival and disease. Journal of Virology, Vol. 79, No. 2, 1036-1044.

Simons, F.A., Vennema, H., Rofina, J.E., Pol, J.M., Horzinek, M.C., Rottier, P.J.M. & Egberink, H.F. 2004. A mRNA PCR for the diagnosis of feline infectious peritonitis. Journal of Virological Methods, 124, 1 1 1-1 16.

Claims

1. A triple helix forming oligonucleotide (TFO), comprising a circular nucleic acid molecule.

2. The TFO according to any of claims 1 to 6, wherein the nucleic acid molecule is RNA, preferably a circular single stranded RNA.

3. The TFO according to any one of the preceding claims, wherein the nucleic acid sequence of the TFO contains at least 50%, 60%, 70%, 80%, 90%, 95%, 98% purine bases.

4. The TFO according to any one of the preceding claims, wherein the TFO comprises two purine rich domains linked by two clamp domains.

5. The TFO according to claim 9, wherein said two purine rich domains independently comprise at least 50%, 60%, 70%, 80%, 90%, 95%, 98% purine bases.

6. The TFO according to claim 9 or 10, wherein the clamp domain comprises two cysteins.

7. The TFO according to claim 9, wherein the two purine rich domains can form with a single stranded target sequence a purine-purine-pyrimidine triple helix.

8. The TFO according to any of the preceding claims for use in the treatment of viral diseases.

9. The TFO according to claim 8, wherein the viral disease is mediated by a single stranded RNA virus.

10. The TFO according to claim 9, wherein the single stranded RNA virus is a corona virus, preferably Feline Infectious Peritonitis Virus (FIPV).

1 1. The TFO according to any of claims 8 to 10, wherein the TFO comprises a nucleotide sequence complementary to a part of the genome of the virus mediating said viral disease, preferably wherein the TFO forms a purine-purine-pyrimidine triple helix with said part of the genome, and thereby blocks viral replication.

12. The TFO according to claim 1 1, wherein the TFO comprises at least one, preferably two sequences complementary to parts of the replicase gene or the virulence gene of the virus mediating said viral disease.

13. A TFO comprising a sequence having a sequence identity of at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 100% compared to any one of SEQ ID Nos 1 to 5.

14. The TFO according to claim 13 for use in the treatment of viral diseases, specifically wherein the viral disease is mediated by a single stranded RNA virus, such as a corona virus, for example Feline Infectious Peritonitis Virus (FIPV).

15. A TFO according to any one of the preceding claims for use in the manufacture of a medicament.

16. The TFO according to claim 15, wherein said medicament is against a viral diseases, preferably wherein said viral disease is mediated by a single stranded RNA virus, such as a corona virus, for example Feline Infectious Peritonitis Virus (FIPV).

17. Method of treating a viral infection, comprising the steps of administering to a subject in need of such a treatment a therapeutically effective amount of a TFO according to any one of claims 1 to 16.

18. The method according to claim 17, wherein the viral infection is a coronavirus, preferably a feline corona virus, such as Feline Infectious Peritonitis Virus (FIPV).

19. The method according to claim 17 or 18, wherein said subject is an animal or human in need of such a treatment, preferably wherein said animal is a pet, such as a cat.

20. The method according to claims 17 to 19, wherein a combination of different TFOs according to claims 1 to 8 are administered to said subject.

21. A pharmaceutical composition comprising a TFO according to any one of claims 1 to 14 together with pharmaceutically acceptable carriers and/or exipients.

22. A pharmaceutical composition according to claim 21, for use in a method according to claims 17 to 20.