Patent Application
20160281089
The present invention concerns the inactivation of viral infectivity in a cell-free environment as well as the preparation of a pharmaceutical agent and a method therefore.
Retroviruses like the Human immunodeficiency virus (HIV)-1 continue to be a global pandemic of enormous consequence to humanity. Current standard-of-care regimens recommended for the treatment of HIV infection include two or more nucleos(t)ide reverse transcriptase inhibitors (NRTI) in combination with non-nucleoside reverse transcriptase or protease inhibitor. Importantly, NRTIs are activated through interactions with the cellular machinery for regulating endogenous nucleoside triphosphate pool.
Protease inhibitors also act inside of infected cells. Although some successes have been demonstrated, many agents in these classes are limited by various factors, including insufficient efficiency, poor tolerability, dependency on cellular enzymes and drug-resistance. A major focus of the search for novel strategies to fight HIV-1 is therefore identification antiviral compounds, which would allow overcoming these limitations.
Known, commonly available anti-viral drugs are also targeting three viral proteins, the Reverse Transcriptase (RT), the protease, and gp41. New antiviral approaches are required, to complement available therapeutics.
Retroviruses replicate the viral RNA by concerted action of the reverse transcriptase (RT) and RNase H, which specifically hydrolyzes RNA in RNA-DNA hybrids (Hansen et al. 1988, EMBO J. 7:239-243; Moelling et al., Nature New Biol. 1971, 234:240-243; Tisdale et al., 1991, J. Gen. Virol. 72:59-66.). Thereby the polypurine tract (PPT) resists to hydrolysis and serves as primer for the second strand DNA synthesis (Volkmann et al., 1995, Nucleic Acids Res. 23:1204-1212; Erratum in: Nucleic Acids Res. 23 3804; Wohrl, B. M. and K. Moelling. 1990, Biochemistry 29:10141-10147; Wohrl, et al., 1991, J. Mol. Biol. 220:801-818.). The PPT is one of the most highly conserved sequences of HIV, which is located in the coding region of the nef gene, adjacent to the unique region at the 3′-end (U3) (Martinez et al., 2002, Cell 110: 563-74). The extended PPT is 25 nucleotides long and consists of two polypurine clusters interrupted by two non-purines (CU), which are located adjacent to an internal A. 5′ of this ACU triplet is the cleavage site of the viral RNase H (Wohrl, B. M., and K. Moelling. 1990, Biochemistry 29:10141-10147). Furthermore, the transcripts of this sequence are important at later stages during replication for recognition by the integrase and integration of the DNA provirus (Reicin et al., 1995, J. Virol. 69:5904-5907).
Thus, ACU is a sequence of extreme functional importance for three viral enzymes, the RT, the RNase H, and the integrase. The distance between the two active centers of the RT and the RNase H is about 18 nucleotides (Sarafianos et al., 1996, EMBO J. 20:1449-1461; Wohrl, B. M., and K. Moelling. 1990, Biochemistry 29:10141-10147). The PPT has the unique capacity to resist RNase H-mediated hydrolysis of the RNA in DNA-RNA heteroduplexes (Müller et al., 1994, J. Mol. Biol. 242:422-429.), probably due to structural peculiarities (Sarafianos et al., 1996, EMBO J. 20:1449-1461; Xiong, Y., and M. Sundaralingam, 1998, Structure 6:1493-1501). The PPT-RNA remains hybridized to minus-strand DNA during reverse transcription and functions as primer for initiation of plus-strand DNA synthesis (Volkmann et al., 1995, Nucleic Acids Res. 23:1204-1212, Erratum in: Nucleic Acids Res. 23 3804). This is a preferred recognition site of the RT/RNase H.
The U.S. Pat. No. 5,849,900 discloses oligodeoxynucleotides (ODN) and oligoribonucleotides (ORN) against the polypurine-tract (PPT) of HIV which were designed for antiviral therapy of an HIV-infected individual. The effects of the disclosed ODNs were demonstrated through premature termination of cDNA synthesis at the PPT.
The effect has been shown to be extremely sequence-specific. Three viral enzymes are required to function correctly at the PPT, therefore this site is considered to be preferred for intervention because of a low chance for the virus to mutate there and become resistant. These results have been published in two papers by Jendis et al 1996, 1998 (AIDS Research and Human Retroviruses 12, 1161, 1996 and AIDS Research and Human Retroviruses 14 (11), 999, 1998).
The strategies of managing retroviral infections known from the state of the art have in common that they are intended for the use in infected cells. Consequently, these strategies are not suitable for the prevention of retroviral infection. To defeat further spreading of retroviral infections there is a strong need for methods and means that are capable of stopping for example HIV transmission before a cell can be entered by the viral particles.
Coming from this state of the art, it is an object of the present invention to provide an alternative method and means for the specific inactivation of retroviruses.
Furthermore it is an object of the present invention to provide a pharmaceutical composition usable as medicinal product corresponding to said alternative method suitable for the inactivation of retroviruses.
In the following we would like to give some more specific definitions of terms used within the context of the present invention:
In connection with the present invention a “cell-free environment” basically means administration “outside a cell”. This means that a method or a pharmaceutical composition according to the present invention is used outside a cell, whereas the use in a vagina or inside a blood vessel is naturally comprised as this represents a body cavity formed of cells, but not the use within a cell. Consequently, also the use within the bloodstream or other body fluids of a human is to be understood as “outside the cell”.
The term “anti-viral” relates to all types of virus defeating measures in connection with viruses that have an RNA or DNA genome replicating through an RNA or DNA intermediate stage. Consequently retroviruses like the Human Immunodeficiency Virus (HIV) are as well comprised as viruses like the Hepatitis B Virus (HBV) as this is a DNA virus replicating through a RNA intermediate stage and being designated due to this as pseudo-retrovirus. Most important in connection with the present invention is on one hand the concerted action of the enzymes ReverseTranscriptase (RT) and RNaseH and on the other hand the presence of a poly(A)-tail as part of a RNA. The invention is not restricted to retroviruses like HIV, but also to all know retroviruses like HIV-I and HIV-II as well as HTLV-I and HTLV-II (human T-cell leukemia virus type 1 and type 2) which could cause infections for humans and animals through RNA, RNA-DNA or a DNA intermediate stage.
The term “oligonucleotides” (ON) summarises ODN (oligodeoxynucleotides) as well as ORN (oligoribonucleotides) or both ODN-ORN (chimera), ONs are 8 to 80 nucleotides in length. The ONs can be single-stranded (antisense) or double-stranded, whereby the double-stranded ON can be partially or fully self-complimentary. The ONs are targeted to a viral sequence and are either fully or partially complementary to the target sequence (some of which are listed below). While oligodeoxynucleotides (referred to as ODN) and oligoribonucleotides (referred to as ORN) or chimeras (ODN-ORN) or combinations thereof may be utilized with a preferred length of 8 to 80 nucleotides it is also intended to use mononucleotides for oligonucleotide synthesis or primer that will be elongated both by RT leading to activation of RNaseH and increasing the destruction of viral RNA (as shown in
In relation with the term oligonucleotides it is further within the scope of invention that also parts of the sequences as shown in table 1 are used as oligonucleotide with the above understanding.
The term “combination” may be understood in connection with the present invention as combining ODN and ORN and mononucleotides in every possible way. The invention is not restricted to the preparation of a mixture of ODN and ORN that is applied after mixing. It is also within the scope of the present invention that a combination means sequential combination of ODN and ORN that is performed by applying one at a time.
The “target sequence” can be the viral RNA genome or a DNA genome or a DNA provirus. The target sequence is preferred a polypurine tract, such as the 3-PPT of HIV. It can be the 5-PPT of HIV, the Primer-binding site (PBS), the packaging site PST or other conserved sequences. A target sequence may also be characterized by a purine-rich sequence not known to be or not being a conserved sequence, like a sixfold consecutive repeat of the purines guanine and arginin in any context with respect to the flanking sequences. Another conserved sequence as target is the poly (A) tail of the HIV-RNA. Also purine rich sequences of HBV RNA are potential targets.
Body fluids will be understood by those skilled in the art as blood, serum, plasma, saliva, tears, sperm, vaginal secretion or any other secret of a body.
The objects of the invention are solved by the features of the independent claims.
In general, the invention can be described as follows: In contrast to known mechanisms and treatments for inactivation of virus functionality the present invention is based on a completely different understanding of the interaction of synthetic oligonucleotides with viral nucleic acids. While so far interaction was directed to cell associated destruction of viral nucleic acid the present invention is based on the surprising effect that viral nucleic acid can be also affected outside a cell. An oligodeoxynucleotide (ODN) or an oligoribonucleotide (ORN) or an ODN/ORN chimera or combination thereof is able to affect the viral RNA also outside of the body or outside of a cell, as it is active inside the virion.
The antiviral action is due to the activation of a viral enzyme, which is present in the virus particle, the Reverse Transcriptase RT/RNaseH. The ODN targeted to the PPT is recognized by the RT/RNase H inside the virus and activates its activities. Thereby the viral RNase H cleaves the viral RNA prematurely, before the RNA is copied into DNA. This irreversibly leads to loss of the genomic information and failure to make a full-length DNA copy for a DNA provirus formation. The effect can be described as a viral suicide, because the virus itself causes the killing of its genome. The RT/RNase H activities are activated prematurely by the ODN. This corresponds to a correct step in viral replication, however prematurely. Mononucleotides activate the RT/RNaseH to generate new hybrids and enhance the destructive effect.
The effect is totally unexpected, because cellular enzymes were considered to perform the destruction of the viral RNA inside the cell, not outside of the cell in the virus particle (and only after DNA synthesis).
Thus, treatment of viral particles can abrogate the viral infectivity. Treatment of virus (HIV) in vitro for 4 hours and subsequent infection of a host cell for test of infectivity demonstrates that the virus has become inactive. The RNA is destroyed and no virus protein (p24) provided (
Preferred according to the invention are the sequences as shown below which are although an object of the invention:
It should be noted that according to the figures, in particular
It is further within the scope of invention that also parts of the sequences as shown in table 1 are used as oligonucleotide, e.g. sequence strands as shown in the figures, in particular “upper” and “lower” sequence strands of
Table 2 shows preferred target regions comprising conserved binding regions for oligonucleotides/oligoribonucleotides according to the invention.
An important improvement for the fight against HIV could be the reduction of transmission during sexual intercourse. This is a recent very important goal, since there may not be a vaccine. Furthermore other virus infections in the vagina increase the risk for transmission of HIV. Even circumcision is considered as a hygiene measure to reduce the rate of transmission, which would be a factor of 2. However, a preferred application of the present invention is a microbicide against viral infections through sexual transmission.
Meanwhile microbicide against Herpes Simplex Virus, HSV, infection of the vagina which is a serious cofactor for the HIV infection, has been described by (Palliser et al., Nature 439, 2006, 89-94). The drug according to the invention is intended for administering into the vagina for antiviral action so that the HSV will not be able to infect. A decrease of HSV infections would strongly decrease the chance for a HIV infection. The formulation of the drug has to avoid any mucosal irritation or lesions in order not to give rise to the opposite effect, such as a better infection instead of prevention.
In the examples below the invention is explained in more detail. To demonstrate the efficiency of the viral inactivation according to the invention, we used a model virus replacing HIV in a small animal, called SFFV (Spleen Focus Forming Virus), because there is no mouse model for HIV. It is a virus similar to HIV, however causing cancer in the spleens. Below we describe the surprising effect that SFFV virus titer in the blood of fully infected mice can be reduced by a single dose of ODN applied to the tail vein, by about 10 fold, depending on the dose. The effect is due to destruction of the virus by the ODN in the blood circulation. This is inside the body, but not inside a cell, especially a viral infected cell.
The reduction of the virus load within the blood stream is completely surprising as the effect of applying oligodeoxynucleotide (ODN) or oligoribonucleotide (ORN) or ODN/ORN chimera or a combination thereof is demonstrably after 4 hours. The so far known effects in a cellular context become detectable after at least 12 hours. This means that the antiviral treatment of the present invention is much more effective than the ones known from the state of the art with the additional advantage that the subject matter of the present invention allows prevention of viral infections and is not restricted to the treatment of already viral infected cells.
Next we demonstrate below that treatment of the virus at the time of infection can abrogate viral infectivity as measured by a twofold increased survival time of 2 out of 5 mice by twofold (
Further, we demonstrate that pretreatment of the virus with the ODN or antisense (asPPT) in a small test tube (the equivalent of a vagina) for 1 to 4 hours and subsequent infection of the mouse can completely abrogate viral infectivity in the body in a dose dependent manner.
This is shown in the
Next we performed an intravaginal treatment with ODNA A of a recombinant retrovirus and recovered the virus after 4 h by lavage. The titer was reduced by a factor of two (
In summary of the above, the surprising results are:
Virus particles can be inactivated by their treatment according to the present invention, whereby
From the above it should be understood that according to the invention the use of an oligodeoxynucleotide or oligoribonucleotide or a chimera or a combination thereof capable of binding at least in part to conserved target regions of viral RNA for the inactivation of viral infectivity outside in a cell-free environment is intended. Preferably the conserved region of the viral RNA, where the sequence of the nucleotides chain is derived from, comprises the sequence and/or complementary sequence of a poly-purine rich tract. In a further embodiment the used nucleotide chain is fully or partially self-complementary and has a preferred length from about 8 to about 80 nucleotides.
Therefore, an object of the invention is the method to use an oligodeoxynucleotide (ODN) or oligoribonucleotide (ORN) or a ODN/ORN chimera or a combination thereof capable of binding to target regions of viral RNA with a purine content of at least 25% or an adenine stretch comprising at least 10 consecutive bases, as microbicide for the inactivation of viral infectivity in a cell-free environment. The ODN, the ORN or the chimera ODN/ORN or the combination thereof is targeted against a region of the viral RNA with a purine content with at least 25% having a length from about 8 to about 80 nucleotides or respectively at least against a poly (A) rich conserved region of the viral RNA or respectively against a region of the viral RNA comprising the sequence and/or complementary sequence or at least a part of the poly-purine rich tract. It may extend beyond the of the poly-purine rich tract. It is preferred if the ODN, the ORN or the combination thereof comprises a single stranded anti-sense sequence, ranging in size from 8-80 nucleotides, preferably an 18 mer, or a partially or full self-complementary sequence, preferably a 54 mer. The nucleotides are capable of binding with at least a portion of the PPT region, however, the nucleotides may extend beyond the PPT. The nucleotides may match the PPT region exactly or may contain several mismatches.
The oligonucleotide need not match the target sequence exactly; it may span only a portion of the PPT and some mismatches may be included. The length of the nucleotide is preferably from about 8 to about 80 nucleotides long. The nucleotides should be a size which is long enough to bind specifically to the target PPT region. It will be understood that e.g. an antisense oligonucleotide may be longer than a triplex-forming oligonucleotide since the former hybridizes via Watson-Crick bonds which may extend further than the polypurine rich region while the latter forms Hoogsten-type bonds which are most efficiently formed in a polypurine rich region. However, mismatches of between 10-20% within a region of complementarity are acceptable.
Further as an alternative according to the invention, the ODN, the ORN, a chimera ODN/ORN or the combination thereof is targeted against a region of the viral RNA with a contiguous sequence of at least 6 guanine (G) or 6 adenine (A) nucleotides in length or against a sequence consisting of 6 nucleotides in length mixed by guanine (G) and adenine (A) nucleotides. Such a nucleotide sequence, e.g. GGGGGG or AAAAAA or as an not exclusive example GAGAGG is independent of the length and its flanking sequences.
To prevent degradation of the used nucleotide chain it is—if necessary—intended that the backbone of the nucleotide chain is stabilized by secondary modifications; preferably the modification comprises a modification of the nucleotide backbone from phosphodiesters to phosphorothioates.
According to the invention sequences Seq.ID NO. 1 to 25 are especially preferred as ODN or ORN or chimera ODN/ORN.
Further, oligodeoxynucleotides comprising a sequence that is capable of binding to the target binding regions according to one of the sequences of Seq.ID NO. 26-40 are preferred as an object of the invention. Furthermore preferred are oligodeoxynucleotides which are complementary to the target binding regions according to one of the sequences of Seq.ID NO. 26-40.
A particular aspect of the invention is the method to use mononucleotide as microbicide for the inactivation of viral infectivity in a cell-free environment. In the presence of monodeoxynucleotides the RT/RNaseH can be activated for cDNA synthesis, e.g. by primer extension at the ODN A and thus increase the length of the hybrid region (
It is a further object of the present invention to use a sequence of oligodeoxynucleotides or oligoribonucleotides or a chimera or a combination thereof according to the method of the invention for the preparation of a pharmaceutical agent for the inactivation of viral infectivity in a cell-free environment, where the used nucleotide sequence is capable of binding to conserved regions of viral RNA, preferably the sequence and/or complementary sequence of a poly-purine rich tract. Such a pharmaceutical composition should be useful for prevention of viral infections after contact with virus containing fluids and/or the reduction of virus load with viruses replicating through a RNA or DNA intermediate stage. A medicinal product like a microbicide should therefore be capable to prevent retroviral infections even if there is direct contact with retroviral contaminated liquids, e.g. at mucosal surfaces.
As already mentioned above, mononucleotides, especially monodeoxynucleotides activate the RT/RNaseH to generate new hybrids and enhance the destructive effect. It is therefore a further object of the invention to present herewith a method to use equimolar amounts of all 4 monodeoxynucleotides adenine (A), cytosine (C), guanine (G) and thymidine (T) for the self synthesis of an oligonucleotide or primer extension as pharmaceutical agent for the inactivation of viral infectivity in a cell-free environment. A corresponding pharmaceutical composition itself is also claimed.
It is also intended according to the present invention, to use at least one of the pharmaceutical preparations as described above in combination with one or more supplementary agents from the group of antiviral, fungicidal or antibacterial agents, anti-cancer agents and/or immunostimulators or immunomodulators. Preferably the pharmaceutical agent is prepared for the application on skin or mucosal surfaces, especially the vaginal mucosa. The method according to the invention is preferably intended for the use on the skin or mucosal surfaces. Consequently it will be understood by those skilled in the art that a pharmaceutical preparation according to the invention comprises any preparation that is suitable for applying a pharmaceutical agent according to the invention and executing the disclosed method of the invention. This means especially forms of administration such as suppositories, lotions, creams, ointments, sprays or liquids, also used in combination with a condom as lubricant.
A pharmaceutical agent prepared according to the invention is especially intended for the prevention of viral infection, especially during sexual intercourse. Although, multi drug resistant HIV patients could benefit from the invention. The invention is further important to prevent mother to child transmission during birth delivery by applying the pharmaceutical composition to the mother shortly before birth delivery.
Further, the invention is of importance for any kind of surgery (also oral and dental surgery) in respect of prevention virus transmission during surgery by reduction of virus load in body fluids (i.e. blood, saliva).
The invention will be described by examples and figures below, where both are meant to illustrate the invention and not to limit it in any way.
To demonstrate the effects of an oligodeoxynucleotide according to the invention, oligodeoxynucleotide A (ODN A), which consists of a 25 mer antisense, and a 25 mer passenger strand, connected by four thymidines (T4) was used. The sequences of the strands are partially complementary. The ODN A was phosphorothioated at each end (3 bases) and in the T4 linker. ODN A was targeted to the extended PPT of HIV-1. We also used a few structural variants of ODN A: ODN T, which has a passenger strand fully complementary to the antisense strand; the single-stranded antisense PPT (asPPT) which lacks the passenger strand and the linker; ODN H and ODN NT, which both have three nucleotide changes at different positions of the passenger strand; ODN CG and ODN D, both having a single nucleotide change at different positions of the passenger strand; and ODN B which has one nucleotide removed from both 5′ and 3′ ends. ODN Sc has the same length and nucleotide composition as ODN A but a randomized sequence of both strands. ODN CO has a secondary structure similar to ODN A but targets sequences downstream of the extended PPT. Antisense oligodeoxynucleotide asEXT, which targets sites outside of the extended PPT, was also used (
We previously showed that the ODN A at high concentrations (1 μM or more) inhibits the RT/RNaseH activity in vitro.12 To check the effect of ODN A at lower concentrations of RT/RNaseH-dependent cleavage of RNA, we tested various concentrations of ODNs in an RNase H cleavage assay. We used synthetic 5′-end-labelled RNA2, which contains the extended PPT and sequences of the viral genome close to the PPT. As can be seen in
To test whether an ODN-mediated activity would preferentially occur at the PPT, we analysed ODNs targeted to the extended PPT (ODN A) or to a region outside of the PPT (ODN CO and an external antisense oligodeoxynucleotide asEXT) in vitro. We used RNA2, which contains the PPT and the binding sites for ODN A, asEXT and ODN CO. As shown in
HIV replication is a complex process which includes reverse transcriptase processivity, RNase H activity for hydrolysis of the viral RNA, and requires the presence of the viral nucleocapsid protein and an appropriate secondary structure of the RNA.14-16 The optimization of all of these conditions may not be fully met in in vitro assays, however more physiological conditions are encountered in HIV virions. Additionally, it has been shown, that phosphorothioated oligodeoxynucleotides can be internalized in cells without any delivery systems.12,17 Therefore, in order to simulate the situation in vivo, we incubated intact virions with ODNs in cell culture medium without any detergents or ODN-carriers. We used a higher concentration of ODNs than in in vitro studies, since the cellular uptake of oligonucleotides is low and the majority of ODNs may not penetrate through the cellular membrane. We assumed that in virions similar to cells only a small portion of ODNs could be internalized.
In our study we used ODN A, ODN CO, asPPT and additional control oligodeoxynucleotides described in
To test whether ODNs may interfere with the RT-PCR assay, used as a readout of RNA degradation, we purified the viral RNA pre-incubated with ODNs under conditions facilitating hybridization. Hybrid complexes were then re-purified and RT-PCR analysis was performed as described in Materials and methods. As shown in
In order to prove that the reduction of viral RNA levels was indeed due to the enzymatic activity of the RNase H domain of the RT/RNaseH, we used Illimaquinone, previously described to specifically inhibit the RNase H activity of HIV RT/RNaseH. We permeabilized virions with 0.1% NP-40 to facilitate the uptake of Illimaquinone and measured the viral RNA degradation by real-time PCR. As shown in
It has been previously shown that some reverse transcription can take place within extracellular HIV virions. Therefore, it is reasonable to assume that following ODN A-mediated cleavage by the RNase H activity of RT/RNaseH, the polymerase activity extends the ODN A as an artificial primer and the RNase H activity degrades the viral RNA in the concerted action. Indeed, incubation of virions with ODN A led to cDNA production starting with ODN A as registered by PCR using ODN A- and viral cDNA-specific primers (
So far we have demonstrated ODN A-mediated HIV RNA degradation in intact virions. As a next step we investigated the antiviral effect of ODNs by testing the infectivity of treated virions in cell culture studies. We used ODN A, ODN T, as PPT and ODN CO. 2×105 C81-66/45 cells were infected with virions pre-incubated with different ODNs at 0.01 MOI for 1, 2 or 4 hours prior to infection. RNA was extracted from infected cells and HIV replication was analysed by real-time PCR 3 days post-infection. The time course analysis revealed that incubation of virions with ODN A for 4 hours was sufficient for complete suppression of viral replication in infected cells (
Pre-incubation of virions with ODN A for 4 hours and infection at low MOI (MOI of 0.1) did not significantly interfere with binding of virions to the host cells. Indeed, analysis of HIV RNA in infected cells 1 hour post-infection showed that virions, pre-treated with ODN A for 4 hours, were taken up by cells only 14% less efficiently compared to virions treated with ODN Sc (
To test the specificity of suppression of viral replication, the effect of ODN A on the infectivity of virions was compared with control ODNs. Virions were pre-treated with the ODNs and HIV replication in infected cells was analysed by real-time PCR, 5 days (
There is no simple HIV mouse model available for replicating HIV. We therefore used the oncogenic replication-defective SFFV, pseudotyped by the replication-competent MuLV helper virus as model virus in mice. SFFV causes spleen foci through its short envelope protein gp55, which activates the Erythropoietin receptor in the absence of the hormone and stimulates cell proliferation. MuLV resembles SFFV in sequence except for an extended gp80 envelope protein. SFFV has a typical retroviral PPT, slightly shorter than that of HIV (
SFFV replicates to high titers in mice, whereby the spleen is a virus-shedding reservoir. The virus stock is recovered from spleen homogenates after full-blown disease, at about day 20 post infection (pi). The clarified supernatant contains around 2×105 focus forming units (FFU) per mil. Infection was normally performed by intravenous (iv) injection of 4×103 SFFV. After 5 days the blood was recovered (100 microliters) for detection of the viral RNA. The equivalent of 0.05 microliters was enough for RNA detection by 40 cycles. One FFU corresponds to about 6×104 RNA copies or 3×104 virus particles.
In this study we used an oligodeoxynucleotide M (ODN M), targeted to the extended murine viral PPT of SFFV, consisting of a 22-mer antisense, and a 22-mer second strand, connected by four thymidines (T4). The sequences of the strands are partially self-complementary, which may allow formation of a hairpin-loop structure (
To test, whether ODN M would have similar effects as ODN A, we tested the ability of ODN M to induce cleavage of SFFV RNA in the presence of the recombinant M-MuLV RT/RNase H. We used synthetic 5′-end-labelled RNA, which contains the extended PPT of SFFV and sequences of the viral genome dose to the PPT. As can be seen in
In order to further characterize antiviral effects of ODNs, we tested ODN M, asPPT and structural analogs of ODN M in SFFV virions, which contain endogenous RT/RNase H and have an appropriate secondary structure of the RNA. Permeabilized virions were incubated with ODNs, viral RNA was then purified, reverse transcribed and the amount of undigested RNA was quantified by real-time PCR using a set of primers harboring the PPT region of the SFFV genome. The result shows the amount of intact RNA in % as bars and amplified PCR products on gels below (
As next step we investigated the antiviral effect of ODNs by testing pre-treated virions for residual infectivity as has been described before for HIV particles. SFFV particles were pretreated with ODN M or its variants and used to infect NIH 3T3 cells. After three days RNA was extracted from infected cells and from the supernatants and SFFV was quantified by real-time RT-PCR and analysis of amplification products (
In the next experiment NIH 3T3 cells were infected with SFFV for 2 h and then treated with three different concentrations each of ODN M, asPPT, and three different siRNAs. The antiviral effect of ODN M is slightly superior to the asPPT but much stronger compared to siRNA under these conditions (
A number of experiments were carried out to evaluate the effect of ODNs on the SFFV mouse model. We treated 5-day-SFFV-infected mice (thick arrow) with ODN M (triangle) and measured the plasma RNA levels 4 hours after the in vivo therapy by real-time RT-PCR (
To elucidate further the antiviral effect of ODN M in vivo we set up different therapeutic regimens. We infected mice with SFFV and then treated them 5 days later twice at an interval of two hours with either ODN M (1 mg), asPPT, ODN A as control, or PBS. The RNA was extracted 4, 8 and 16 hours later for PCR analysis (
In the next study, we used a single injection as treatment protocol and assessed the antiviral effect of both ODN M and asPPT in a short time-course. Plasma was taken from mice at 0.5, 1, 2, and 4 hours after the single treatment (
In order to test, whether the SFFV titer increases again after ODN M-treatment, and whether it remains sensitive for ODN M, we treated 5-day-SFFV-infected mice at 3 days intervals with a double dose within 2 hours intervals. Plasma was taken before and 4 hours after treatment and assessed for viral RNA levels (
To obtain a more continuous effect of ODN M and asPPT on SFFV replication we treated the SFFV-infected mice every 12 hours (
Taken together these results demonstrate that ODN M has a strong antiviral effect against SFFV detectable 4 hours after beginning of the therapy. Depending on the regimen of treatment, the antiviral efficiency can be transient or longer lasting.
Next we evaluated the prophylactic effect of ODN M on SFFV replication. For this we established 5 groups of 5 mice each. We pre-incubated SFFV with different ODNs or PBS for 2 hours and then injected the two together intraperitoneally at time 0. Then the mice were treated at 2, 4, and 24 hours post-infection by i.v. route. After 5 days, plasma from the mice was analyzed for viral RNA levels. Mice treated with ODN M (group 3) showed a 5-fold decrease of RNA levels compared to the control mice that were infected and treated with PBS, while mice treated with ODN A showed a 30% reduction in RNA expression level (
We also monitored survival of SFFV-infected mice treated with and without ODN M at times 0 and 1 hour, in this case DBA/2 mice and by i.p. route (
In another experimental setting we assessed the effect of an i.v. therapy early after infection. We infected mice with SFFV and treated them 2, 6, and 10 hours later with ODN M, asPPT or PBS. The PCR results obtained from the blood samples show that this treatment prevented the establishment of the SFFV-infection for the whole period of time tested (
In order to analyse the efficiency of the ODN A in the vagina, we used a recombinant retrovirus vector (FUGW) and placed the virus with and without ODN A into the vagina. We recover the virus by lavage and tested the RNA copies by real-time PCR. As can be seen (
(A) The sequences of the extended polypurine tract, PPT, and a site downstream to the PPT of the viral RNA are shown in complex with ODN A and ODN CO accordingly. ODNs consist of an antisense strand and passenger strand linked by four thymidines. Watson-Crick bonds are shown by vertical bars. The sequence of the PPT within the extended PPT is indicated in bold. Antisense oligodeoxynucleotide asEXT targets sites outside of the extended PPT. Relative positioning of ODN A, ODN CO and asEXT on synthetic RNA2 is schematically shown on the lower panel. The box represents the extended PPT and the white stripe symbolises the cleavage site for the RT/RNaseH at the 3′ terminus of the PPT within the extended PPT.
(B) The sequences of ODN A and its variants are depicted in hypothetical partially self-complementary hairpin-loop structures. Curved lines symbolize the linker consisting of four phosphorothioated thymidines. ODN Sc has a randomized sequence of both strands and serves as a control for non-specific action of phosphorothioated oligonucleotides. ODN CO targets a region on HIV RNA outside of the PPT. All the other ODNs are designed to target the extended PPT. Abbreviations: Sc—scrambled, B—one nucleotide removed from both 5′ and 3′ ends of ODN A, T—multiple substitutions in the passenger strand for complete complementarity to the antisense strand, H—triple substitution in the passenger strand for partial complementarity to the antisense strand, NT—triple substitution in the passenger strand in the site complementary to CCCCCC site of the antisense strand, D—single substitution in the passenger strand in the site complementary to AGT site of the antisense strand, CG—single substitution in the passenger strand in the site complementary to the TCT site of the antisense strand. The asEXT was used for primer-extension experiment. Nucleotide changes made compared to ODN A are indicated in bold.
(A) Cleavage assay. 10 nM of in vitro transcribed 5′-labeled RNA was hybridized with 10, 100 and 1000 nM of ODN A and incubated in the presence of RT/RNaseH. The cleavage products were analyzed by denaturing PAGE as described in Materials and methods. (B). Primer extension assay. Synthetic RNA2 was hybridized with ODN A, antisense primer (asEXT) or both primers combined, and the primer extension was performed as described in Materials and Methods. Elongation products were analysed by 8 M urea/10% PAGE. (C) Cleavage assay. RNA/ODN hybrids, obtained using 10 nM of RNA2 and 10 nM of ODN A, asPPT or ODN CO were incubated with RT/RNaseH and cleavage products were analyzed by denaturing PAGE as described in Materials and methods. Cleavage or extension products are presented schematically to the right of each blot. Cleavage sites are indicated by arrows and labelled cleavage products are shown by black lines. Dots on extension products indicate the incorporated labelled dATPs.
(A) Schematical representation of the sample preparation for real-time PCR
(B) 0.5 μM of ODNs were incubated with intact HIV virions in RPMI cell culture medium for 6 hours and RNA extracted from the treated virions was analyzed by real-time PCR as described in Materials and methods. Each bar represents the mean±SD of three independent experiments
(C) ODNs do not interfere with RT-PCR reaction. Purified viral RNA was incubated with ODNs in conditions facilitating hybridization. Hybrid complexes were then re-purified and RT-PCR analysis was performed as described in Materials and methods.
Permeabilized HIV virions were incubated without (A) or with (B) RT/RNaseH (0.05 units/μl), 50 nM ODN A and 150 μM of Illimaquinone, a selective inhibitor of the RNase H activity of HIV RT/RNaseH for 30 min at 37° C. Then viral RNA was purified and real-time PCR analysis was performed. Each bar represents the mean±SD of three independent experiments.
(C) Intact virions were incubated with 0.5 μM of ODNs for 6 hours in cell culture medium. Total viral RNA was then extracted and analyzed by agarose gel electrophoresis.
(D) Virions were incubated with 0.5 μM of ODNs for 6 hours in cell culture medium. Viral RNA was then extracted, DNase-treated and RT-PCR was performed using primers covering Env-region or PPT-region of HIV RNA. Numbers on the scheme refer to the coordinates of primers on HIV RNA.
(E) Virions containing monodeoxynucleotides were incubated with 0.5 μM of ODNs for 6 hours in cell culture medium to allow primer extension. Viral nucleic acids were then extracted, RNase-treated and PCR was performed using primers specific for viral cDNA and ODN A. 80 bp is the size of the extendes ODN A.
(A) HIV virions were incubated with 0.5 μM of ODNs for 1, 2 or 4 hours. C81-66/45 cells were infected with pre-incubated virions, at 0.01 MOI. RNA was extracted from infected cells and HIV replication was analysed by real-time PCR, 3 days post-infection (p.i.).
(B) The same experiment as in (A) was performed with ODN T, ODN CO, asPPT and ODN A pre-treated virions.
(C) Comparative efficiency of ODNs in virions. ODNs were incubated with intact HIV virions in cell culture medium for 1, 2 or 4 hours, then the RNA was extracted from the treated virions and analyzed by real-time PCR as described in Materials and methods.
(D) C81-66145 cells were infected with virions pre-incubated with 0.5 or 5 μM of ODNs for 4 h, at an MOI of 1. RNA was extracted from infected cells and HIV replication was analysed by real-time PCR, 3 days post-infection.
(E) C81-66/45 cells were infected with virions pre-incubated with ODN A and ODN Sc for 4 h, at an MOI of 0.01. RNA was extracted from infected cells and HIV replication was analysed by real-time PCR, 1 h and 24 h post-infection. (F) The same experiment as in (E) was performed except for using ODN T, ODN B and ODN A, 1 hour post-infection.
C81-66/45 cells were infected with virions pre-incubated with ODN A, ODN T, ODN CO or as PPT for 4 h, at MOI of 0.01. RNA was extracted from infected cells and HIV replication was analysed by real-time PCR, 5 days p.i. (A), and 20 days p.i. (B); by measuring p24 production in the medium of the infected cells (C) or by RT-PCR, 11 days p.i. (D). Each bar represents the mean±SD of three independent experiments. Syncytia formation is observed in the cells infected with control ODN-treated and not ODN A-treated virions 15 days post-infection (Data not shown).
a. The SFFV extended PPT sequence with RNase H cleavage site (arrow) is shown. ODNs targeted to the PPT are shown as hairpin-looped structures. Vertical lines indicate base-pairing. The antisense strand (lower strand) is fully complementary to the PPT and linked by four thymidines (curved line) to the passenger strand (upper strand). ODN A targets the PPT of HIV-1 (HIV-1 PPT).
b. Structural organizations of M-MuLV and HIV RT/RNase H are depicted schematically. The 80 kDa RT/RNase H of M-MuLV is a monomer while RT/RNase H of HIV-1 is a heterodimer of p66 and p51 and cleaved RNase H.
a. 5′-labeled synthetic PPT-RNAs of SFFV (left) and HIV-1 (right) were annealed and cleaved with recombinant M-MuLV or HIV-1 RT/RNase H in the absence or presence of ODN M or ODN A. Cleavage products were analyzed by 8M urea-containing PAGE (6%). Filled arrowheads correspond to the specific RT/RNase H cleavage sites, open arrowheads to additional cleavage sites. RNase T1-digested RNA (RNase T1) served as marker. M indicates the Decade Marker.
b. Titration of recombinant M-MuLV RT/RNase H in the presence of ODN M (50 nM) and 5′-labeled SFFV-PPT-RNA. Increasing amounts of MuLV RT/RNase H (1, 20, and 120 units in 20 μl assays) were used (left). 5′-labeled SFFV-PPT-RNA or control RNA without PPT (Con.) were hybridized with the ODNs and treated with and without MuLV RT/RNase H for in vitro cleavage as indicated and analyzed by 8M urea-containing PAGE (10%, right).
a. Cell-free SFFV virions (1×103 FFU in 20 μl) were permeabilized and incubated with the indicated ODNs (5 μM). RNA was extracted three days post-treatment and analyzed by quantitative real-time RT-PCR shown as bars (top) and RT-PCR shown as amplifications products on a 1.5% agarose gel (bottom).
b. NIH 3T3 cells were infected with SFFV pretreated with various ODNs. After three days the cells and supernatants were harvested and analyzed for SFFV RNA levels by quantitative real time RT-PCR (top) and RT-PCR (bottom) using GAPDH for comparison.
c. NIH-3T3 cells were infected with SFFV for 2 hours and then treated with three decreasing concentrations of ODN M, asPPT and three different PPT-specific siRNAs. RNA was isolated from supernatants three days post-infection and analyzed by real time RT-PCR.
a. ODN treatment of plasma from SFFV-infected mice in vivo and ex vivo. Different experimental regimens were utilized for in vivo (upper line) and ex vivo (lower line) studies. Symbols represent intravenous (i.v.) SFFV infection (⇓), mock-infection (), ODN treatment (τ), and bleeding (↓).
PCR was performed as indicated. 1. Mice infected with 200 μl containing 4×103 FFU SFFV for 5 days were treated i.v. with ODN M (1 mg corresponding to about 20 μM ODN M in the blood of a mouse) or PBS 4 hours post-treatment, plasma (100 μl) was recovered and analyzed by real time RT-PCR for SFFV RNA. 2. Plasma from a 5-day-SFFV-infected mouse was recovered and 100 μl were treated with ODN M (20 μM) for 4 hours at 37° C. ex vivo and then analyzed. 3. Plasma from a mock-infected mouse was treated with ODN M (20 μM) for 2 hours and then treated with 4×103 FFU in 20 μl for 4 hours. 4. Plasma from mock-infected mice was incubated with a combination of SFFV (4×103 FFU in 20 μl) and ODN M (20 μM) for 4 hours and then analyzed. The RNA levels were determined by real-time RT-PCR in the four experimental scenarios and are shown to the right.
b. Effect of ODN treatment following SFFV-infection in vivo. Mice infected with 200 μl containing 4×103 FFU SFFV for 5 days were treated with ODN M (1 mg), asPPT (0.4 mg), ODN A (1 mg) or PBS. Real time PCR for SFFV RNA was performed with plasma of the treated mice. 1. Mice were treated with ODNs twice at time 0 and 2 h. Viral RNA levels were analyzed at the beginning of the treatment (0 h) and 4, 8, and 16 hours post-treatment. 2. Treatment of 5-day-infected mice with ODNs at 0 h followed by bleeding at 0, 0.5, 1, 2, and 4 hours. 3. Infected mice were treated twice at two-hour intervals on days 0, 3, and 6 with ODNs. Bleeding was performed before and 4 hours after each double-therapy and SFFV RNA determined. 4. Treatment of 5-day-infected mice with ODNs every 12 hours up to 60 hours. Bleeding was performed prior to treatment.
a. SFFV virions pre-treated with ODNs for 2 hours were injected i.p. into mice. Subsequent treatments were at 2, 4 and 24 h post infection. 5 groups of 5 mice each were used: negative control (1), infection with PBS (2), ODN M (3), or ODN A treatment (4) and mock-infection with ODN M treatment (5). Bleeding was performed five days post-infection for analysis of the viral RNA. The relative RNA levels standardized to group 2 of fully infected mice (bars) as well as RT-PCR products of the individual mice (gel analysis) are shown.
b. Five mice each received 300 μl SFFV (1:100) i.p. supplemented with or without ODN M and 1 h later a second dose of ODN M (1 mg each). Survival was monitored until day 68. The spleen weights at death are indicated as numbers on the graph.
c. Mice infected with SFFV (4×102 FFU) i.v. received triple-treatments with ODNs at 2, 6 and 10 hours post-infection. Real-time RT-PCR analysis (graph) and RT-PCR (gel analysis) was performed with RNA from plasma beginning at day 5 and at the days indicated.
To demonstrate the effect of ODN, 20 μl containing either 105 Infectious Units, IU, or 104 IU FUGW-virus were used and applied with 25 μM ODN for 4 h in the vagina of C57BL/6 mice. The incubation was followed by a lavage of the vagina. Then lavage samples from the vagina were collected for RNA extraction using QIAamp viral RNA kit. RNA were eluted in 30 μl elution buffer. For reverse transcription, 2 μl RNA were used in 12.5 μl volume and 3 μl of the cDNA were used for real time PCR. Virus reduction is dependent on the ratio of ODN A to virus titer and the reduction is 2 fold for 104 IU.
With respect to mucosal conditions like the secretion in relation to the current hormone status, it is very likely that an optimization of the application form or dose will result in a much higher reduction of virus.
| Number | Date | Country | Kind |
|---|---|---|---|
| 05090299.8 | Oct 2005 | EP | regional |
| 05090303.8 | Nov 2005 | EP | regional |
| 06090079.2 | May 2006 | EP | regional |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12091605 | Sep 2008 | US |
| Child | 15086012 | US |
