Patent Application
20090326043
The present application claims priority under 35 USC 119(b) to European patent application EP 08075554.9, filed Jun. 13, 2008, which is incorporated herein by reference in its entirety.
The invention relates to an anti-viral therapeutic that inactivates the Human Immunodeficiency Virus (HIV) RNA by siDNA. The siDNA antiviral therapeutic is effective in the treatment and inactivation of cell free virus particles before infection and/or in the treatment and prevention of HIV infections inside the cell. The invention exploits the HIV RNase H activity of the reverse transcriptase which is essential for viral replication, causing premature cleavage and degradation of the viral RNA genome. This is achieved by partially double-stranded, hairpin-loop-structured oligodeoxynucleotides. These oligonucleotides comprise one strand which is fully complementary to the 3′-polypurine tract PPT of HIV RNA and creates a local DNA/RNA hybrid as substrate for the virion-associated RNase H. The oligonucleotide contains a second strand partially complementary to the antisense strand, which protects against nucleases. Both strands are connected by a linker of four thymidines. The presence of G-clusters can assist higher order structure formation within or between siDNA molecules and enhances the anti-viral function. Triple-helix formation between siDNA and the target RNA is a preferred effect. The siDNA is superior to siRNA because the formation of RNA-DNA hybrids is preferred over double-stranded DNA or double-stranded RNA, which forms as a tertiary structure in RNA genomes, siDNA is easier to synthesize and is more stable than siRNA, and siDNA leads to HIV particle inactivation outside the cell before infection, whereas siRNA has no such activity. It can be combined with other siDNAs targeted against various virus strains in a cocktail; it can also be combined with siRNA to target early and late steps in viral replication.
The publications and other materials referenced throughout the present application, including, among others patents, are incorporated herein by reference in their entireties. US Patent Publication 2009-0117179 to Moelling is specifically incorporated herein by reference in its entirety.
Human Immunodeficiency Virus (HIV) is a lentivirus of the retrovirus family, and can lead to acquired immunodeficiency syndrome (AIDS), a condition in which the immune system fails leading to susceptibility to a wide range of diseases. HIV infection is pandemic in humans and until the present time no cure or vaccine has been developed. Current anti-retroviral drugs are expensive and often ineffective in counteracting the disease, thus there is an enormous need for the development of new antiviral therapeutics for the treatment and/or prevention of HIV infection.
There are several ways to inactivate viral RNA genomes or RNAs such as messenger mRNAs inside the cell. One of these is the well-known antisense strategy, whereby a single stranded DNA oligonucleotide is directed to a target RNA and hybridizes by Watson-Crick based pairing. The RNA-DNA hybrid is recognized by an enzyme designated as ribonuclease H or RNA H, whereby H indicates the RNA-DNA hybrid substrates. It cleaves the RNA within the hybrid region (illustrated in
A second mechanism is based on a triple helix, whereby the target strand may be single or double stranded DNA and the third strand binds to this double stranded structure. Again one strand hybridizes to the target strand by Watson-Crick base pairing, whereas the third strand binds to Hoogsteen base-pairing. A triple helix is characterized by its resistance to cleavage by cellular enzymes and leads to inhibition of expression by unknown mechanisms, possibly translation arrest. The role of a triple helix inside the cell is unclear (illustrated in
A third mechanism is based on inactivating a target RNA by double stranded RNA, the so-called ‘short interfering’ siRNA. Here one strand of the siRNA is poly-hybridized to the target RNA by Watson Crick base pairing, the so-called antisense strand. A second RNA strand, the passenger strand, is fully complementary to the antisense strand and only transiently required for inactivation of the RNA. It is removed in a so-called RISC-complex. This mechanism of inactivation of the target RNA by siRNA is called silencing. The enzyme required for RNA silencing is designated as Argonaute. The discovery of this mechanism was awarded with the Nobel Prize in 2006. In all cases gene inactivation is achieved by small oligonucleotides about 20 nucleotides in length, which may or may not contain a linker region between the two nucleic acid strands (illustrated in
A fourth mechanism is the use of short interfering DNA (siDNA) to inactivate viral RNA genomes (illustrated in
Furthermore, it could be shown by the inventor that siDNA is able to induce silencing of an oncogenic retrovirus, the Spleen Focus-Forming Virus and prevent infection, cause delay of disease progression and lead to increased survival time (Matzen et al. Nature Biotechnol. 25, 669-674 (2007)). Furthermore the inventor showed in several cases that siDNA is superior to a single-stranded antisense effect (Jendis et al, AIDS Research and Human Retroviruses 12, 1161-1168 (1996), AIDS Research and Human Retro-viruses 14, 999-1005, (1998), Moelling et al, FEBSLetters, 580, 3545-3550 (2006), Matzen et al. Nature Biotechnol. 25, 669-674 (2007)). Thus the effect of siDNA has been demonstrated in several cases.
The four mechanisms described are all distinct from each other.
One problem to be solved with the present invention is to present an effective antiviral therapeutic against cell-free HIV particles and HIV infections using siDNA. This problem may be solved by the features of the independent claims.
An object of the invention is therefore to present siDNA oligonucleotides, capable of binding to one or more RNA target regions of HIV, as an antiviral therapeutic for the treatment and inactivation of cell free virus particles before infection and/or for the treatment and prevention of HIV infections inside the cell. The siDNA oligonucleotides comprise, consist essentially of or consist of an antisense-strand homologous to the viral RNA target and a second strand, partially complementary to the antisense-strand.
Another object of the invention is therefore to present siDNA oligonucleotides, capable of binding to one or more RNA target regions of HIV, as an antiviral therapeutic for the treatment and inactivation of cell free virus particles before infection and/or for the treatment and prevention of HIV infections inside the cell. The siDNA oligonucleotides comprise, consist essentially of or consist of an antisense-strand fully or almost fully homologous to the viral RNA target and a second strand, partially complementary to the antisense-strand forming a partially double stranded hairpin-loop-structured oligonucleotide, comprising G-clusters consisting of or including at least two G nucleotides in succession to allow tetrade or tetramer formation or tetra-helices or higher-ordered structures within the same siDNA oligonucleotide molecule (cis conformation) or through interaction with another siDNA oligonucleotide molecule (trans conformation).
Another object of the invention is to provide a method, respectively the use of siDNA oligonucleotides, or a combination of two or three siDNAs, as an antiviral therapeutic for the treatment and inactivation of cell free virus particles before infection and/or for the treatment and prevention of HIV infections inside the cell, capable of binding to RNA target regions of HIV or different HIV variants, wherein different siDNA oligonucleotides may be combined in a cocktail as pharmaceutical agent.
A further object of the invention is to provide a pharmaceutical composition as an antiviral therapeutic for the treatment and inactivation of cell free virus particles before infection and/or for the treatment and prevention of HIV infections inside the cell, comprising said siDNA oligonucleotides capable of binding to RNA target regions of HIV as an antiviral therapeutic.
The present invention describes a mechanism for the inactivation of an RNA target, preferentially of viral origin, such as retroviruses or retrovirus-like viruses or elements, using a short partially double stranded DNA. This so-called short DNA (sDNA) forms a hairpin loop structure. One arm serves as antisense arm targeted to the RNA of interest. A local RNA-DNA hybrid is formed, which is a substrate for RNase H. In the case of retroviruses, such as HIV, the RNase H is of viral origin, coded for the viral genome and is even present is cell-free virus particles. It is required inside the cell immediately after infection and therefore carried into the cell by the virus particle. As in the above-mentioned cases, the antisense arm is about 20 to 30 nucleotides long and the second DNA strand has been selected by Hoogsteen base pairing rules. The retroviral RNase H is pre-maturely activated by the siDNA for self-destruction of the HIV RNA genome. The viral RNA is inactivated at the site where the local RNA-DNA hybrid forms due to cleavage by the viral RNase H.
The RT/RNase H is located inside the virus particles and is carried into the cell during infection. During RT-mediated reverse transcription RNA-DNA hybrids are generated. The RNase H removes the RNA in the hybrids, but leaves the polypurine tract (PPT) RNA intact. The PPT is required as primer for initiation of second strand DNA synthesis. An oligodeoxynucleotide (ODN) that specifically binds the PPT of HIV-1 forms a local RNA-DNA hybrid that mimics a natural replication intermediate. This hybrid activates RNA hydrolysis by the HIV RT/RNase H and destroys the viral RNA template before DNA synthesis. A partially double-stranded oligo inhibits HIV-1 replication in infected cells, including drug-resistant strains of HIV. Furthermore, because the RT/RNase H is associated with virus particles, they can be inactivated outside of the cell and this renders the virus non-infectious. The effect of such oligos is sequence-specific and a partially double-stranded ODN is more inhibitory than a single-stranded antisense ODN. G-tetrades also appear to be important in the function of the oligos.
Therefore, siDNA oligonucleotides, capable of binding to one or more RNA target regions of HIV are claimed as antiviral therapeutics, wherein the siDNA oligonucleotides comprise, consist essentially of or consist of an antisense-strand homologous to the viral RNA target and a second strand, partially complementary to the antisense-strand forming a hairpin-loop structure. The siDNA oligonucleotides correspond to one or more HIV target regions of at least 20 nucleotides in length, wherein the antisense strand of siDNA is fully or almost fully (more than 80%, more preferably more than 90%) homologous to the target HIV viral RNA. Further, the second strand of the siDNA oligonucleotides is connected to the antisense strand through a thymidine linker, preferred—but not exclusively—4 nucleotides in length, and wherein, furthermore, the second strand is partially (40-60% homology) complementary to the antisense-strand and may be able to form triple helices by non-Watson-Crick base pairing with the viral RNA target strand.
Conserved target RNA regions are preferred due to the lower variation of DNA sequence between strains or variants, thus creating a higher likelihood of siDNA-RNA target pairing. Targeting a subunit of the transcriptase, and thus its silencing, would also reduce viral RNA replication efficiently. Furthermore, siDNA can also be targeted to cellular mRNAs coding for proteins essential for virus replication and indirectly prevent HIV replication.
Also a preferred embodiment of the invention is the use of a combination of two or three siDNAs as described herein as an antiviral therapeutic capable of binding to RNA target regions of HIV. HIV has a high genetic variability due to its fast replication cycle, high mutation rate and the potential generation of recombinant strains. Because of this a further preferred embodiment is the use of a cocktail of different siDNA oligonucleotides in a pharmaceutical agent to target different HIV variants that have or may have infected the patient.
The siDNA can be applied to an infected cell or an infected individual with a transducing agent. The transducing agent may be selected from the following group: the virus itself, a replicating HIV particle, which carries the siDNA into the cell during the process of infection, a liposome, transmembrane carriers, peptides. Most preferred is a cell transfection by using a lipid-based transfection reagent like Lullaby which is known from siRNA silencing. Other lipid based transducing agents are preferred, too.
It is an unexpected unique embodiment of the invention that siDNA acts early on the virus replication cycle and holds promise for the prevention of infection. The mechanism employed by this invention is a novel anti-viral approach in that virus particles can be inactivated outside the host cell, rendering HIV susceptible to treatment before infection of the host immune system.
A pharmaceutical composition prepared according to the invention is especially intended for the prevention of viral infection (by inactivation of the virus particle itself (outside the cell, before infection)), especially during sexual intercourse. 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). Another mode of application could be Pre-Exposure Prophylaxis (PrEP)—a treatment that would counteract a major route of infection in the third world.
One significant advantage of the invention over siRNA treatment is that inactivation of virus particles outside the cell cannot be accomplished by siRNA. The treatment of RNA viruses by siRNA is dependent on cell infection. No other HIV antiviral treatment known at this time shows destructive activity against cell free HIV particles.
This invention provides surprising and unexpected success in the inactivation of HIV both in vitro and in vivo via a novel anti-viral mechanism, as demonstrated in the examples below. As demonstrated in vitro, the invention leads to a significant reduction of plasma HIV RNA levels and a subsequent reduction in the infectivity of treated virions. As demonstrated in vivo, infection of a lentiviral HIV-analogue and HIV infection of a humanised immune deficient (huPBL/SCID) mouse model are reduced after treatment with the claimed antiviral therapeutic. Due to induced self-destruction of HIV—observed with clinical samples—premature activation rather than inhibition of a viral enzyme is an effective tool for antiretroviral control, and provides significant advantages over existing inhibition-based HIV therapeutics.
Under the term “homology” the following should be understood: An siDNA oligonucleotide according to the invention comprises, consists essentially of or consists of an antisense-strand which is more than 80% homologous, including more than 90% homologous, to the viral RNA target and a second strand, which is partially complementary to the antisense-strand forming a partially double stranded hairpin-loop-structure. The term “partially complementary” means a homology between 40 and 60% within the hairpin-loop, including about 50%
Under the term “G-cluster” the following should be understood: “G-clusters” according to the invention are motifs within a sequence of an siDNA oligonucleotide. A G-cluster is defined by at least 2 G nucleotides in succession (“GG”). Several G-clusters lead to tetrade or tetramer formation or tetra-helices or higher-ordered structures within the siDNA oligonucleotide molecule. Each G-cluster is separated from another G-Cluster by 1 to 20× nucleotides, wherein X is A, T, C or a single G, followed by A, T or C. General examples (among others) according to said definition are
G-tetrade formation may be beneficial consisting e.g. of GGXXGGXXXXGGG etc, whereby X represents basically other nucleotides than “GG”, such as A, T or C or a single G, as long as the single G is followed by A, T or C. The siDNA oligonucleotides according to the invention contain G clusters to allow tetrade or tetramer formation or tetra-helices by those sequences. The hairpin-loop structure may comprise or consist preferably of up to 9 non-self-complementary sequences at the 3′- and 5′-ends, followed by 6 base-pairing, 2 non-pairing, 3 pairing, 2 non-pairing, 3 pairing sequences, followed by 4Ts. A homology or sequence identity of 40 to 60% is preferred.
An isolated and purified siDNA oligonucleotide according to the invention also comprises, consists essentially of or consists of an antisense-strand which has more than 80% sequence identity to the viral RNA target and a second strand, which is partially identical to the antisense-strand forming a partially double stranded hairpin-loop-structure. The term “partially identical” means a sequence identity between 40 and 60% within the hairpin-loop, including about 50%.
The term “sequence identity” refers to a measure of the identity of the nucleotide sequences. In general, the sequences are aligned so that the highest order match is obtained. “Identity”, per se, has recognized meaning in the art and can be calculated using published techniques. (See, e.g.: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).
Whether any particular nucleic acid sequence, say an antisense-strand, is more than 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the viral target can be determined conventionally using known computer programs such as DNAsis software (Hitachi Software, San Bruno, Calif.) for initial sequence alignment followed by ESEE version 3.0 DNA/protein sequence software (cabot@trog.mbb.sfu.ca) for multiple sequence alignments. The term “partially identical” means a sequence identity between 40 and 60% within the hairpin-loop. The term “almost fully identical” means more than 80%, more preferably more than 90% identical.
The tetrade or tetramer formation or tetra-helices or higher-ordered structures within the siDNA oligonucleotide molecule results of an interaction of G-clusters within the same siDNA oligonucleotide molecule (so-called cis conformation) or through inter-action with another siDNA oligonucleotide molecule (so-called trans conformation). Both conformations (cis or trans) are possible.
Preferred specific examples according to the invention are siDNA oligonucleotides comprising the sequences SEQ ID NO 1 or SEQ ID NO 21 (framed sequence motifs are able to form tetrads, tetramers or tetra-helices or higher-ordered structures; bold nucleotides indicate G-cluster):
The invention is based on the cognition that a DNA strand has a thermodynamic preference to form an RNA-DNA hybrid over double-stranded DNA or double-stranded RNA. Thus the invention is based on the unexpected result that siDNA is of high preference (in contrast to siRNA) to form RNA-DNA hybrids; siDNA is therefore of advantage and a preferred object of the invention. Furthermore siDNA acts against the newly infecting incoming viral RNA, 2 to 3 days before siRNA. Thus a virus infection can be prevented by siDNA only. In contrast thereto, interferon-induction is a risk for siRNA and reduced or absent for an RNA-DNA hybrid or double-stranded DNA. Furthermore, siDNA is preferred due to the fact that DNA is easier to synthesize and more stable.
Also, an siDNA-containing pharmaceutical agent can also contain ribonucleotides, siDNA/RNA chimeras (as ODN AM, SEQ ID NO 21), or can be combined with siRNAs. However, the RNase H cleavage site needs to consist of/comprise a local RNA-DNA hybrid.
siDNA is also superior to a simple antisense Oligodeoxynucleotide, because it is more stable during uptake and inside the cell, and is therefore more effective. It acts earlier than antisense DNA, and can target incoming RNA, while antisense DNA targets mRNA. siDNA targets viral RNA before replication while siRNA is effective only late during replication targeting the mRNA.
Furthermore, siDNA oligonucleotides can be stabilized by base modifications. Such base modifications entail 2′-O-methylated nucleotides and phosphorothioates at the ends and in the central linker regions to protect against nucleases and to increase stability and longevity both in vivo and in pharmaceutical preparations. Other chemical modifications are also envisaged that may improve the stability of the siDNA oligonucleotides.
Materials and Methods used in the Following Examples
We obtained HIV-infected blood from 20 patients from the Zurich University hospital with a broad range of history, HAART, ART or no therapies, high or low viral loads and CD4 counts (see
Plasma was extracted from 10 ml of blood of HIV-1 infected patients (available through the Department of Infectious Diseases of the University Hospital, University of Zurich, Switzerland) using the BD Vacutainer CPT method (Becton Dickinson, USA). Overview of the patients' isolates from Zurich is presented in
Primary Peripheral Blood Mononuclear Cells (pPBMCs) and plasma were extracted from fresh blood obtained from healthy donors (available through the Blood Donation Center, Zurich, Switzerland) using Ficoll Paque Plus methods (GE Health Technologies, USA). Sucrose-purified virions from patient 8898 used for cleavage assays were a kind gift from Dr. J. Boeni from the Swiss National Center for Retroviruses, Zurich, Switzerland.
The ODN A (SEQ ID NO 1) consists of a 25 mer antisense strand of PPT, and a 25 mer passenger strand, connected by four thymidines (
ODN AM (SEQ ID NO 21) is the methylated version of the ODN A (
The ODNs were purchased from Operon (Germany) or Integrated DNA Technology (USA).
Viral RNA was isolated from plasma or cell supernatants using the QIAamp Viral RNA Mini Kit (Qiagen, Germany) according to the manufacturer's directions.
Determination of HIV RNA by qRT-PCR
HIV-1 RNA levels were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using RNA extracted from plasma or cell-culture supernatants as indicated in the legends. Isolated viral RNA was reverse transcribed using High-Capacity cDNA Archive Kit (Applied Biosystems, USA) and qRT-PCR was then performed using the Taqman Universal PCR master mix and the ABI 7300 instrument from Applied Biosystems. Location of the PCR primers and probes on the HIV-1 genome are schematically indicated in
HIV-1 virions, derived from 20 μl of patient plasma were incubated with different concentrations of ODNs. At indicated time points viral RNA was purified and the amount of undigested RNA was quantified by qRT-PCR using sets of primers covering the PPT region of the HIV genome, nef or UTR. Samples (3 μl) obtained from ARRRP were diluted to 20 μl in RPMI containing 20% FBS and treated similarly. qRT-PCR was performed using nef-primers.
100 μl of HIV-containing plasma from patients (Zurich isolates) or 5 μl of the African isolates were treated with either 1 or 5 μM of ODNs for 1 or 4 hours. 1×106 Phyto-hemagglutinin (PHA)-activated PBMCs were infected with pre-treated HIV-1 virions and grown in culture at 37° C. for 3 days in a total volume of 5 ml of RPMI-1640 with 20% FBS in the presence of interleukin 2 (IL-2, 20 U/ml, Zeptometrix, USA). At day 3, 3 ml of RPMI-1640, 20% FBS and IL-2 were added. At day 7 HIV RNA levels were analyzed in the supernatants using gag-specific qRT-PCR (
RNA isolated from HIV-1 infected patients and cell culture supernatants was reverse transcribed using random primers and High-Capacity cDNA Archive Kit (Applied Biosystems, USA) and the cDNA was sequenced in the HIV-1 PPT-flanking genomic regions using forward primer CCTCAGGTACCTTTAAGACCAATGAC (SEQ ID NO 8) and reverse primer CCCCTGGCCCTGGTGTGTA (SEQ ID NO 9). Thermal cycle sequencing was performed on a thermocycler (Touchgene from Genius, USA) for 25 cycles with either the forward or reverse primer using 10 ng of DNA templates. Purified sequencing products were run on an automated ABI 3100 genetic analyzer (Applied Biosystems). Sequences of both strands were analyzed and edited individually using the software package Sequencer 4.7 (Gene-Codes, USA), assembled as contigs with Sequence Assembler (Applied Biosystems) and subsequently aligned through Clustal-W algorithm of MacVector 7.1 software program (Accerlerys, USA).
HIV-PPT RNA was in vitro transcribed, dephosphorylated and 5′-phosphorylated. Different ODNs (10 nM) and HIV-PPT RNA (10 nM) in RT buffer were annealed (2 min 90° C., 10 min 37° C.) and cleaved with 0.1 U/μl RT/RNase H (GE Healthcare, USA) for 30 min at 37° C. The samples were subjected to electrophoresis in 10% polyacrylamide containing 8 M urea, together with HIV-PPT RNA partially digested with RNase T1 (Ambion) as described by the manufacturer.
FUGW is a lentiviral self-inactivating vector with a VSV-G coat, which allows transduction of many cell types but does not replicate. This lentiviral vector contains the PPT sequence that is identical to that of HIV. For generating high titer lentiviral vectors, 7×106 human embryonal kidney (HEK) 293T cells in 10 cm plates were cotransfected with cFUGW, pCMVΔR8.9, and pHCMV-G (5 μg each) using lipofectamine 2000 (Invitrogen). Six hours post-transfection 8 ml fresh medium replaced the original medium. Two days post-transfection the culture media was collected and filtered through a 0.45 μm filter. The filtrated medium was then ultracentrifuged twice for 90 min using Polyallomer tubes. Titers of viral preparations were assessed by infecting 5×105 C81-66 cells with serial dilution of the virus and determining the fraction of GFP-positive cells one day after infection by FACS. Titers of 5-9×107 transducing units (TU) per ml were routinely obtained.
FUGW virions (104 TU) were either treated with different concentrations of ODN A (0-25 μM) for titration or different ODNs for 4 h at 37° C. These virions were also titrated using either 104 IU or 105 IU with 5 μM ODN A for also 4 h at 37° C. Viral RNA was then extracted using the viral RNA mini kit (Qiagen) and eluted with 50 μl elution buffer. Then 5 μl RNA was used for cDNA synthesis in a reaction volume of 12.5 μl and 5 μl of the cDNA was used for qPCR.
Both cell lines VK2/E6E7 and Ect1/E6E7 were purchased from American Type Culture Collection (ATCC). The VK2/E6E7 (ATCC CRL-2616) cell line was established from normal vaginal mucosal tissue taken from a premenopausal woman undergoing anterior-posterior vaginal repair surgery. The ectocervical Ect1/E6E7 (ATCC CRL-2614) cell line was established from normal epithelial tissue taken from a premenopausal woman undergoing hysterectomy for endometriosis. These two cell lines were seeded in a 24-well-plate at a density of 1×105 cells/well in Keratinocyte-Serum Free medium (Invitrogen) supplemented with 0.1 ng/ml human recombinant epidermal growth factor (EGF, Sigma-Aldrich), 0.05 mg/ml bovine pituitary extract (Invitrogen), and additional 44.1 mg/ml calcium chloride (final concentration 0.4 mM). Cells were infected with 104 IU of FUGW resulting in multiplicity of infection (MOI) of 0.1, for 2 days at 37° C. Cells were washed twice with PBS and then genomic DNA (gDNA) was extracted using the blood DNA mini kit (Qiagen) and eluted in 30 μl elution buffer. For qPCR analysis, 100 ng of total gDNA was used in a reaction volume of 25 μl. Primary vaginal cells extracted from mouse vaginal lavage fluid were cultured in epithelial cell medium (ECM). ECM consists of equal volumes of phenol-red-free DME (Sigma-Aldrich) and Ham's F-12 medium (Invitrogen) supplemented with 10% FCS, 100 mg/ml streptomycin, 100 IU/ml penicillin, 1 mmol/ml L-glutamine, and 10 ng/ml EGF. The vaginal cells at a density of 5×104 cells in 500 μl volume were infected with 104 FUGW TU corresponding to a MOI of 0.2 for 1 day at 37° C. Cells were washed twice with PBS and then gDNA was extracted and eluted in 30 μl elution buffer. For PCR, 40 ng of total gDNA was used in a reaction volume of 25 μl.
Six- to eight-week old female C57BL/6 mice purchased from Harlan (Zeist) were used throughout these studies. Animal studies were performed according to Swiss Animal Rights in the animal facilities of the Institute of Medical Virology, university of <Zurich, with permission by the Zurich-Veterinary-Office (213/00). Mice were kept in conventional conditions with full access to food and water. All mice were treated s.c. with 2.5 mg of progestin (Depo-Provera, Pfizer) to synchronize the estrus cycle. One week later, the progestin-treated mice were anesthetized with isoflurane (ABBOTT AG) and challenged with an intravaginal inoculum of 20 μl 3% carboxymethyl cellulose sodium (CMC) medium (Sigma-Aldrich) containing 104 IU FUGW with or without 25 μM ODNs in. Four hours later the mice were euthanized and vaginal lavage with 100 μl of sterile PBS was performed. Viral RNA was then extracted from these vaginal lavage fluids using the viral RNA mini kit (Qiagen).
For qRT-PCR RNA was reverse transcribed into cDNA using the High Capacity cDNA Archive Kit (Applied Biosystems) according to the manufacturer's instructions. Primers and Probes flanking the PPT sequence of FUGW are: 5′-GAGGAGGTGGGTTTTCCAGT-3′ (forward) (SEQ ID NO 10), 5′-GGGAGTGAATTAGCCCTTCC-3′ (reverse) (SEQ ID NO 11), and -FAM-5′-ACCTTTAAGACCAATGACTTACAAGGCAGC-3′-TAMRA (probe) (SEQ ID NO 12); for mouse glyceraldehyde-3 phosphate dehydrogenase (mGAPDH), 5′-CTTCACCACCATGGAGAAGGC-′3 (forward) (SEQ ID NO 13), 5′-GGCATGGACTGTGGTCATGAG-′3 (reverse) (SEQ ID NO 14). FAM-5′-CCTGGCCAAGGTCATCCATGACAACTTT-3′-TAMRA (SEQ ID NO 15); for human glyceraldehyde-3 phosphate dehydrogenase (hGAPDH), 5′-GTTCCAATATGATTCCACCC-3′ (forward) (SEQ ID NO 16), 5′-GAAGATGGTGATGGGATTTC-′3 (reverse) (SEQ ID NO 17), FAM-5′-CAAGCTTCCCGTTCTCAGCC-TAMRA-3′ (probe) (SEQ ID NO 18). All primers and probes were purchased from Microsynth. The cycling conditions were: 50° C. for 2 min (1 cycle), 95° C. for 10 min (1 cycle), 95° C. for 15 sec and 60° C. for 1 min (50 cycles). The results are presented in the figures as FUGW RNA or DNA corresponding to absolute copy numbers per assay.
Viral RNA levels after treatment were compared using analysis of variance for repeated measures with Bonferroni post-hoc test applied to logarithmically trans-formed data. SPSS 13.0 (SPSS Inc. IL) was used for statistical analyses.
The statistical significance of the antiviral activity of ODN A and ODN AM in virions and in vitro was determined by student's T-test and the results were expressed as mean±SEM. (error bars in graph). These P-values were for two-tailed significance test. For the in vivo studies, viral RNA levels after treatment were compared by analysis of variance with Bonferroni post-hoc test applied to logarithmically trans-formed and PBS normalized data using SPSS 13.0 (SPSS Inc. IL). Differences were considered to be significant at P<0.05.
Analysis of Humanized SCID Mice with HIV In Vivo
For the evaluation of an antiviral effect of ODN A in vivo, we tested the inhibition of viral growth in a mouse model, with human peripheral blood lymphocyte grafted to severe combined immunodeficiency mice, the huPBL/SCID-mouse model (Balzarini et al, 1996, Mol Pharmacol, 50:394-401). We designed a regimen, which ensured a high ODN A concentration during the test period by continuous treatment. HIV and ODN A were applied together at day 15 after engrafting SCID mice with huPBL, followed by administration of ODN A every one to two days up to day 28, when HIV is usually detectable in HIV-infected huPBL-SCID mice. Three groups were used: HIV-infected mice treated with ODN A (n=6) or PBS (n=3) as negative control as well as non-infected, non-treated mice (n=4). No visible toxicity or unusual behavior or appearance was observed for any of the mice. At day 28 the mice were sacrificed and the p24 levels in plasma were determined by a p24 ELISA.
An ODN has been shown to have in vivo efficacy against a murine oncogenic retro-virus. The virus causes splenomegaly in mice and we were able to demonstrate reduction of disease progression. However, a murine oncogenic retrovirus does not reflect the situation of primary blood-borne HIV. We designed this ex vivo study to be as close to the human situation, it builds on the unique property that virus can be targeted before entering a cell. It therefore intervenes earlier than the inhibitors of reverse transcriptase, which only work inside of a cell.
The invention leads to surprising and unexpected data, in particular through clinical studies using plasma of HIV-1 infected patients and the antiviral potential of ODN. Here we demonstrate that HIV particles present in the plasma of infected individuals from Zurich and HIV field isolates from Africa including two drug-resistant strains can be treated before entering a cell with an ODN for activation of the viral RNase H and self-destruction of the virus. Plasma of 20 HIV-1-infected patients from Zurich and 10 HIV-1 isolates from Africa and drug-resistant strains were processed for ex vivo treatment. HIV-RNA of cell-free virions treated with ODN in the plasma was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (
ODNs were used to target the RNase H in HIV particles. The ODNs are designed to bind to the PPT of HIV-1 IIIB. As controls we used a scrambled ODN Sc or buffer PBS. The sequences of the PPT and ODNs are shown in
In order to verify the molecular mechanism of the treatment, HIV-PPT RNA together with the ODN A was allowed to form a local hybrid as substrate for the RNase H and cleavage of the RNA moiety. This is demonstrated with in vitro transcribed radioactively end-labeled RNA and virion-associated RT/RNase H from a sucrose gradient-purified patient-derived HIV preparation. The cleavage site of the viral RT/RNase H was verified with recombinant RT/RNase H and proven to correspond to the natural cleavage site, 5′ of the ACU sequence (
In order to establish the conditions required for treatment of HIV in blood, we isolated plasma from the blood of healthy donors, spiked it with the cell culture-adapted strain HIV-1 IIIB, and treated it with ODNs for up to 24 hours in vitro. The amount of non-hydrolyzed RNA was determined and shown to be significantly reduced with time and dose (
20 HIV-1-containing blood samples of patients from Zurich with different histories were analyzed in this study immediately after isolation. The samples were supplied with information on plasma viral loads and CD4+ counts determined prior to this study (
The levels of initial viral RNAs of the patients varied. Several parameters contribute to the variability, such as the patients' histories, the sequences of the primer binding sites used for the PCR reaction, degree of the complementarity of the ODN A to the PPTs (see below) as well as components of the blood.
In order to reduce some of the variations, we tested the samples for infectivity as a second independent read-out, which correlates with viral RNA loads. To establish the conditions for infectivity of HIV-1-containing plasma, we treated one sample (patient 3) with ODN A for 4 h, infected normal human pPBMCs and cultured them for 7 days (
In order to examine the antiviral effect of ODNs in the absence of blood factors and differences in viral titers, we used the supernatants from these infected cell cultures (first passage), matched the viral inputs to identical RNA copy numbers, treated them with ODNs and infected human pPBMCs. The supernatants (second passage) were analyzed after 7 days, showing even more pronounced the ODN A-mediated inhibition (
The PPT sequences targeted by the ODN A were determined by using RT-PCR-amplified products from selected patients (
We then analyzed well-characterized HIV-1 African primary field isolates and strains resistant against the protease inhibitor Saquinavir (Saqi) or the nucleoside analogue AZT (Azt) from the AIDS Reagent Program. These samples differed from Zurich isolates, since they were supplied after passaging in primary PBMCs. Therefore we present them as a separate group.
After demonstrating dose- and time-dependence with an HIV-1 isolate from Rwanda (Rw,
The viral load in HIV-infected plasma samples from patients can be reduced by activation of viral RNase H by an ODN, demonstrating the effectiveness of the invention as an antiviral treatment. This leads to self-destruction of the virus particles. All ODN A-treated samples had a statistically significant decrease of the mean value of viral RNA compared to the control ODN Sc- and PBS-treated groups. Cell-free virions in plasma contained significantly less intact HIV-RNA upon treatment with ODN (p=0.000006), and their infectivity was decreased 52-fold (p=0.0014). In 39% of the Zurich samples infectivity was reduced more than 10-fold, in 33% more than 100-fold and in 28% more than 1000-fold (
Human immunodeficiency virus (HIV) has been shown to undergo self-destruction upon treatment of cell-free virions with partially double-stranded oligodeoxynucleotides targeting the polypurine tract (PPT) of the viral RNA in the virus particle. The ODN forms a local hybrid with the PPT activating the viral RNase H to prematurely cleave the genomic RNA. Here the self-destruction of a recombinant lentivirus harboring the PPT of HIV in a mouse vagina model is described. It can be shown a decrease in viral RNA levels in cell-free virus particles and reduced reverse transcribed complementary DNA (cDNA) in virus-infected human and primary murine cells by incubation with ODNs. In the vagina simultaneous, prophylactic or therapeutic ODN-treatments led to a significant reduction in viral RNA levels.
We tested an application of ODN against a lentiviral vector applied to the mouse vagina as a model for sexual transmission. The lentiviral vector FUGW (Flap, ubiquitin promoter, GFP and WRE vector) contains the HIV-PPT sequence, which is essential for its single replication round. Here we demonstrate that FUGW present in the mouse vagina or in human vaginal or cervical cell lines can be treated with ODN A. We applied chemical modifications of the ODN such as phosphorothioates or 2′-O methyl groups to protect against nucleases and increase stability. In all cases, statistically significant reduction of FUGW virus RNA copies was observed with ODNs compared to their respective randomized sequence serving as negative controls. The approach we are using differs from previous ones, since it is based on the activation of a retroviral enzyme for destruction of the virus, instead of inhibition.
In order to establish an animal model for the antiviral activity of ODN we used the lentiviral vector FUGW (
To demonstrate ODN A-mediated hydrolysis by the RT/RNase H, we cleaved labelled PPT-RNA, which contains 167 nucleotides of the PPT region of FUGW. The PPT-RNA was hybridized to ODNs and incubated with RT/RNase H for 0.5 h at 37° C. In parallel the PPT-RNA was sequenced by RNase T1 to determine the specific cleavage site. Only ODN A and ODN AM, but not ODN S2 or ODN S2M led to specific cleavage 5′ to ACU, which is the natural cleavage site (
To test for cleavage of viral RNA in lentiviral particles, purified FUGW virions were treated with different concentrations of ODN A for 4 h at 37° C. Then the viral RNA was purified and determined by qRT-PCR using primers flanking the PPT. These primers do not yield an amplification product, when the genomic RNA has been cleaved by RT/RNase H upon binding of ODN A to the PPT. The viral RNA levels in the virions were significantly reduced in a dose-dependent manner. With 5 μM ODN A the reduction was 71% (p<0.01) and was used for further experiments (
FUGW transduces cells through one round of replication leading to provirus-containing cells, which then express GFP. However, no new virus progeny is produced from these cells, since FUGW is replication defective.
In order to analyze the effect of ODNs in human vaginal cells, we transduced the human vaginal (VK2/E6E7) and cervical (Ect1/E6E7) cell lines with FUGW. The cells were tested in the absence and presence of different ODNs. Genomic DNA (gDNA) was purified from the infected cells after 2 days incubation at 37° C. and analyzed by FUGW-specific qPCR (
Next we used murine primary vaginal cells (VC), which were obtained by lavage of mouse vagina and infected them with FUGW using different concentrations of ODN A from 0.05 to 5 μM (
In order to establish the conditions for ODN treatment in the in vivo model, we applied three increasing doses of FUGW (103, 104 and 105 IU) into the vagina in 20 μl containing 3% carboxymethyl cellulose, in order to prevent leakage of the virus. After 4 h infection, a vaginal lavage was performed, viral RNA purified and genomic FUGW RNA detected by qRT-PCR using PPT-flanking primers. The lavage fluid contained FUGW RNA levels, which correlated with the dose of the applied lentivirus (
In order to exclude the possibility that ODN A interfered with the RT-PCR system, we performed a control. We used purified FUGW RNA from 104 TU and added increasing ODN A concentrations and subsequently purified the amount of RNA. It was then tested by RT-PCR and amplification was only weakly affected and this effect was independent on the amount of ODN A (
For the evaluation of ODN-mediated inhibition of FUGW in vivo we decided to apply three different treatment regimens, namely co-application, prophylactic and therapeutic regimens. First FUGW and 50 μM ODN A were co-applied and FUGW RNA levels were determined after 2 and 4 hours. A significant four- to sevenfold decrease (P<10−6) of viral RNA levels in the presence of ODN A was observed (
Here we are demonstrating the role of the RNase H in the antiviral effect of ODNs in the mouse vagina. We first demonstrated the cleavage of the PPT-RNA in presence of ODN A and ODN AM in vitro and mapped the cleavage site (
In the lentiviral mouse vaginal model, we are evaluating different therapeutic regimens. We are treating mice first by applying FUGW and ODNs simultaneously (
The data of all in vivo experiments in the mouse vagina are summarized and statistically analyzed in Table 1. The number of mice, the mean RNA level and standard error, the lower and upper bound of the 95% confidence interval as well as P-values for each combination of ODNs are shown. P<0.05 are considered as significant. From the distribution of differences the percentage of responders with a more than 10-fold reduced FUGW RNA levels were calculated (n.a., not applicable).
The use of ODN to counteract HIV infection is a novel approach, which is based on the activation rather than the inhibition of a retroviral enzyme. The activation of the viral RNase H and thereby induced self-destruction of the RNA by treatment of virus particles through this approach demonstrates an antiviral therapeutic for the inactivation of viral infectivity outside of the cell. One of the most prominent examples where this is relevant would be prevention of sexual and mother-to-child transmission.
To further characterize the ODN we analyzed firstly induction of resistant mutants by ODN A and secondly its effect on HIV in a humanized mouse model. Rapidly emerging drug resistant mutants are a problem, yet we are targeting the highly conserved PPT which coevolved with the reverse transcriptase (RT) and hypothesized that it might be less prone to mutations. To analyze this we determined the dose-dependence of the antiviral activity of ODN A on the human T-cell line C81/66-45. The cells were infected with HIV-1 for 30 minutes, washed, treated with increasing concentrations of ODN A (0.2, 1, and 5 μM final concentration) or the scrambled control ODN S2 (5 μM), and viral growth was allowed to continue for 4 days at 37° C. (
Then we tested the effect of a continuous treatment with ODN A to further reduce the viral load. We established conditions for serial passages to maintain HIV without treatment and then performed serial passages in the presence of 0.1 or 2.5 μM ODN A and PBS as control (
In order to test, whether ODN A treatment induced mutations we performed medium-term serial passages for three months in the presence of ODN A. We applied the same regimen as before for each passage, used 50% of the supernatant at day 6 for the next passage, and applied in parallel to ODN A also an inhibitor to determine whether we could induce resistant HIV mutants and possibly escape mutations. The RT inhibitor Foscarnet known to induce specific mutations in its target pol gene of the RT was used as a positive control and PBS as negative control. Sixteen passages were performed. Following an initial drop in the viral load in the supernatants for ODN A and Foscarnet the titer reached a plateau after passage 10 (data not shown). Samples of passages 1, 5, 7, 10, and 14 were analyzed for nucleotide variations in the 3′ PPT region and the pol gene. We did not detect any mutants in the 3′ PPT or its flanking regions as well as in pol for ODN A and PBS treatment (
For the evaluation of an antiviral effect of ODN A in vivo, we tested the inhibition of viral growth in the huPBL/SCID-mouse model. Based on the results described above and published previously we designed a regimen, which ensured a high ODN A concentration during the test period by continuous treatment. HIV and ODN A were applied together at day 15 after engrafting SCID mice with huPBL, followed by administration of ODN A every one to two days up to day 28, when HIV is usually detectable in HIV-infected huPBL-SCID mice (
Mutagenesis of HIV during serial passage in combination with ODN and the effect of ODN against HIV in a humanized mouse model were analyzed. ODN A inhibits growth of HIV in vitro in a single passage by 100- to 1000-fold. When applied continuously a more than 105-fold reduction is achieved within two passages at 2.5 μM, and in four passages at 100 nM, without using a transfection reagent. Interruption of ODN A treatment allowed virus to grow—but it remained sensitive and could be suppressed again (
This demonstrates an unexpected advantage over other HIV treatments such as Foscarnet. Using ODN mutagenesis of HIV does not occur as rapidly, and thus HIV remains sensitive to treatment. The in vivo efficacy of ODN A and sequence stability of the PPT are important prerequisites for further possible applications of ODN A. Since we are specifically inactivating the cell-free virus particles with ODNs, a mode of application could be Pre-Exposure Prophylaxis (PrEP) e.g. in form of a microbicide—against a major route of infection in the third world.
| Number | Date | Country | Kind |
|---|---|---|---|
| 08075554.9 | Jun 2008 | EP | regional |
