Patent Application
20100311809
The invention relates to a vector system a and a composition for treatment of virally infected cells.
Enteroviruses such as coxsackievirus, poliovirus and echovirus are small non-enveloped viruses belonging to the picornavirus family. They possess a single-stranded RNA genome in positive orientation that acts directly as mRNA in infected cells.
Picornaviruses are separated into nine distinct genera and include many important pathogens of humans and animals. The diseases they cause are varied, ranging from acute “common-cold”-like illnesses, to poliomyelitis, hepatitis to chronic infections in livestock like food-and-mouth disease. Two main categories are enteroviruses and rhinoviruses.
Thus, Picornaviruses are of high clinical relevance. However, currently there is no specific therapy available.
Coxsackievirus B3 (CVB3), a member of the enterovirus group of the Picornaviridae, is one of the most commonly identified infectious agents associated with acute and chronic myocarditis, and can also mediate infectious pancreatitis and meningitis. The virus is especially critical for newborn and babies.
Acute enterovirus myocarditis may not lead to initial mortality. In some cases, acute myocarditis can persist chronically and develop into a dilated cardiomyopathy (DCM), which is one of the most frequent causes of heart transplantation. In biopsies of DCM patients both persistent and latent enterovirus infections were detected. Cultured human foetal heart cells infected with CVB-3 showed completely lysed myocytes within a few days, whereas myocardial fibroblasts survived and multiplied. Continuous production of CVB-3 indicated a carrier state infection of human myocardial fibroblasts.
Currently, enterovirus myocarditis is treated non-specifically by conventional supportive methods, since no effective antiviral therapy is available. CVB load, replication and persistence are directly associated with cardiac injury and progression of the disease. Direct cytopathogenic effect of CVB in vitro, and the induction of cardiac injury in immunodeficient mice in vivo, supports the significance of direct virus-mediated cardiac injury in disease pathogenesis. Specific targeting of CVB in viral myocarditis will therefore, not only abrogate virus-mediated direct cardiac damage, but will also block immune response-mediated damage by blocking viral spread to uninfected tissue.
The host cell receptor for group B coxsackieviruses is the coxsackievirus-adenovirus receptor (CAR). This transmembrane protein is involved in the formation of tight junctions in the endothelium and in cell adhesion. Group B coxsackieviruses were shown to bind initially to the Decay Accelerating Factor (DAF) as a co-receptor, which activates intracellular signalling and transports the virus to the tight junctions, where it becomes internalized by CAR.
The CAR mediates cellular attachment for adenoviruses subtypes A and C-F and is essential to permissive infection of all 6 serotypes of CVB. CAR is a member of the immunoglobulin superfamily consisting of two extracellular Ig-like domains (D1 and D2), a transmembrane domain, and an intracellular tail of variable length. The N-terminal D1 domain has been shown to bind both adenovirus fiber knob protein and the canyon structure of CVB capsids.
Soluble decoy viral receptors have been found to efficiently inhibit the infection of rhino-, measles- and adenoviruses.
Various soluble variants of CAR (sCAR) have been detected, which originate by alternative splicing. Soluble (s) CAR proteins inhibit CVB infection of susceptible target cells in vitro and in vivo. The interaction of CVB3 with the sCAR leads to formation of altered (A) particles which are characterized by loss of VP4 from the virion shell and coincident irreversible loss of infectivity. It can be assumed that sCAR acts as a decoy and saturates epitopes on the virus surface that are essential for the interaction with the cellular receptor.
However, severe side effects with increased cardiac inflammation and heart injury have been observed following treatment of CVB3-infected mice with CAR4/7, a native sCAR variant with an intact CVB3-binding D1 domain, half of the D2 domain, and 23 amino acid long C-terminus.
Previous investigations have shown that dimeric sCAR, expressed as an immunoglobulin Fc-region fusion protein, has reduced systemic clearance and increased virus neutralizing capacity relative to monomeric sCAR and do not induce undesirable side effects.
To enhance solubility and extend half-life, the extracellular domain of CAR was fused to the Fc domain of human immunoglobulin G1 (IgG). Basically, sCAR-Fc proved to be a potent antiviral tool as it was suitable to protect cells and animals from CVB-3 infection. Under therapeutic conditions, however, when animals were treated with sCAR-Fc after CVB-3 infection, the antiviral efficiency decreased substantially.
Another promising new strategy for the inhibition of viruses is the application of RNA interference (RNAi). This evolutionary conserved mechanism of post-transcriptional gene silencing is triggered by double-stranded RNA molecules, which induce sequence-specific degradation of a target RNA.
In mammalian cells, double-stranded RNA molecules shorter than 30 nucleotides, known as small interfering RNAs (siRNAs), are usually employed to trigger RNAi without inducing an unspecific interferon response. The siRNAs become incorporated into a protein complex referred to as the RNA-induced silencing complex (RISC), in which the antisense strand of the siRNA acts as a guide to the target RNA, while the sense strand is degraded. After binding of activated RISC, cleavage of the target RNA by the Argonaut 2 protein is initiated. For the design of active siRNAs, thermodynamic features of the duplex as well as accessibility of the target region have to be taken into consideration.
Among other applications, RNAi has been found to efficiently inhibit viruses and clinical trials to treat infections with the respiratory syncitial virus, the human immunodeficiency virus and the Hepatitis B Virus have already been initiated. Successful application of RNAi for various enteroviruses was reported, including the inhibition of poliovirus, enterovirus 71, and CVB-3.
Just like for the sCAR-Fc approach, pre-treatment with RNAi efficiently protected cells from CVB-3 infections, but the antiviral activity was substantially lower when the curative approach was carried out with an ongoing CVB-3 infection.
In view of the above it is therefore one object of the invention to create an in vivo delivery system that would express a transgene, preferably a soluble receptor protein with high expression levels and without having undesirable side effects.
It is another object of the invention to provide a vector system and a composition for treating virally infected cells, especially cells infected with a virus of the Picornavirus family.
According to an exemplary embodiment of the invention the vector system comprises at least one viral vector and at least one regulable expression cassette inserted in said viral vector. The viral vector system facilitates a high and steady expression of the transgene. The regulable gene expression cassette governs the transgene expression. Simultaneously it provides the possibility of turning off the transgene expression in order to avoid potential side effects.
It is preferred that the at least one regulable expression cassette comprises at least one transactivator, at least one promoter and at least one nucleotide sequence coding for a transgene. Thus, all regulation systems are located on one single vector genome.
The cassette can be inducible, preferably by Doxycycline or any other known applicable inducer.
The expression cassette can be inserted into any region of the viral vector, preferably into the E-1 region of said vector.
The transactivator is preferably a second generation tetracycline-depended reverse transactivator (rtTA-M2) and the promoter a second generation tetracycline-depended response promoter (tight 1). The nucleotide sequence encodes preferably a soluble receptor protein or a part of it.
In an exemplary embodiment the vector system comprises two expression cassettes, whereby one expression cassette is regulated in a constitutive manner and/or a second expression cassette is regulated in an inducible manner.
It is also preferred that at least one expression cassette, especially the constitutive cassette, comprises at least one transactivator, preferably a second generation reverse tetracycline transactivator rtTA-M2, and at least one promoter, preferably a CMV promoter or a tissue specific promoter.
It is furthermore preferred that at least one expression cassette of the vector system, especially the inducible cassette, comprises at least one promoter, preferably a second generation tetracycline response promoter tight1, and at least one nucleotide sequence coding for a transgene, preferably for a soluble receptor protein or at least a part of a soluble receptor protein.
It is also possible to use instead of the components of the Tet-ON regulable system (rtTA-M2 and tight 1) the components of the Tet-OFF regulable system by using the tetracycline depending transactivator (tTA). It is also conceivable to use a transpressor as for instance the tetracycline transcriptional surpressor (tTs) instead of the transactivator.
The at least one transgene nucleotide sequence encodes for a soluble receptor protein or parts of it. The soluble receptor protein is preferably selected from a group comprising soluble Coxsackie-Adenovirus-receptor sCAR, rhinovirus receptor ICAM-1, human herpes virus receptor CD46, enterovirus receptor CD55, human poliovirus receptor, HIV receptor CD4 and HIV co-receptors CCR5 and CXCR4.
In one exemplary embodiment the transgene nucleotide sequence enodes for a fusion protein. The fusion protein comprises preferably a domain of the soluble receptor protein as the extracellular domain of the human soluble Coxsackie-Adenovirus-receptor (sCAR), rhinovirus receptor ICAM-1, human herpes virus receptor CD46, human poliovirus receptor, enterovirus receptor CD55, HIV receptor CD4 and HIV co-receptors CCR5 and CXCR4 and the Fc-domain of the human IgG1 or the C4b binding protein (C4 bp) α chain.
The regulable expression cassette is inserted into the vector either in tandem or in opposite direction.
In an exemplary embodiment the vector system comprises a first constitutive expression cassette comprising a CMV promoter and a second generation reverse tetracycline transactivator rtTA-M2 and a second inducible expression cassette comprising a second generation tetracycline response promoter tight1 and nucleotide sequence coding for a sCAR-Fc fusion protein according to sequence 1 or a sequence inverse to sequence 1.
The said transgene nucleotide sequence is advantageously codon optimized. This allows for a higher species specific transgene expression.
In one exemplary embodiment of the invention the translation and expression of the transgene is regulated by Doxycycline. It is also possible to induce expression of the transgene by addition of suitable antibiotics, nuclein acid molecules, as siRNA and other regulatory biomolecules.
The vector system is exemplary a non-leaky vector, i.e. the expression of transgene is either completely switched on in the presence of an inducer or completely switched off in the absence of an inducer.
After transduction of an organism with said vector and after induction the vector systems enables the expression of the transgene in a rate up to 500 ng in a ml blood plasma of an organism, preferably up to 700 ng/ml, preferably up to 1000 ng/ml, preferably up to 1500 ng/ml, preferably up to 2000 ng/ml, preferably up to 2500 ng/ml, preferably up to 2700 ng/ml, preferably up to 3000 ng/ml.
The vector system according to the invention is applicable as a medicament.
In one exemplary embodiment the vector system is applicable for treatment of cells infected with a virus of the Picornavirus family, especially in humans and newborn. The vector is preferably used for the treatment of meningitis, myocarditis, pancreatitis, hand, foot and mouth disease and Bornholm disease.
In an exemplary embodiment the vector system is applicable for treatment of CVB infected cells, preferably infected cardiac or pancreatic cells. The vector system can be used for in vitro and/or in vivo treatment of virally infected cells, preferably CVB infected cells, and most preferably CVB3 infected cells.
The vector system is also applicable for treatment of cells infected with adenovirus, especially cells infected with adenovirus A, C-F.
The treatment of viral infected cells, preferably infected cardiac or pancreatic cells, can also be carried out in combination with other viral inhibiting agents, preferably siRNA.
The applied siRNA is obtained synthetically or via expression from a vector, e.g. a plasmid or viral vector. siRNA can be expressed from a single vector. siRNA can also be expressed from the a vector comprising the nucleotide sequence of the siRNA and the soluble receptor protein.
It is most preferred to use a combination of the present viral vector with siRNA2 according to sequence 2 and siRNA4 according to sequence 3 for the treatment of infected cardiac cells.
The vector system is administered in vitro or in vivo before, simultaneously or after infection of the virally infected cells.
In an exemplary embodiment the infected cells are treated in vitro with an amount of the vector upto a MOI of 1 to 10, preferably upto a MOI of 3 to 8, most preferably upto a MOI of 5. Induction is preferably carried out with 1 to 1000 ng/ml doxycycline, preferably 100 to 800 ng/ml doxycycline, most preferably 500 ng/ml doxycycline.
In a further exemplary embodiment the vector dosage comprises 1×1010 to 1×1015 vector particles for in vivo, whereby for the treatment in mice 1×1010 to 3×1010 particles of viral vector are used and for in vivo treatment of human 1011 to 1015, preferably 1013 vector particles are used.
The vector system is preferably based on a viral vector selected from the group comprising an adenoviral vector, a replication deficient adenoviral vector, an adeno-associated virus (AAV), a retrovirus vector, a reovirus vector, a herpes vector or a lentiviral vector having at least one deletion in at least one gene.
It is also of an exemplary advantage that a codon-optimized viral vector is used. It is possible to codon-optimize the CAP gene of the adeno-associated virus (AAV) in order to increase its expression and thus optimize the packaging.
In one exemplary embodiment the transgene, preferably a soluble protein is synthesized using a vector system having the above described features.
The viral vector system according to the invention enables the systemic release of a soluble receptor protein, preferably sCAR-Fc in the liver under the tight control of an inducible promoter. An adenoviral vector (AdV) was constructed that only expressed a soluble receptor protein, preferably sCAR-Fc in the presence of doxycycline (Dox). This vector is able to block viral infections, especially CVB3 infection in vitro and CVB3 infection and myocarditis in vivo, using haemodynamic and histological measurements to monitor cardiomyopathy post-CVB3 infection as shown in the examples.
Thus, therapeutic efficacy in treating CVB3 myocarditis with sCAR-Fc delivered from a pharmacologically regulated adenoviral vector is detectable. The treatment is highly efficient, without clinically observable side effects and leads to improved haemodynamics and heart function in CVB3-infected animals. Thus, combination of sCAR-Fc approach with gene therapeutic methods represents a new approach for treatment of cardiac CVB3 infections.
The object of the invention is also solved by providing a composition.
According to an exemplary embodiment of the invention the composition comprises a vector system having the above described features and antiviral siRNAs.
The applied siRNA is obtained synthetically or via expression from a vector, e.g. a plasmid or viral vector. siRNA can be expressed from a single vector. siRNA can also be expressed from the a vector comprising the nucleotide sequence of the siRNA and the soluble receptor protein.
The composition is applicable as a medicament.
In one exemplary embodiment the composition is applicable for treatment of cells infected with a virus of the Picornavirus family, especially in humans and newborn. The composition is preferably used for the treatment of meningitis, myocarditis, pancreatitis, hand, foot, mouth disease and Bornholm disease.
In an exemplary embodiment the composition is applicable for treatment of CVB infected cells, preferably infected cardiac or pancreatic cells. The composition can be used for in vitro and/or in vivo treatment of virally infected cells, preferably CVB infected cells, most preferably for CVB3 infected cells.
The composition is also applicable for treatment of cells infected with adenovirus; especially cells infected with adenovirus A, C-F.
In one exemplary embodiment the siRNA of the composition comprises siRNA2 comprising sequence 2 and siRNA4 comprising sequence 3.
It is preferred that the composition is administered to the cells before, simultaneously or after viral infection of the cells. The composition is advantageously applicable for the treatment of chronic infections of cardiac cells.
In one exemplary embodiment the composition comprises 1×1010 to 1×1015 particles of viral vector and 1 to 100 000 μg siRNA, preferably 100 to 10 000 μg siRNA, most preferably 500 to 5000 μg siRNA. For the in vivo treatment in mice 1×1010 to 3×1010 particles of viral vector are used and for in vivo treatment of human 1011 to 1015, preferably 1013 vector particles are used.
The composition is exemplary obtained by mixing the viral vector and the siRNA.
The mixing is advantageously carried out immediately before administering the composition in vitro or in vivo. In this case the viral vector and the siRNA are stored separately before mixing. The viral vector is preferably stored in form of a solution comprising 1×1010 to 3×1015 particles of viral vector, preferably 1011 to 1013 particles of viral vector. The siRNA is preferably stored in form of a solution comprising 1 to 100 000 μg siRNA, preferably 100 to 10 000 μg siRNA, most preferably 500 to 5000 μg siRNA.
If the siRNA is obtained by expression from a vector the vector is stored in solution or in a bacterial or viral host known to a person skilled in the art.
The object of the invention is also solved by a method of treating infections caused by a virus of the Picornavirus family, especially in humans and newborn, using a vector system with the above described features and/or a composition with the above described features.
The method is applicable preferably for treating meningitis, myocarditis and pancreatitis, hand, foot and mouth disease and Bornholm disease.
In an exemplary embodiment the method is applied for treatment of CVB infected cells, preferably infected cardiac or pancreatic cells. The method can be used for in vitro and/or in vivo treatment of virally infected cells, preferably CVB infected cells, most preferably for CVB3 infected cells.
The method is also applicable for treatment of cells infected with adenovirus, especially cells infected with adenovirus A, C-F.
In an exemplary embodiment of the invention the viral vector and the siRNA are administered separately. In this case the viral vector is administered in a concentration of 1×1010 to 1×1015 particles, whereby for the treatment in mice 1×1010 to 3×1010 particles of viral vector are used and for in vivo treatment of human 1011 to 1015, preferably 1013 vector particles are used. siRNA is used in a concentration of 1 to 100 000 μg siRNA, preferably 100 to 10 000 μg siRNA, most preferably 500 to 5000 μg siRNA.
In another exemplary embodiment the viral vector and the siRNA are administered simultaneously. The applied concentrations are 1×1010 to 1×1015 vector particles for in vivo, whereby for the treatment in mice 1×1010 to 3×1010 particles of viral vector are used and for in vivo treatment of human 1011 to 1015, preferably 1013 vector particles are used. siRNA is used in a concentration of 1 to 100 000 μg siRNA, preferably 100 to 10 000 μg siRNA, most preferably 500 to 5000 μg siRNA.
While both of the individual treatments, application of siRNAs and expression of a soluble receptor protein, especially in form of the fusion protein sCAR-Fc from the viral vector system only led to a moderate decrease of the viral load, the combination of both approaches in the composition according to the invention resulted in a strong synergistic antiviral effect. This is shown in the examples. Initially, a 4-log reduction of the virus titer was achieved, and even at the end of the experiment (day 13) the viral load was diminished by 3-logs, whereas the single treatments had completely lost their inhibitory activity.
Only the combination of treatment with the present viral vector, especially the viral vector AdG12, and siRNAs, especially siRNA against CVB-3 in form of the composition according to the invention is suitable to achieve both, an increase of cell viability and a substantial reduction of the virus titer. Both antiviral agents act in a synergistic manner. While sCAR-Fc expressed from the viral vector traps the virus extracellularly, siRNAs induce degradation of virus genomes that are present in the cells either at the beginning of the experiment or by entering the cells after circumventing the extracellular shield.
Examples of embodiments are explained in further detail by means of the following figures and examples.
Coxsackievirus B3: In vitro and in vivo experiments utilized the genetically characterized, cardiovirulent Nancy strain of CVB3. Methods detailing virus propagation and titration of CVB3 in HeLa cells, as well as storage at −80° C., prior to infection of cells or animals were as previously described (Yanagawa B, Spiller O B, Proctor D G et al. Soluble recombinant coxsackievirus and adenovirus receptor abrogates coxsackievirus b3-mediated pancreatitis and myocarditis in mice. J Infect Dis 2004; 189:1431-9).
Cells: Human myocardial fibroblast (HMF) cell line (immortalized; HMF1226K/I), HEK293 and HeLa cells (Wisconsin strain; courtesy of Dr. R. R. Ruckert, Madison) were propagated in monolayer culture in Minimal Essential Medium containing 5% heat inactivated fetal calf serum (FCS), 1% antibiotic/antimycotic, gentamycin and non-essential amino acids. Cell lines were propagated at 37° C. in a humidified atmosphere with 5% carbon dioxide.
Coxsackievirus infection of HMF cell line and cell viability assay: For the lytic infection assays, HMF cells were first transfected with siRNAs and/or transduced with AdG12 and inoculated with CVB-3 at a multiplicity of infection (m.o.i.) of 1 plaque forming unit (pfu) per cell in medium without FCS four hours thereafter for 30 minutes and maintained in cell culture medium. To generate persistently infected HMF cells, nearly confluent cells were inoculated with CVB-3 at an m.o.i. of 30. Medium was changed every other day. More than 90% of cells died within one week. Single cell clones grew up slowly and during the second week the cells were passaged. Henceforth, the infected cells were propagated by passaging twice a week in medium with a reduced FCS content of 2%. Virus titer in the supernatant was controlled regularly. After storage in liquid nitrogen and subsequent re-culturing, cells still produced high virus titer. For most of the experiments, the infected cells were seeded in 96-well (half area) plates and maintained for more than one week without passaging. As a measure of cytopathic effects induced by the CVB-3 in these non-subcultured HMF cells, cell viability was determined at several time points after treatment using the Cell Proliferation Kit II (Roche, Mannheim, Germany) according to the manufacturer's instructions. Measured absorbance at 492 nm thus correlates directly to cell viability.
Development of Adenoviral Vector, transduction and induction: sCAR-Fc was generated by fusion of the extracellular domain of human CAR with the carboxy terminus of human IgG1 Fc coding region. sCAR-Fc was cloned into the plasmid pZS2-CMV-rtTA downstream of the second-generation reverse tetracycline (tet)-dependent transactivator rtTA-M2 in two opposite directions. For generation of a Dox-regulated sCAR-Fc the Dox responsive tight1 promoter was inserted upstream of sCAR-Fc resulting in the adenoviral shuttle plasmids pAdG12-sCAR-Fc and pAdR4-sCAR-Fc. pAdG12-sCAR-Fc and pAdR4-sCAR-Fc were linearized with XbaI and ligated to the 5′ long arm of XbaI-digested E1-E3-adenovirus 5 mutant RR5. Transfection into HEK293 cells and propagation was carried out as described (Marienfeld U, Haack A, Thalheimer P et al. ‘Autoreplication’ of the vector genome in recombinant adenoviral vectors with different E1 region deletions and transgenes. Gene Ther 1999; 6:1101-13) generating the adenoviral vectors termed AdG12 and AdR4.
HMF cells were transduced with adenoviral vector at a concentration of 10 m.o.i. by addition of the required amount to the medium. Immediately after transduction the sCAR-Fc protein production was induced by adding Dox (1.5 μg/ml). Every second or third day Dox (and medium) was refreshed. When combined with siRNA double-transfections, AdG12 was transduced during the first and Dox was added after the second transfection.
siRNAs and transfection: siRNAs with two nucleotide overhangs used in this study were purchased from MWG Biotech (Ebersberg, Germany). Both, sRNA2 (target sequence CUA AGG ACC UAA CAA AGU U, Sequence 2) and siRNA4 (target sequence GUA CAG GGA UAA ACA UUA C, Sequence 3), are directed against the 3D RNA dependent RNA polymerase (3Dpol) of CVB-3 (GenBank acc. no. M33854; target nucleotides 6315-6333 and 6735-6753, respectively). As a control, an siRNA from Qiagen (Hilden, Germany) with no known homology in the human and viral genome was used. For transfection, HMF cells were seeded in 24-well plates at a density of 1.2×105 cells per ml in a volume of 500 μl without antibiotics. The next day, cells were transfected with 12.5 nM siRNA 2 and 4 or 25 nM control siRNA and 2 μl Lipofectamine™ 2000 (Invitrogen, Karlsruhe, Germany) per well, following the manufacturer's instructions.
The persistently infected cells were plated in 96-well (half area) plates at a density of 105 cells per ml in a volume of 50 μl. These cells were transfected twice on the same day with the siRNA concentrations denoted above using 0.125 μl Lipofectamine™ 2000. The supernatant was replaced by medium two hours after the first transfection and the second transfection mixture was left on the cells for about 20 hours.
Cell Cultures, Northern Blot, Western Blot, Virus Plaque Assays, IgG ELISA: HeLa (human cervical carcinomas) cells and HEK293 (human embryonal kidney) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL, Karlsruhe, Germany) supplemented with 10% FCS and 1% penicillin/streptomycin. Northern and Western analysis and virus plaque assays were carried out as described (Fechner H, Pinkert S, Wang X et al. Coxsackievirus B3 and adenovirus infections of cardiac cells are efficiently inhibited by vector-mediated RNA interference targeting their common receptor. Gene Ther 2007; 14:960-71). Human IgG ELISA (Bethyl Laboratories Inc., Montgomery, Tex., USA) for detection of the Fc-tail of sCAR-Fc was performed according to supplier's introductions.
Determination of CVB-3 titer. The amount of infectious CVB-3 in the supernatant of infected HMF cells was determined on HeLa cells by an agar overlaid plaque assay as described. Shortly, the at least ten-fold diluted samples were incubated for 30 min on HeLa monolayers. Subsequently, cells were overlaid with agar containing Eagle's MEM. After incubation in a humidified atmosphere for two days, cells were stained with neutral red and virus titers were determined by plaque counting.
Detection of soluble CAR-Fc (SCAR-Fc). For analysis of sCAR-Fc expression, the supernatants of induced cultures were collected at different time points and stored at −20° C. sCAR-Fc protein levels were determined by the use of the Human IgG Enzyme Linked Immuno Sorbent Assay (ELISA) Quantitation Kit (Bethyl Laboratories, Montgomery, Tex., USA). Following the manufacturer's instructions, a MaxiSorb™ (Nunc, Langenselbod, Germany) 96 well plate was coated with a Goat anti-human IgG for one hour. During the blocking step the collected samples were diluted 1:10 and 100 μl were transferred to the reaction plate. After an additional incubation for one hour and an intensive washing, a Goat anti-human IgG-HRP conjugate in a 1:150.000 dilution was added to each well. Following the addition of a Tetramethyl Benzidine (TMB) substrate and a sulfuric acid, the oxidized product can be measured in a plate reader at 450 nm. As a calibrator, human reference serum in a working range of 3.9 ng/ml-500 ng/ml were used in each assay in duplicate. For calculation of results we used the calibrator as a standard curve with a four parameter logistic curve-fit.
Murine CVB3 myocarditis: AdG12 was injected into the vena jugularis of 6-8 weeks old Balb/c mice. Two days following AdV injection, mice were infected with 5×104 pfu of CVB3 intraperitoneally. Dox (200 μg/ml) was orally administered to the mice via drinking water two days before CVB3 infection (preinfectious approach), concurrent or 1 d after CVB3 infection (therapeutic approach). Seven days post-CVB3 infection, the haemodynamic parameters of the mice were analysed as described (Fechner H, Sipo I, Westermann D et al. Cardiac-targeted RNA interference mediated by an AAV9 vector improves cardiac function in coxsackievirus B3 cardiomyopathy. J Mol Med 2008, 86:987-997), then blood was taken and organs were harvested for histopathological analysis. CVB3 positive-strand genomic RNA in tissues was detected by in situ hybridization using single-stranded 35S-labeled RNA probes as described (Klingel K, Hohenadl C, Canu A et al. Ongoing enterovirus-induced myocarditis is associated with persistent heart muscle infection: Quantitative analysis of virus replication, tissue damage, and inflammation. PNAS 1992; 89:314-8) or standard plaque assay for CVB3 as described (Fechner H, Sipo I, Westermann D et al. Cardiac-targeted RNA interference mediated by an AAV9 vector improves cardiac function in coxsackievirus B3 cardiomyopathy. J Mol Med 2008, 86:987-997).
Statistics: Statistical analysis was performed by Student's t test (for data meeting parametric criteria) or Mann-Whitney U test (for non-parametric data analysis). Values are presented as the mean±the standard deviation, where n represents the number of independent experiments. Differences were considered significant at values of p<0.05.
In order to achieve Doxycycline (Dox)-dependent sCAR-Fc expression two adenoviral vectors (AdV) were constructed. Each AdV contains two expression cassettes, one cassette for constitutive expression of the second generation reverse tetracycline transactivator rtTA-M2, the other for expression of sCAR-Fc from the improved second generation tetracycline (Tet) response promoter tight1. The expression cassettes were inserted either in tandem orientation (AdR4) or in opposite orientations (AdG12) into the E1 region of an E1-E3-adenovirus 5 backbone (see
To analyse Dox-dependent regulation of AdV mediated sCAR-Fc expression, HeLa cells were transduced with AdR4 or AdG12 and cultured in the presence or absence of Dox. Northern blot analysis found sCAR-Fc mRNA expression to be strictly Dox-dependent, while rtTA-M2 expression was constitutively high (
sCAR-Fc protein was only detectable in AdG12 transduced cells and in the cell culture supernatant in the presence of Dox (
For safety reasons, non-leaky, induction confined expression of sCAR-Fc is an important feature of this gene therapy approach. As early as 24 h after transduction of HeLa cells with AdG12, in the presence of Dox, sCAR-Fc was detectable in the cell culture supernatant and achieved maximum extracellular concentration two to three days after transduction (
Next the sCAR-Fc mediated inhibition of CVB3 in vitro as a function of AdG12 dose, Dox concentration and the dose of CVB3 was studied. Transduction of HeLa cells with 5 MOI of AdG12 and induction with 500 ng/ml Dox for 48 h were sufficient to prevent CVB3 infection in sCAR-Fc expressing cells completely. Under these tranductional conditions sCAR-Fc expression levels reached a maximum of 29.4 μg/ml in the cell culture supernatant. sCAR-Fc expressed from AdG12 could efficiently block CVB3 doses of up to 2.5 MOI (data not shown). Therefore, sCAR-Fc expressed by AdG12-transduced cells efficiently inhibited CVB3 infection of these cells and secreted sCAR-Fc levels were directly related to initial AdG12 MOI and Dox concentration.
To assess the potential of AdG12 to suppress ongoing infections as this represents the typical situation encountered the clinical setting, HeLa cells were transduced with AdG12 and sCAR-Fc expression was induced with Dox at different times relative to CVB3 infection. Expression of sCAR-Fc 48 h and 24 h before CVB3 infection resulted in complete inhibition of CVB3 infection in the transduced cells. The inhibitory efficiency of sCAR-Fc was gradually reduced the later the sCAR-Fc expression was induced. However, even if sCAR-Fc expression was induced 24 h after infection CVB3 progeny virus number was still reduced by about 106-fold compared to controls without sCAR-Fc (
To examine the effect of AdG12 mediated sCAR-Fc expression on the progression of CVB3-induced myocarditis first sCAR-Fc expression kinetics following intravenous administration of 3×1010 particles of AdG12 to Balb/c mice was determined. sCAR-Fc serum concentrations in AdG12 (+Dox) transduced animals roughly doubled from day 2 (254±29 ng/ml) to day 5 (464±159 ng/ml), then decreased at day 8 (147±60 ng/ml), but expression did not decrease further when measured at day 14 (141±51 ng/ml). In the absence of Dox, sCAR-Fc serum levels were indistinguishable from levels in untransduced control mice (data not shown). Histopathological examination of liver and heart samples did not show any signs of tissue damage and inflammation at various time points (not shown).
Based on sCAR-Fc in vivo expression kinetics determined above, the ability of AdG12 transduced mice to inhibit CVB3-mediated myocarditis was performed. Mice were transduced with AdG12 and sCAR-Fc expression induced and maintained via permanent oral Dox administration. The AdG12 doses was reduced to 1×1010 virus particles per mouse as in the initial experiment with 3×1010 particles AdG12 the sCAR-Fc serum levels distinctly exceeded therapeutical relevant levels that were already found below 100 ng/ml. Two days after vector transduction animals were infected with 5×104 pfu CVB3 (
To document whether absence of pathological changes of the heart correlates with cardiac CVB3 infection we performed radioactive in situ hybridization experiments to visualize the presence of plus strand CVB3 RNA at the cellular level at a high sensitivity. Animals of AdG12 (+Dox) CVB3 infected group did not show any CVB3-infected cells in the heart (
In a next step the efficacy of sCAR-Fc gene therapy in a therapeutic approach was analyzed. Mice were transduced with AdG12 and Dox was applied for induction of sCAR-Fc either concurrent with CVB3 infection two days after transduction or one day after CVB3 infection at day three after transduction (
For a first assessment of the antiviral potential of both strategies, uninfected human myocardial fibroblasts (HMF) were initially pre-incubated with 12.5 nM of each of the siRNAs 2 and 4, both of which are directed against the viral 3DPol, and inoculated with 1 m.o.i. of CVB-3 four hours thereafter. Virus titer on subsequent days was determined by titration of culture supernatants on confluent HeLa cells. A reduction of more than 1-log was observed after 24 hours and lasted for at least three days (
The combination of sCAR-Fc expressing adenoviral vector and antiviral siRNAs yielded an additive increase of the inhibitory activities resulting in an almost 7-log reduction of the virus titer. Closer statistical analysis revealed that the antiviral activity of the combination of sCAR-Fc and siRNAs was significantly higher than the inhibitory effect of the sCAR-Fc expressing vector in the presence of a control siRNA on day 2 of the experiment.
In the next step, the antiviral potential of both siRNAs and AdG12 in HMF cells with an ongoing CVB-3 infection was tested. For this purpose, the persistently infected cells were transfected with siRNAs 2 and 4 twice a day on two consecutive days. After the treatment, the virus titer decreased by 1-log(
To investigate the time course of sCAR-Fc expression in persistently infected HMF, the amount of protein in the supernatant was quantified by a human IgG ELISA. For these quantifications, supernatants of cells transduced with AdG12 and induced by the addition of Dox were collected and measured (data not shown). The amount of detected protein corresponded to approximately 10 to 100 ng Fc-domains per ml supernatant. The protein level dropped drastically after day 4 of the experiment and could be restored by a second transduction with the AdG12 on day 6. Initially, hardly any difference was observed between cells, which were only transduced with AdG12 in the presence of Dox, and cells, which underwent additional treatment with siRNAs 2 and 4 (data not shown). Owing to the improved cell viability, higher sCAR-Fc levels were detected for the double-treated cells at later time points. Taken together, the time course of secreted sCAR-Fc levels in the supernatant was comparable in both types of experiments and prolonged high-level expression of sCAR-Fc can be achieved by a second transduction of the cells.
In order to compensate for the loss of antiviral impact the HMF were transfected and/or transduced cells again on day six of the experiment. As can be seen in
According to these results the combination strategy with siRNAs and AdG12 is considerably more efficient in inhibiting CVB-3 in persistently infected HMF cells than either of the single approaches. Furthermore, repeated treatments are required to maintain inhibition.
In an next step the question was tackled whether it will be necessary to repeat the double treatment or if it might be sufficient only to use the adenoviral vector for the second administration. To address this question, persistently infected HMF cells were initially treated with both siRNAs and AdG12. Cultures that do not undergo a second round of treatment loose viability (
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2007 052 005.2 | Oct 2007 | DE | national |
This application is a National Phase patent application of International Patent Application Number PCT/EP2008/009058, filed on Oct. 27, 2008, which claims priority of German Patent Application Number 10 2007 052 005.2, filed on Oct. 26, 2007.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2008/009058 | 10/27/2008 | WO | 00 | 6/24/2010 |
