WO2012045473A1

WO2012045473A1 - Viruses for the treatment of fibrosis

Info

Publication number: WO2012045473A1
Application number: PCT/EP2011/005030
Authority: WO
Inventors: Jennifer Altomonte; Olivier Ebert
Original assignee: Technische Universität München
Priority date: 2010-10-07
Filing date: 2011-10-07
Publication date: 2012-04-12

Abstract

The present invention relates to viruses for use in a method of treatment of fibrosis and in a method of selectively inducing apoptosis of collagen-producing fibroblasts or collagen-producing fibroblast-like cells, e.g. activated hepatic stellate cells (HSCs) or activated fibroblasts, such as activated pulmonary fibroblasts, wherein said fibrosis is associated with a pathogenic mechanism which involves the proliferation of collagen-producing fibroblasts or collagen-producing fibroblast-like cells.

Description

Viruses for the treatment of fibrosis

The present invention relates to viruses for use in the treatment of fibrosis. The fibrosis is associated with a pathogenic mechanism which involves the proliferation of collagen- producing fibroblasts or collagen-producing fibroblast-like cells. In one aspect, the present invention relates to viruses for use in a method of treatment of liver fibrosis and in a method of selectively inducing apoptosis of activated hepatic stellate cells (HSCs). In another aspect, the present invention relates to a method of treatment of liver fibrosis and to a method of selectively inducing apoptosis of activated hepatic stellate cells (HSCs). In a further aspect, the present invention relates to viruses for use in a method of treatment of pulmonary fibrosis and in a method of selectively inducing apoptosis of activated pulmonary fibroblasts. In yet a further aspect, the present invention relates to a method of treatment of pulmonary fibrosis and to a method of selectively inducing apoptosis of activated pulmonary fibroblasts.

Liver fibrosis (or hepatic fibrosis) is the excessive accumulation of extracellular matrix proteins including collagen that occurs as wound-healing response to most types of chronic liver injury. Chronic liver injury can result from persistent viral, toxic, autoimmune, metabolic, or cholestatic impairments. Long-term alcohol abuse and chronic hepatitis C virus infection are the most prominent underlying factors responsible for liver cirrhosis in Europe and North America (Colombo, 1993). If left untreated, hepatic fibrosis progresses to cirrhosis, a condition which is characterized by fibrotic nodule formation and liver contraction. The fibrotic response underlies virtually all of the complications of end-stage liver disease, including portal hypertension, ascites, encephalopathy, and metabolic dysfunction, as well as the development of hepatocellular carcinoma (HCC).

During the fibrogenic process, hepatic stellate cells (HSCs) differentiate from the quiescent to the activated form. Activated HSCs are characterized by a loss of intracellular vitamin A-rich fat droplets and a change to a myofibroblast phenotype coinciding with expression of a-smooth muscle actin (ctSMA). These transdifferentiated HSCs promote extracellular matrix (ECM) remodeling by secreting matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs), resulting in the degradation of the normal matrix and replacement with interstitial collagen (primarly type I) and scar matrix. In addition, HSCs migrate and proliferate in response to a variety of cytokines and growth factors elicited during hepatic injury to further promote the progression of fibrosis. These hepatic changes cause a distortion of the normal liver architecture and lead to compensated liver function.

Over the last 25 years, much progress has been made in understanding the mechanism of liver fibrogenesis, and as a result, it is now believed that fibrosis and cirrhosis are reversible processes. Despite this growing body of evidence, the clinical management of cirrhosis has fallen behind, and the success of available therapies has yet to be demonstrated. While it is known that the major mechanism for regression of fibrosis involves apoptosis of activated HSCs, the challenge for a successful and safe antifibrotic therapy is specific targeting of activated HSCs, without collateral effects on quiescent HSCs or myofibroblasts present in other tissues. Unfortunately, the majority of drugs under investigation have resulted in only minor antifibrotic effects, with a general lack of specificity on the HSC activation pathway (Poynard et al., 2000; Rambaldi et al., 2000).

When the cirrhotic condition progresses to the development of HCC, the condition presents an even greater challenge for clinicians. HCC is a complex condition with limited therapeutic options, even in the context of a healthy liver. The presence and degree of cirrhosis, which is reflected by liver function, can greatly determine the possibility for curative, or even palliative therapies. In the absence of treatment, the prognosis for early and advanced HCC is poor, with the median survival being 6-9 months and 1-2 months, respectively (Bosch et al., 2004). Even in patients who are diagnosed early, the course of the disease is often fatal due to the underlying liver cirrhosis, since there is currently no available therapy with proven efficacy for simultaneously treating HCC and cirrhosis other than liver transplantation. Tumor resection in the context of cirrhosis and portal hypertension is not effective, as the recurrence rate in a cirrhotic liver is extremely high, and patients with advanced cirrhosis and/or multifocal tumor nodules exceeding the inclusion criteria at the time of HCC diagnosis are not candidates for surgical resection (Belghiti et al., 1991 ; Figueras et al., 1997). Transarterial chemoembolization (TACE), which is a promising palliative therapy for HCC, is only indicated for patients with well-preserved liver function due to the possibility of severe complications and poor prognosis after administration in patients with limited hepatic reserve (Farinati et al., 1996; Olivo et al., 2009). Therefore, liver transplantation is considered the only curative treatment option for HCC in the context of cirrhosis; however, the supply of livers available for transplantation is extremely limited, and patients often face waiting periods of one year or more. Furthermore, in cirrhotic patients with HCC, recurrence rates after transplantation are high (Iwatsuki et al., 1991 ; Selby et al., 1995), and there is an indication that operative mortality during liver transplantation is also higher for cirrhotic patients (Pichlymayr et al., 1989). Therefore, due to the ever-increasing incidence of cirrhosis and subsequent HCC, and the obvious limitations of currently available therapies, novel and effective treatments are urgently needed.

The proliferation of activated fibroblasts is not a phenomenon exclusive to hepatic fibrosis, but rather it is a common theme in the pathogenesis of fibrosis in several major organs, such as the lungs (pulmonary fibrosis).

Pulmonary fibrosis, which can occur idiopathically or as a secondary effect of other diseases or conditions, such as autoimmune diseases, sarcoidosis, inhaled environmental pollutants, or cigarette smoking, is characterized by the replacement of the normal lung parenchyma with fibrotic scar tissue. This process results in significantly impaired lung function and oxygen diffusion capacity, and can lead to pulmonary hypertension and increased risk for pulmonary emboli and heart failure. Although five million people are affected worldwide by pulmonary fibrosis, the treatment options are extremely limited. To date, there are no available medications with proven efficacy, and lung transplantation remains the only therapeutic option indicated for severe cases. Due to the extremely limited availability of transplantable lungs, coupled with the serious complications associated with the procedure, this condition represents a major unmet medical need, and new therapeutic agents for pulmonary fibrosis are urgently required.

Accordingly, it was an object of the present invention to provide for new antifibrotic substances/agents for effective and safe therapy of fibrosis, in particular liver fibrosis and/or pulmonary fibrosis. It was also an object of the present invention to provide for new antifibrotic substances/agents which allow specific targeting of activated HSCs or of activated fibroblasts, e.g. activated pulmonary fibroblasts, without collateral effects on quiescent cells, e.g. quiescent HSCs or myofibroblasts present in other tissues.

The objects of the present invention are solved by a virus for use in a method of treatment of fibrosis wherein said fibrosis is associated with a pathogenic mechanism which involves the proliferation of collagen-producing fibroblasts or collagen-producing fibroblast-like cells. In one embodiment, said fibrosis is liver fibrosis or pulmonary fibrosis.

In one embodiment, said virus is an RNA virus.

In one embodiment, said fibrosis manifests itself in the formation of an excess of fibrous connective tissue or in the formation of abnormal fibrous connective tissue in or around an organ. Such fibrosis is typically associated with a pathogenic mechanism which involves the proliferation of collagen-producing fibroblasts or collagen-producing fibroblast-like cells. The term "associated with a pathogenic mechanism", as used herein, is meant to refer to a scenario, wherein the pathogenic mechanism may be the underlying cause of the disease or may simply accompany said disease. In a preferred embodiment, this term is intended to mean that the disease is accompanied by such pathogenic mechanism. In another embodiment, it is intended to mean that the disease is caused by such pathogenic mechanism. The term "fibroblast-like cells" is meant to refer to cells which resemble fibroblast cells in one or several characteristics related to the fibroblast's capability to synthesize the extracellular matrix (ECM) and/or collagen. Just like fibroblasts, fibroblast-like cells are involved in the secretion of constituents or precursors of the extracellular matrix. Just like fibroblasts, fibroblast-like cells are involved in the synthesis of one or several of: collagens, glycosaminoglycans, reticular and elastic fibers, glycoproteins found in the extracellular matrix and other constituents/components of the extracellular matrix.

Examples of fibrosis which are associated with a pathogenic mechanism which involve the proliferation of collagen-producing fibroblasts or collagen-producing fibroblast-like cells are pulmonary fibrosis, such as idiopathic pulmonary fibrosis, hepatic fibrosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis of the lungs, nephrogenic systemic fibrosis, Crohn's disease, keloid formation, systemic sclerosis, such as scleroderma, and arthrofibrosis.

The genetic material of a virus is made from either DNA or RNA. Accordingly, an RNA virus is a virus that has RNA (ribonucleic acid) as its genetic material. RNA viruses are also referred to as riboviruses.

In one embodiment, said RNA virus is a negative-strand RNA virus.

A negative-strand RNA virus (also known as antisense-strand RNA virus) is a virus whose genetic information consists of a single strand of RNA that is complementary to mRNA (messenger RNA), and thus must be converted to positive/sense RNA by an RNA polymerase before translation. Negative-strand RNA viruses are viruses belonging to Group V of the Baltimore classification system.

In one embodiment, said negative-strand RNA virus is selected from the group of vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), measles virus, mumps virus, Sendai virus (SeV) and influenza viruses. In a preferred embodiment, said negative-strand RNA virus is selected from the group of vesicular stomatitis virus (VSV), Newcastle disease virus (NDV) and measles virus.

In an even more preferred embodiment, said negative-strand RNA virus is a vesicular stomatitis virus (VSV).

In one embodiment, said virus is a wildtype virus or a recombinant virus.

The term "wildtype virus" as used herein is meant to refer to the phenotype and/or genotype of the typical form of a virus as it occurs in nature, i.e. without any artificial changes or modifications.

In one embodiment said virus is provided in the form of a vector. In such cases, the term "wildtype virus" thus refers to a vector carrying all genes of the virus in an unmodified form. In one embodiment, said vector consists of said virus.

The term "recombinant virus" as used herein is meant to refer to a virus produced by recombining pieces of DNA or RNA using recombinant DNA or RNA technology. Recombinant negative-strand RNA viruses can be generated using a "reverse genetics" system as described previously (Ebert et al., 2003). More specifically, the term "recombinant virus" is meant to refer to viruses where one or more viral gene(s) have been modified (e.g. by mutation, deletion, insertion) and/or which express one or more non-viral gene(s) (i.e. transgenes), wherein, preferably, said transgenes are selected from the group comprising IFN-a, IFN-β, srhcB, M3, MMPl, MMP8, relaxin and a reporter gene, such as betagalactosidase (β-Gal) and green fluorescent protein.

IFN-ct/-P refers to interferon-a and interferon-β. srlicB refers to "super-repressor to NF-κΒ". NF-KB is a transcription factor, which regulates the expression of various down-stream genes associated mainly with inflammation and proliferation. Because NF-κΒ is induced in response to VSV infection, resulting in the rapid recruitment of inflammatory cells and thereby inhibiting viral replication and spread, it is hypothesized that blocking this response could be beneficial when trying to treat a fibrotic liver or lung, in which inflammation is a common feature. A reduction of hepatic inflammation would not only reduce associated toxicities, but it could also enhance the ability of VSV to replicate in activated HSCs.

M3 encodes a broad-range chemokine binding protein derived from the heterologous murine gammaherpesvirus, which has been shown to effectively suppress the chemotaxis of inflammatory cells in vivo.

The remaining genes MMP1, MMP8 and relaxin encode for matrix metalloproteinase-1, matrix metalloproteinase-8, and relaxin, respectively. These genes are all involved in the degradation of collagen, and it is hypothesized that incorporation of these genes into the viral (e.g. VSV) vector would further promote a reduction of fibrotic content in the liver or lung.

In one embodiment, said virus does not contain any exogenous agent or express any exogenous gene, wherein said exogenous agent or exogenous gene is not IFN-a, IFN-β, srlKB, M3, MMP1, MMP8 or relaxin.

In one embodiment, said virus does not contain any exogenous agent or express any exogenous gene at all, i.e. said virus is not used as a shuttle for such exogenous agents or genes.

The term "exogenous agent" as used herein is meant to refer to a non-viral substance, which includes chemical compounds, drugs, toxins, non-viral nucleic acid molecules (i.e. non-viral DNA and RNA, including small regulatory RNAs such as siRNAs or miRNAs) and non-viral polypeptides. The term "non-viral" refers to the used virus.

The term "exogenous gene" (also referred to as transgene) as used herein is meant to refer to a nucleic acid molecule encoding a non-viral polypeptide/protein. In one embodiment, said virus is a recombinant vesicular stomatitis virus (rVSV). Preferably, said recombinant vesicular stomatitis virus (rVSV) is selected from the group of rVSV(MA51) and wildtype VSV or rVSV(MA51) expressing one or more of the following transgenes: IFN-ct (rVSV-IFN-a), IFN-β (rVSV-IFN-β), srlicB (rVSV-srli B), M3 (rVSV-M3), MMP1 (rVSV-MMPl), MMP8 (rVSV-MMP8) and relaxin (rVSV- relaxin). rVSV(MA51) is a mutant vector, in which the endogenous matrix protein (M) has been modified such that amino acid 51 has been deleted. This modified vector is generally regarded as safer than the wildtype virus, due to its inability to shut down host translation, which allows for a potent induction of the anti-viral interferon (IFN) system.

In one embodiment, said virus selectively induces apoptosis of collagen-producing fibroblasts or collagen-producing fibroblast-like cells, e.g. activated hepatic stellate cells (HSCs) or activated fibroblasts, such as activated pulmonary fibroblasts.

In one embodiment, activated hepatic stellate cells are characterized by at least one of: a loss of intracellular vitamin A-rich fat droplets and a change to a myofibroblast phenotype coinciding with expression of ct-smooth muscle actin (aSMA). In one embodiment, activated hepatic stellate cells are characterized by all of the aforementioned characteristics.

In one embodiment, activated pulmonary fibroblasts are characterized by at least one of: phenotype of proliferation, migration, and the expression of a-smooth muscle actin (aSMA), fibronectin, and collagen I. In one embodiment, activated pulmonary fibroblasts are characterized by all of the afore-mentioned characteristics.

In one embodiment, said liver fibrosis is associated with chronic liver injury, portal hypertension, ascites, encephalopathy, metabolic dysfunction, liver cirrhosis and/or hepatocellular carcinoma (HCC). In one embodiment, said pulmonary fibrosis is idiopathic pulmonary fibrosis. In another embodiment, said pulmonary fibrosis is associated with autoimmune disorders, sarcoidiosis, Wegener's granulomatosis, viral infections, inhalation of environmental pollutants, or smoking.

In one embodiment, said chronic liver injury is caused by persistent viral, toxic, autoimmune, metabolic and/or cholestatic impairments, in particular by long-term alcohol abuse and/or chronic hepatitis C virus infection.

In one embodiment, said liver fibrosis is associated with HCC.

The objects of the present invention are also solved by a virus as defined above for use in a method of selectively inducing apoptosis of collagen-producing fibroblasts or collagen- producing fibroblast-like cells, e.g. activated hepatic stellate cells (HSCs) or activated fibroblasts, such as activated pulmonary fibroblasts.

The objects of the present invention are further solved by the use of at least one virus as defined above for the manufacture of a medicament for the treatment of fibrosis, wherein said fibrosis is associated with a pathogenic mechanism which involves the proliferation of collagen-producing fibroblasts or collagen-producing fibroblast-like cells. In one embodiment, said fibrosis is liver fibrosis or pulmonary fibrosis.

The objects of the present invention are solved by the use of at least one virus as defined above for the manufacture of a medicament for selectively inducing apoptosis of collagen-producing fibroblasts or collagen-producing fibroblast-like cells, e.g. activated hepatic stellate cells (HSCs) or activated fibroblasts, such as activated pulmonary fibroblasts.

The objects of the present invention are also solved by a method of treatment of fibrosis, wherein said fibrosis is associated with a pathogenic mechanism which involves the proliferation of collagen-producing fibroblasts or collagen-producing fibroblast-like cells. In one embodiment, said fibrosis is liver fibrosis or pulmonary fibrosis, wherein said method comprises the step of administering an effective amount of at least one virus as described above to a person in need thereof.

In one embodiment, said liver fibrosis is associated with chronic liver injury, portal hypertension, ascites, encephalopathy, metabolic dysfunction, liver cirrhosis and/or hepatocellular carcinoma (HCC).

In one embodiment, said liver fibrosis is associated with HCC.

In one embodiment, said pulmonary fibrosis is idiopathic pulmonary fibrosis. In another embodiment, said pulmonary fibrosis is associated with autoimmune disorders, sarcoidosis, Wegener's granulomatosis, viral infections, inhalation of environmental pollutants, or smoking.

The term "effective amount" as used herein is meant to refer to an amount of at least one virus as described above sufficient to selectively induce apoptosis of collagen-producing fibroblasts or collagen-producing fibroblast-like cells, e.g. activated stellate cells (HSCs) or activated fibroblasts, such as activated pulmonary fibroblasts, and to result in an antifibrotic effect. This amount may vary depending on the particular virus used, the age and condition of the subject being treated, or the extent of fibrosis, and the like.

Said effective amount of at least one virus as described above may be administered via the oral, intravenous, intra-arterial, intramuscular, subcutaneous, intranasal, intradermal, intraperitoneal, or suppositories routes or by implantation, or by intratracheal administration for the treatment of pulmonary fibrosis. Administration can also be achieved using a combination of routes. In a particularly preferred embodiment, said effective amount is administered intra-arterial ly, preferably via the hepatic artery, or intravenously, preferably via the hepatic portal vein for the treatment of hepatic fibrosis.

In one embodiment, said effective amount of at least one virus as described above is admixed with at least one pharmaceutically acceptable carrier, excipient and/or diluent which is preferably sterile. Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art and may include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. Proper formulation is dependent on the route of administration chosen, as is known in the art. In embodiments for (intra-arterial or intravenous) injection/infusion, said effective amount of at least one virus as described above may be formulated in aqueous solutions, or in physiologically compatible solutions or buffers such as Hank's solution, Ringer's solution, mannitol solutions or physiological saline buffer (e.g. PBS).

In one embodiment, said virus is VSV and is sequentially administered on days 0, 2 and 4.

In one embodiment, more than one virus as defined above (i.e. two or three or four or even more different viruses) are administered, wherein preferably, said viruses are administered successively.

The objects of the present invention are also solved by a method of selectively inducing apoptosis of collagen-producing fibroblasts or collagen-producing fibroblast-like cells, e.g. activated hepatic stellate cells (HSCs) or activated fibroblasts, such as pulmonary fibroblasts, said method comprising the step of contacting said collagen-producing fibroblasts or collagen-producing fibroblast-like cells, e.g. activated HSCs or activated fibroblasts, such as activated pulmonary fibroblasts with at least one virus as described above.

In one embodiment, said contacting is performed via administration as defined above. The present inventors have surprisingly found that viruses can be used for safe and effective treatment of fibrosis, wherein said fibrosis is associated with a pathogenic mechanism which involves the proliferation of collagen-producing fibroblasts or collagen-producing fibroblast-like cells, e.g. liver fibrosis or pulmonary fibrosis. They have shown that the observed reversal of fibrotic progression involves several aspects: viruses according to the present invention selectively target activated fibroblasts, e.g. activated hepatic stellate cells or activated pulmonary fibroblasts through activation- specific viral replication and subsequent apoptosis;

infection with viruses according to the present invention causes an increase of IFN-a in the liver or the lung; and

infection with viruses according to the present invention causes a decrease in TIMP-1, aSMA, TGF-, and Pro-collagen, and thereby leads to a decrease in fibrotic content and improved liver staging or lung staging.

The present inventors have used vesicular stomatitis virus (VSV) as an exemplary virus - however, the results obtained can be extrapolated to other viruses, in particular to other negative-strand RNA viruses, since their mechanism of replication (leading to a robust viral replication specifically in activated HSCs) is similar to that of VSV. Particularly preferred viruses according to the present invention are VSV, the Newcastle disease virus (NDV) and the measles virus.

The term "liver staging" as used herein is meant to refer to the (fibrotic) state of the liver expressed by a score, which is determined by liver biopsy and subsequent histological analysis. There exist several different scoring systems. In the Ishak classification system, a score of 0 refers to "no liver scarring", a score of 1 or 2 refers to "minimal liver scarring around liver blood vessels", a score of 3 refers to "scarring extended out from liver blood vessels", a score of 4 refers to "scarring that forms 'bridges' between blood vessels" and a score of 5 or 6 refers to "extensive scarring or cirrhosis". Another scoring system is a 0- 4-scoring system, which was also sometimes used by the present inventors, wherein 0= normal liver, 1= expansion of portal tract, 2= expansion of portal tract with septa, 3= porto-portal and porto-central bridges, and 4= cirrhosis. However, the present invention should not be understood as being limited to this specific or any other scoring system.

The experiments performed by the present inventors show that the viruses according to the present invention are an effective tool for clearance of collagen-producing fibroblasts or collagen-producing fibroblast-like cells, such as activated HSCs or pulmonary fibroblasts and subsequent regression of fibrosis, while sparing quiescent HSCs and quiescent pulmonary fibroblasts and resulting in no observable hepatic or pulmonary or systemic toxicity. According to the experimental data presented herein, not only are the viruses associated with a reduction of activated HSCs or pulmonary fibroblasts, but there is also a clear shift in signaling toward fibrotic regression, which could lead to the resolution of fibrosis even in those areas in the liver or lung in which HSCs or pulmonary fibroblasts are not directly infected by the viruses.

Furthermore, the inventors have shown that the viruses according to the present invention are safe and effective agents for the treatment of fibrosis associated with hepatocellular carcinoma (HCC), i.e. for the simultaneous treatment of fibrosis/cirrhosis and HCC.

FIGURES

Reference is now made to the figures wherein

Figure 1 shows the specificity of VSV for activated hepatic stellate cells (HSCs). A. Quiescent (serum starved) or TGF- -activated LX-2 cells and quiescent (day 2 post- plating) or activated (day 10) primary human HSCs were infected with rVSV-P-Gal at an MOI of 0.01, and aliquots of the conditioned medium were subjected to TCID50 analysis for determination of viral titers after 24 hours. For both cell types, statistically significant increases in titers were observed in activated cells (p < 0.05). B. Quiescent (serum starved) or TGF- -activated LX-2 cells and quiescent (day 2 post-plating) or activated (day 10) primary human HSCs were infected with rVSV-P-Gal at an MOI of 0.1, and lactate dehydrogenase (LDH) in the supernatant was measured at 8, 24, and 48 hours post-infection to determine cytotoxicity. C. Quiescent-like (day 2 post-plating) (a) or activated (day 10) primary human HSCs treated with either PBS or rVSV at an MOI of 0.01 for 48 hours were stained with propidium iodide and subjected to FACS analysis of sub-Gl populations for determination of apoptotic cells. Additional cells were subjected to Hoechst staining for determination of nuclear blebbing associated with apoptotic cells. Arrows indicate nuclear blebbing.D. Quiescent (day 2 post-plating) (a) or activated (day 10) primary human HSCs treated with either PBS (b) or rVSV-p-Gal (c) for 8 hours were subjected to immunofluorescent staining for SMA using a Cy3-labeled secondary antibody. Counterstaining was performed using DAPI for detection of nuclei. Representative photomicrographs are shown at 40x magnification. E. Gelatin zymography was performed from supernatants from quiescent or activated LX-2 (left panel) or primary human HSCs (right panel) after 8 hours treatment with PBS or rVSV- β-Gal;

Figure 2 shows the mechanism of VSV specificity for activated HSCs. A. IFN-β and ISRE promoter activation in primary human HSCs was quantified using luciferase reporter plasmids. Quiescent or activated cells were grown in 24-well plates and co- transfected with pIFN-p-Luc or pISRE-Luc and pRL (constitutively active Renilla), and stimulated with pI:C or IFN and VSV 24 hours post-transfection. After overnight incubation, fold-inductions were calculated by normalization to Renilla expression and comparison to mock-stimulated cells. IFN protection assay results are presented in the right panel. Quiescent or activated HSCs were plated in 24-well dishes and pre-treated overnight with increasing doses of IFN as indicated and infected with rVSV- -Gal at an MOI of 0.1 for 24 hours. Aliquots of supernatant were subjected to TCID50 analysis for determination of viral titers. Means of triplicate experiments are presented with error bars indicating standard deviation. B. 24-well dishes of activated LX-2 or primary human HSCs were treated with a panel of cell-cycle inhibitors or DMSO for 24 hours. Inhibitors were refreshed, and all wells were infected with rVSV-p-Gal at an MOI of 0.1 and incubated overnight. Aliquots of supernatant were, subjected to TCID50 analysis. Error bars indicate standard deviations of experiments performed in triplicate. C. Quiescent or activated LX-2 cells were treated with DMSO or a panel of cell cycle inhibitors for 24 hours. Cells were harvested and stained with propidium iodide and subjected to FACS analysis for determination of percentage of cells in each phase of the cell cycle; D. Activated LX-2 cells were transfected with siRNAs targeting cyclin B l or topoisomerase-2a, or with scramble siRNA, and then infected with VSV at an MOI of 0.01 overnight. Aliquots of the medium were subjected to TCID50 analysis of viral titers. To confirm siRNA knock-down of the respected gene expression, cell lysates were subjected to Western blot analysis using antibodies specific for the respective genes.

Figure 3 shows regression of hepatic fibrosis after in vivo administration of VSV. A. Rats harboring TAA-induced hepatic fibrosis were treated by intra-hepatic arterial infusion of either PBS (a, b) or rVSV- -Gal (c, d) and sacrificed on day 1 post-treatment. Paraffin-embedded liver sections were visualized by Masson's trichrome stain, and representative fields of view were photographed at 2x (a, c) or lOx (b, d) magnification. Stage 4 fibrosis (probable or definite cirrhosis) is exemplarily shown in the PBS-treated upper panel, as evidenced by fibrous bridges connecting portal tracts and central veins with formation of multiple nodules. The VSV-treated bottom panel is representative of stage 2 fibrosis, in which connective tissue connects neighboring portal tracts, but the overall architecture is preserved. B. Anillin blue staining without counterstaining was used to detect connective tissue. Slides were scanned at 20x magnification, and the resulting images were analyzed using commercial software to quantify areas of positive staining. The relative area of specific anillin stained regions were calculated as a ratio of the total area to represent fibrotic contents in PBS vs. VSV treated liver sections. Bottom panel: anillin ratios were plotted against Ishak staging scores for consecutive liver sections to determine a correlation of the two staging methods. The best-fit line was drawn, with an r² of > 0.8, indicating a strong correlation. C. The collagen contents of fibrotic livers treated with PBS or rVSV-P-Gal were compared using a commercially available kit for quantifying concentrations of acid-pepsin soluble collagen from solubilized samples of flash-frozen liver tissue. VSV therapy caused a significant reduction in soluble collagen contents (p < 0.05), resulting in approximately one-third the amount observed in PBS-treated livers; Figure 4 shows that VSV therapy leads to decreased SMA expression in vivo. A. Homogenates of snap-frozen sections of normal liver or TAA-induced fibrotic liver harvested 24 hours after intra-hepatic arterial infusion of PBS or VSV were subjected to Western blot analysis for SMA expression. Blots were stripped and re-probed with an antibody for tubulin to control for loading variations. Semi-quantification of band intensities normalized to tubulin was performed using ImageJ software. Three representative liver homogenates from each treatment group are shown. B. Rats harboring TAA-induced fibrotic livers were treated with either PBS or rVSV-P-Gal, and paraffin sections were prepared from tissues harvested 24 hours post-treatment. Immunohistochemical staining was performed with an antibody specific for SMA, and representative sections were imaged at 5x (left panel) or 20x (right panel) magnification. C. Normal or TAA-induced fibrotic livers were treated with rVSV-P-Gal, and analyzed by double-immunofluorescent staining against -Gal and SMA at 24 hours post-treatment. Counterstaining was performed with DAPI to visualize nuclei, and representative sections were photographed at 20x magnification. Merged images are shown in the bottom panel;

Figure 5 shows increased TUNEL staining in fibrotic livers following VSV therapy. Rats harboring TAA-induced hepatic fibrosis were treated with PBS or rVSV- -Gal via trans- arterial infusion of the hepatic artery. A. On day 3 post-treatment, animals were euthanized, and liver sections were paraffin-embedded and subjected to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) using a commercially available kit. Sections were counterstained with DAPI for localization of nuclei, and representative sections are shown at 20x magnification. B. Liver sections obtained on day 3 post treatment with PBS (left panel) or VSV (right panel) were subjected to immunohistochemical staining for NK cell marker (ANK61). The number of positively stained cells was quantified. P value < 0.01.

Figure 6 shows modulation of mRNA expression following VSV administration. TAA- induced fibrotic livers in rats were treated by trans-arterial infusion of PBS or rVSV-β- Gal via the hepatic artery. Liver samples snap frozen 24 hours post-treatment were subjected to mRNA purification and subsequent cDNA preparation using commercially available kits. Aliquots of cDNA were used for quantification of relevant genes associated with fibrotic progression by real-time PCR, as indicated. PBS values were set to 1, and VSV values were calculated as a fold-expression with respect to PBS. Means from each treatment group are shown, with error bars representing standard deviations.

Figure 7 shows VSV replication and cytotoxicity in primary mouse lung fibroblasts.

Primary fibroblasts were isolated from mouse lung and differentially cultured to achieve a quiescent or activated phenotype. Cells were treated with either control buffer (PBS) or rVSV at a multiplicity of infection (MOI) of 0.01 for 48 hours. A. Aliquots of conditioned medium were harvested at 24 or 48 hours post-infection and subjected to TCID50 analysis of virus titers. B. Cells were photographed at 200x magnification to reveal differences in viability among quiescent and activated cells treated with PBS or VSV after 48 hours. Representative images are shown. C. Cells were immunofluorescently stained for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and counter-stained with DAPI to reveal cell nuclei, which appear in blue. TU EL-positive cells are green. Images are representative. D. Cells were stained with propidium iodide (PI) and annexin V and subjected to FACS analysis. Data were obtained by gating for PI negative and annexin V positive populations.

Figure 8 shows Reduction of aSMA expression in activated lung fibroblasts after VSV infection. Activated primary mouse lung fibroblasts were either treated with control buffer (PBS) or rVSV at an MOI of 0.01 for 24 hours. A. Cells were harvested and mRNA was extracted for analysis aSMA expression by quantitative real-time PCR. B. Cells were immunofluorescently stained for aSMA (green) and co-stained for phalloiden (red) and DAPI (blue). Representative images are shown at 250x magnification.

EXAMPLES The present invention shall now further be described by means of the following examples which are intended to illustrate and not to limit the present invention:

1. VSV replication and subsequent cell killing is specific for activated, and not quiescent HSCs

To compare the relative sensitivities of activated and quiescent HSCs to VSV infection, LX-2 and primary HSCs were prepared and infected at an MOI of 0.01 for 24 hours. TCID50 analyses of conditioned supernatants revealed a significant differential in virus replication, with titers reaching 2-3 logs higher in activated, as compared to quiescent, cells (Figure 1 A). Furthermore, to determine if this augmented VSV replication resulted in increased cytotoxicity in activated HSCs, lactate dehydrogenate (LDH) assays were performed (Figure IB). For the above and the following experiments recombinant VSV, namely wildtype VSV expressing β-Gal (rVSV-p-Gal), was used.

2. VSV infection results in an inactivation of differentiated HSCs at early time-points post-infection

To investigate the type of cell death undergone by activated HSCs, and to differentiate necrotic versus apoptotic death, the present inventors performed FACS analysis of propidium iodide (PI) stained primary human HSCs. The sub-Gl populations were quantified to determine the percentage of apoptotic cells. A significant increase in apoptosis was observed in HSCs infected after 10 days in culture, while a negligible amount of apoptosis could be quantified in cells infected after only 2 days in culture, indicating that the ability of VSV to induce apoptosis was directly correlated with degree of activation (Fig 1C, top panel). To further confirm this phenomenon, primary human HSCs were subjected to Hoechst staining and subsequent immunofluorescent analysis at 0 and 48 hours post-infection with VSV. As expected, chromatin condensation and nuclear condensation were apparent only in the HSCs which were infected after 10 days in culture (Fig 1C, bottom panel), confirming that, not only does VSV induce cytotoxicity specifically in an activation-specific manner in HSCs, but the cell death is apoptotic in nature.

To investigate the effects of VSV treatment of activated HSCs at early time-points postinfection, immunofiuorescent staining for SMA, a marker for activated HSC, was performed in primary human HSCs. While quiescent cells expressed very low levels of SMA as expected, activated HSCs infected with VSV for 8 hours expressed much lower amounts of the marker as compared to PBS-treated cells at the same time-point (Figure ID). To further examine this issue, gel zymograms were performed to compare matrix metalloproteinase (MMP) activities in the supematants of quiescent cells versus activated cells treated with PBS or VSV for 8 hours. MMP-2, an enzyme which degrades type IV collagen, the major structural component of basement membranes, is known to be secreted from activated HSCs and is upregulated in fibrotic liver tissue. In fact, a significant increase in MMP-2 activity was observed in the supematants of activated HSCs treated with PBS, as compared with quiescent cells. After 8 hours of VSV infection, MMP-2 activity was reduced to nearly that of quiescent cells (Figure IE), further indicating a VSV-induced inactivation of HSCs. These results were reproducible both in LX-2 cells, as well as in primary human HSCs.

3. Interferon signaling is not the major determinant of VSV specificity for activated HSCs

To elucidate the mechanism by which VSV exerts its specificity for activated, as opposed to quiescent, HSCs, first the robustness of interferon (IFN) signaling in both cell phenotypes was investigated. To this end, reporter assays were performed, in which firefly luciferase is driven by the IFN- or interferon stimulated response element (ISRE) promoters. While both quiescent and activated LX-2 and primary HSCs demonstrated induction of IFN- and ISRE in response to polyI:C or IFN and virus in the medium, implying a functional IFN induction and response, respectively, there was a slight impairment of IFN- signaling in .the activated cells (Figure 2A). To determine if this impairment was responsible for the augmented VSV replication in activated cells, an IFN protection assay was performed. Both quiescent and activated LX-2 and primary HSCs were pre-treated overnight with increasing doses of universal type I IFN, and then infected with VSV. Surprisingly, even relatively low concentrations of IFN were sufficient for attenuation of VSV titers in activated HSCs, and no significant differences in titers were detected upon comparison of activated and quiescent cells at any dose of IFN. Therefore, it was concluded that, despite some degree of impairment in IFN signaling in activated HSCs, this factor does not seem to be the major determinant of specificity of VSV for activated cells.

4. Specificity of VSV replication for activated HSCs is associated with cell-cycle progression

Since proliferation is an important feature of activated HSCs it is intriguing to speculate if perhaps some component of cell cycle progression was essential for VSV replication. To test this hypothesis, a panel of cell cycle inhibitors was used to pre-treat activated LX- 2 and primary HSCs prior to VSV infection. TCID50 analysis of conditioned supernatants revealed a significant attenuation of viral replication in those cells treated with roscovitine, etoposide and the JNK2 inhibitor (Figure 2B), while no other tested inhibitor had an appreciable affect on virus growth. To further elucidate the mechanism by which the selected inhibitors interfered with VSV replication, FACS analysis of propidium iodide stained cells was performed after 24 hours of treatment with each cell cycle inhibitor. Interestingly, while most of the inhibitors tested resulted in arrest of activated HSCs in Gl phase, roscovitine, etoposide and JNK2 inhibitors were unique in that they caused a significant accumulation of cells in G2 (Figure 2C).

To confirm this finding siRNAs against genes associated with G2-S progression were used. To this end, activated LX-2 cells were transfected with siRNA targeting cyclin Bl and topoisomerase-2cc prior to virus infection. As a control, a scramble siRNA was transfected. After confirming that the protein expression of cyclin B l and topoisomerase-2a was inhibited by the respective siRNA (Fig. 2D, bottom panel), TCID50 analysis of aliquots of the supernatant after overnight infections was performed. Indeed, viral titers were significantly inhibited in cells treated with siRNAs against genes involved in G2 cell cycle progression in comparision to the scramble control (Fig. 2D, top).

5. In vivo therapy of TAA-induced fibrosis with VSV results in improved liver staging, a reduction of intra-hepatic fibrotic content, and a reduction of soluble collagen in the liver

Based on the specificity of VSV for activated HSCs observed in vitro, the present inventors hypothesized that VSV therapy could result in anti-fibrotic effects in vivo. To test this idea, they established a rat model of hepatic fibrosis by long-term exposure to thiacetamide (TAA) in drinking water. Orthotopic single-nodule HCC tumors were implanted by injection of syngeneic McA-RH7777 cells into the liver capsules of Buffalo rats to simulate the clinical case of HCC with underlying hepatic fibrosis. As a control, HCC cells were similarly implanted into the livers of healthy age-matched Buffalo rats who were fed with normal drinking water for the same time period and subsequently had normal liver function. To compare virus replication and oncolytic effects within tumors implanted in normal versus fibrotic livers, PBS or rVSV~Gal was injected via the hepatic arteries of both groups of rats, and tumor and liver tissue was extracted on day 1 or 3 post-treatment for histological examination and TCID50 analyses of viral titers. As anticipated, VSV replicated to similar intratumoral titers, which translated to comparable percentages of tumor necrosis, irrespective of underlying liver function (data not shown). Similarly, analysis of serum chemistries (GPT, BUN, creatinine) demonstrated only transient elevations on day 1 post-treatment, which returned to normal levels on day 3 and did not differ significantly between animals harbouring normal or fibrotic livers (data not shown).

Next, histological sections of liver were subjected to Ishak classification to determine the fibrotic stages in response to treatment. While PBS-treated liver sections were assigned a mean score of 3 due to the presence of porto-portal and porto-central bridges, VSV- treated sections were down-graded to a mean score of 2, which represents a less advanced stage of fibrosis (Figure 3A). To obtain a quantitative assessment of fibrotic progression, an anillin staining on liver sections was performed to visualize connective tissue and then morphometric analysis was applied to quantify the fibrotic contents of livers in response to treatment with PBS or rVSV-P-Gal. As anticipated, trans-arterial infusion of VSV led to a decrease in fibrotic tissue within the liver as compared to that of PBS-infused animals (Figure 3B). In order to validate this experimental protocol, and to compare this data with the previously determined Ishak scores, to two sets of values were plotted against each other. Indeed, the r² value for the resultant best-fit line was greater than 0.8, indicating that the two sets of data were positively correlated.

As a third measure of fibrotic progression, the collagen contents of fibrotic livers treated with PBS or rVSV- -Gal were compared using a commercially available kit for quantifying concentrations of acid-pepsin soluble collagen from solubilized samples of flash-frozen liver tissue. From this assay, it was determined that VSV therapy caused a significant reduction in soluble collagen contents (p < 0.05), resulting in approximately one-third the amount observed in PBS-treated livers (Figure 3C). Together, the improvement in fibrotic staging, decrease in fibrotic content, and reduction in soluble collagen content in response to oncolytic VSV therapy, all indicate that the virus has a beneficial effect in fibrosis reversal.

6. Trans-arterial infusion of VSV results in a reduction of SMA expression by HSCs

To ascertain the effect of VSV treatment on SMA expression, first liver homogenates were subjected to Western blot analysis. As predicted, samples prepared from PBS- treated fibrotic livers demonstrated significantly increased amounts of SMA compared with normal control livers. However, this increase was almost completely abrogated following VSV infusion (Figure 4A). Semi-quantification of this data, using tubulin as a loading control, revealed that the difference in SMA expression in the livers of PBS and rVSV-P-Gal treated fibrotic livers was statistically significant (p = 0.01). To further support these findings, paraffin-embedded liver sections from the same rats were processed for immunohistochemical staining to visualize SMA protein. As expected, a qualitative difference among PBS- and rVSV-P-Gal -treated livers was observed, as evidenced by a clear reduction in, not only the number of positively stained cells, but also the intensity of staining on day 1 post-rVSV- -Gal administration (Figure 4B). Similar findings were also observed on day 3 (data not shown). In order to determine whether or not this effect on SMA expression was directly due to infection of activated hepatic stellate cells, a double-immuno-fluorescent staining of these same tissues was performed. Because the rVSV vector expressed a -Gal reporter gene, SMA expression and VSV infection could be detected using specific antibodies for SMA and -Gal, respectively, and appropriate fluorescently labeled secondary antibodies. Interestingly, a distinctive co- localization of -Gal with SMA-positive cells was observed (Figure 4C), indicating that VSV infects and replicates in activated HSCs with high specificity. No specific accumulation of virions was detected in normal control liver sections, which expressed very low levels of basal SMA.

7. VSV treatment in TAA-induced fibrotic livers is associated with an increase in apoptotic cells

To analyze apoptosis in TAA-induced fibrotic livers, a commercially available immuno- fluorescent TUNEL staining assay was employed. Results demonstrated the existence of increased numbers of apoptotic cells in those livers treated with VSV (Figure 5). Based on the observation that the apoptotic cells coincided with the locations of extracellular collagen deposits, it is consistent with the probability that these cells were most likely activated HSCs. These data suggest that replication of VSV in activated HSCs leads to apoptosis, an important mechanism associated with repression of fibrosis.

8. An increase in NK cell accumulation corresponds with rVSV treatment in fibrotic livers.

Natural killer (NK) cells have been implicated in the induction of apoptosis in activated HSCs. Since a significant intratumoral infiltration of NK cells to sites of VSV replication had been observed by the present inventors, immunohistochemical staining of fibrotic liver tissue on day 1 post-VSV or PBS therapy was performed to quantify the accumulation of NK cells in response to therapy. Interestingly, a significant increase in the number of NK cells in VSV-treated livers was observed (Fig. 5B), and the accumulation was especially apparent along the connective tissue. This observation could provide a secondary mechanism for apoptosis of activated HSCs not directly infected by VSV.

9. In vivo administration of VSV results in therapeutic modulation of key genes associated with fibrotic progression

To investigate the effects of anti-fibrotic VSV therapy at an mRNA level, quantitative real-time PCR from cDNAs prepared from snap-frozen fibrotic liver sections from rats treated with either PBS or rVSV-p-Gal on days 1 and 3 post-treatment was performed. Consistent with a repression of the fibrotic condition, qPCRs revealed significant decreases in SMA, TGF-, Pro-collagen, and TIMP-1 mRNA, as well as an increase in IFN- in response to VSV therapy (Figure 6). These results indicate that, not only is VSV associated with a reduction of activated HSCs, as indicated by the reduction in SMA mRNA, but there is also a clear shift in signaling toward fibrotic regression, as evidenced by the decrease in TGF-, TIMP-1 and pro-collagen, which could lead to the resolution of fibrosis even in those areas in the liver in which HSCs are not directly infected by VSV.

10. VSV replicates and causes apoptosis preferentially in activated mouse lung fibroblasts.

To compare the relative permissiveness of quiescent versus activated lung fibroblasts, primary cells from mouse lung were isolated and differentially cultured, either in starvation medium or in serum-rich medium containing TGF-β, to induce quiescent and activated phenotypes, respectively. Cells were then infected with rVSV at a multiplicity of infection (MOI) of 0.01 , and aliquots of the conditioned medium were harvested at 24 and 48 hours post-infection for determination of viral titers. While viral replication was clearly attenuated in quiescent fibroblasts, the activated cells seemed to support robust propagation of VSV, with titers reaching nearly 10⁷ TCID50/ml by 48 hours, representing a 2 log differential in comparison with quiescent cells (Figure 7A). To determine the effect of viral replication on cytotoxicity, the cells were examined microscopically and photographed at 200x magnification. A significant cytopathic effect (CPE) could be observed in activated fibroblasts treated with VSV, while the infected quiescent cells appeared to be quite viable and were nearly indistinguishable from buffer treated cells (Figure 7B). To further confirm these results, and to ascertain whether or not the cell death induced in activated cells by VSV infection was apoptotic in nature, two independent assays for apoptosis were performed. First, immunofluorescent staining for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed, which revealed a significant upregulation of TUNEL-positive cells specifically in activated fibroblasts treated with VSV (Figure 7C). This was substantiated by FACS analysis of propidium iodide (PI) and annexin V stained cells with gating for annexin V positive and PI negative, which demonstrated a significant increase in apoptotic cell populations in activated fibroblasts after VSV treatment, while quiescent cells revealed only a marginal increase in apoptosis (Figure 7D).

1 1. VSV infection results in a decrease in aSMA expression in activated lung fibroblasts.

As ct-smooth muscle actin (aSMA) is a marker for activated fibroblasts, the effect of VSV infection on the expression levels of aSMA in vitro was investigated. Primary mouse lung fibroblasts were differentially cultured to achieve quiescent and activated phenotypes, and were then treated with control buffer or rVSV at an MOI of 0.01 for 24 hours. Aliquots of cells were prepared either for mRNA analysis by quantitative realtime PCR (qPvT-PCR) or for immunofluorescent staining using an antibody against aSMA. Analysis of mRNA expression levels of aSMA normalized to an internal housekeeping control (GAPDH) revealed a significant reduction after rVSV treatment (p < 0.01) (Figure 8A). In addition, a reduction in aSMA staining in activated fibroblasts treated with VSV was observed, which was partially due to the significant CPE observed in these cells (Figure 8B). The data presented here provide evidence that the antifibrotic properties of VSV demonstrated previously in a hepatic fibrosis model can be extended to the case of pulmonary fibrosis. Here it is shown that VSV replicates preferentially in activated, as opposed to quiescent, primary mouse lung fibrosis and results in apoptotic cell death specifically in these cells. This is further supported by a down-regulation of aSMA in activated fibroblasts following VSV therapy, indicating a possible reversal of the activated phenotype prior to cell death. Taken together, these data support the notion that VSV exploits the proliferative nature of activated fibroblasts, independently of their tissue of origin, in order to propagate selectively in these cells while sparing normal quiescent cells.

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The features of the present invention disclosed in the specification, the claims and/or in the accompanying drawings, may, both separately, and in any combination thereof, be material for realizing the invention in various forms thereof.

Claims

1. A virus for use in a method of treatment of fibrosis, wherein said fibrosis is associated with a pathogenic mechanism which involves the proliferation of collagen-producing fibroblasts or collagen-producing fibroblast-like cells.

2. Virus according to claim 1, wherein said fibrosis is liver fibrosis or pulmonary fibrosis.

3. Virus according to claim 1 , wherein said virus is an RNA virus, preferably a negative- strand RNA virus.

4. Virus according to claim 3, wherein said negative-strand RNA virus is selected from the group of vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), measles virus, mumps virus, Sendai virus (SeV) and influenza viruses, preferably from the group of vesicular stomatitis virus (VSV), Newcastle disease virus (NDV), measles virus.

5. Virus according to claim 4, wherein said negative-strand RNA virus is a vesicular stomatitis virus (VSV).

6. Virus according to any of the foregoing claims, wherein said virus is a wildtype virus or a recombinant virus.

7. Virus according to any of the foregoing claims, wherein said virus is provided in the form of a vector.

8. Virus according to any of the foregoing claims, wherein said virus does not contain any exogenous agent or express any exogenous gene, wherein said exogenous agent or exogenous gene is not IFN-ct, IFN-β, srlicB, M3, MMP1 , MMP8 or relaxin.

9. Virus according to any of the foregoing claims, wherein said virus is a recombinant vesicular stomatitis virus (rVSV).

10. Virus according to claim 9, wherein said recombinant vesicular stomatitis virus is selected from the group of rVSV(MA51) and wildtype VSV or rVSV(MA51) expressing one or more of the following transgenes: IFN-a, IFN-β, srli B, M3, MMP1, MMP8 and relaxin.

1 1. Virus according to any of the foregoing claims, wherein said virus does not contain any exogenous agent or express any exogenous gene at all.

12. Virus according to any of the foregoing claims, wherein said virus selectively induces apoptosis of collagen-producing fibroblasts or collagen-producing fibroblast-like cells, e.g. activated hepatic stellate cells (HSCs) or activated fibroblasts, such as activated pulmonary fibroblasts.

13. Virus according to any of claims 2-12, wherein said liver fibrosis is associated with chronic liver injury, portal hypertension, ascites, encephalopathy, metabolic dysfunction, liver cirrhosis and/or hepatocellular carcinoma (HCC), and wherein said pulmonary fibrosis is idiopathic pulmonary fibrosis or is a pulmonary fibrosis associated with autoimmune disorders, sarcoidosis, Wegener's granulomatosis, viral infections, inhalation of environmental pollutants, or smoking.

14. Virus according to claim 13, wherein said chronic liver injury is caused by persistent viral, toxic, autoimmune, metabolic and/or cholestatic impairments, in particular by long-term alcohol abuse and/or chronic hepatitis C virus infection.

15. A virus as defined in any of claims 2 to 1 1 for use in a method of selectively inducing apoptosis of collagen-producing fibroblasts or collagen-producing fibroblast-like cells, e.g. activated hepatic stellate cells (HSCs) or activated fibroblasts, such as activated pulmonary fibroblasts.