Novel mechanism for controlling RNA virus replication and gene expression

Abstract

To provide a novel mechanism to control RNA virus replication and gene expression using a conditional protease approach using a specific protease inhibitor for regulation, more specifically, to provide a single-stranded RNA virus, preferably of the order Mononegavirales, comprising a polynucleotide sequence encoding at least one protein essential for viral transcription and / or replication, a protease and a cleavage site for the protease.SOLUTION: The protease can be inhibited using a protease inhibitor and hence the protease and the cleavage site for the protease form a regulatable switch. By changing the insertion site of the regulatable switch from an INTRA- to an INTER-molecular location in the at least one protein essential for viral transcription and / or replication, the effect of the protease inhibitor can be altered from an ON switch to an OFF switch. RNA virus may further encode a heterologous protein, the expression of which is then regulated by regulating viral activity. The ON switch may also be used in an RNA virus to directly regulate heterologous protein expression. Further provided are in vivo and in vitro uses of the virus with conditional viral activity or heterologous protein expression.SELECTED DRAWING: None

Description

Field of the Disclosure

[0001] The present invention relates to a novel mechanism for controlling RNA virus replication and gene expression using a conditional protease approach, employing a protease-specific regulatory inhibitor. More specifically, the present invention relates to a single-stranded RNA virus, preferably a Mononegavirales single-stranded RNA virus, comprising a polynucleotide sequence encoding at least one protein essential for viral transcription and / or replication, a protease, and a cleavage site for said protease. The protease can be inhibited using a protease inhibitor, and thus the protease and the cleavage site for said protease form a regulatable switch. By changing the insertion site of the regulatable switch from an intramolecular to an intermolecular position in at least one protein essential for viral transcription and / or replication, the effect of the protease inhibitor can be changed from an ON switch to an OFF switch. The RNA virus may further encode a heterologous protein, the expression of which can then be regulated by controlling viral activity. The ON switch in the RNA virus can be used to directly regulate the expression of the heterologous protein. Furthermore, in vivo and in vitro uses of said viruses for conditional viral activity or heterologous protein expression are provided. [Background technology]

[0002] Recombinant viruses have shown great potential as efficient gene therapy vectors, viral immunotherapy, and oncolytic viruses in cancer virotherapy. These three distinct therapeutic modalities generally utilize gene overexpression, gene knockdown using RNA interference, and suicide gene delivery. In therapeutic settings, temporal control of transgene or viral gene expression constitutes both a safety switch and an efficacy dial. While modifiers of DNA virus activity, such as regulatable promoters (e.g., the Tet system), are well established, these mechanisms are unable to regulate most RNA viruses (with the exception of retroviruses).

[0003] Aptazymes are a mechanism for tightly controlling RNA viruses (Ketzer et al., PNAS, 2014, 111(5): E554-62). Aptazymes consist of an RNA structure that responds to small compounds (aptamers) and an enzymatically active RNA (ribozyme). This mechanism was found to regulate the spread of RNA measles viruses by acting as an OFF switch on their fusion proteins. However, it did not control viral transcription or replication. For measles virus, effective inhibition of viral spread required the placement of aptazymes in both the 3' UTR and 5' UTR of the viral fusion protein, resulting in a 1000-fold reduction in viral progeny. The inhibition occurred at a later stage in the viral replication cycle, i.e., the fusion step. Apparently, even a small amount of fusion protein is sufficient to promote viral spread. Thus, a reduction of 1 / 1000 occurred only with multistage infection assays (low MOI of 0.0001) and a long observation period of 8 days. Single insertion of aptazymes into either the 3' UTR or 5' UTR did not significantly reduce titers.

[0004] Following a different approach, OFF-switch control of measles virus RNA replication was demonstrated by a small molecule-assisted shutoff (SMASh) tag fused at the C-terminus to the viral P protein, which controls protein degradation (Chung et al., Nature Chemical Biology, 2015, 11:713-722).

[0005] We attempted to develop a regulatable system based on conditional proteolysis. This system involves cloning a small human immunodeficiency virus (HIV) protease (99 amino acids) adjacent to the cleavage site of the HIV protease into different loci in the genome of the RNA vesicular stomatitis virus (VSV) as a model protease. HIV protease is active as a homodimer and acts as an aspartyl protease. In HIV, this protease cleaves the polyprotein translated from the positive-strand genome into functional proteins. HIV protease is essential for the viral replication cycle, and several protease inhibitors have been approved by drug regulatory agencies, with novel inhibitors currently under development. One such protease inhibitor is amprenavir, which binds to the catalytic center between HIV protease homodimers, thereby attenuating its function.

[0006] Vesicular stomatitis virus (VSV), a negative-sense single-stranded RNA virus and the prototype of the Rhabdoviridae family, has been widely explored as a vaccine vector, oncolytic virus, and tracking tool. Despite its widespread use in basic viral science and therapeutic virus development, VSV, an RNA virus, has not yet been shown to be externally regulatable. The VSV RNA genome contains five viral genome components, in the following order from 3' to 5': the nucleoprotein (N protein), followed by the phosphoprotein (P protein), matrix protein (M protein), glycoprotein (G protein), and finally the polymerase or large protein (L protein). All VSV genes are transcribed sequentially by the VSV polymerase using the same insertion site at the 3' end (upstream of the N protein) where transcription is initiated. The genes are interspersed with intergenic regions, allowing transcription of several viral mRNAs from a single RNA genome. The first viral protein, the N protein, spans the viral RNA genome and interacts with the viral polymerase complex formed by the P and L proteins. The M protein forms the viral capsid and inhibits cellular translation by blocking nuclear pores. The G protein promotes cell attachment and entry, and its membrane fusion properties constitute another virulence factor. Clinical development of VSV has been limited due to potential neurotoxic side effects observed in experimental animals. Summary of the Invention

[0007] For the first time, we present a regulatory switch that conditionally controls the activity of an RNA virus in the presence of an exogenously administered clinically approved compound. Changing the insertion site of the switch from an intramolecular to an intermolecular location allows the compound's effect to be converted from an ON switch to an OFF switch. These regulatory elements offer a novel safety measure for RNA viruses currently being considered for development as therapeutic viruses in the fields of oncology and vaccination. Furthermore, they rely on the presence of an administered drug to provide environmental safety protection in the event of viral shedding during treatment. In the case of the ON switch, an autocatalytically active protease, such as HIV protease, is inserted into the intramolecular insertion site, i.e., within the open reading frame, of essential proteins, such as the P protein and / or L protein, of prototypic negative-strand RNA viruses such as VSV. Addition of a protease-specific inhibitor, such as an HIV protease inhibitor, prevents cleavage of these essential viral proteins, allowing viral polymerase activity to proceed. Furthermore, we identified a novel insertion site in the L protein that has only a minor effect on viral replication. In the case of the OFF switch, an autocatalytically active protease inserts into the intermolecular insertion site and fuses to an essential protein, generating a nonfunctional fusion protein. Autoproteolysis releases functional essential proteins, such as the L protein of VSV, a prototypical negative-strand RNA virus. Addition of specific protease inhibitors, such as HIV protease inhibitors, prevents cleavage of the dysfunctional polyprotein and thus inhibits viral polymerase activity.

[0008] In one aspect, a single-stranded RNA virus is provided, comprising a modified viral genome including a polynucleotide sequence encoding at least one protein essential for viral transcription and / or replication, a protease, and a cleavage site for the protease, wherein (a) the at least one protein essential for viral transcription and / or replication comprises an insertion at an intramolecular insertion site comprising at least the protease and, optionally, a cleavage site for an additional protease, or (b) the at least one protein essential for viral transcription and / or replication is encoded as a fusion protein comprising a protease fused to its N-terminus or C-terminus, separated by the cleavage site for the protease. Optionally, the virus may further encode a heterologous protein. In one option (a), also referred to herein as an ON switch, the protease cleaves the at least one protein essential for viral transcription and / or replication at the cleavage site for the protease at the intramolecular insertion site. In the other option (b), also referred to herein as an OFF switch, the protease cleaves at a protease cleavage site located at the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication encoded by the fusion protein, thereby releasing the at least one protein essential for viral transcription and / or replication. In one embodiment of option (b), the fusion protein does not comprise the amino acid sequence of SEQ ID NO: 30. The protease may be any protease, as long as it is inhibited using a protease inhibitor.

[0009] In one embodiment, at least one protein essential for viral transcription and / or replication is an RNA-dependent RNA polymerase or a protein of a polymerase complex comprising an RNA-dependent RNA polymerase or a nucleocapsid, preferably selected from the group consisting of a polymerase cofactor (such as the P protein or a functional equivalent thereof), a polymerase (such as the L protein), and a nucleocapsid (such as the N protein).

[0010] Preferably, the single-stranded RNA virus is a negative-sense single-stranded RNA virus, more preferably a negative-sense single-stranded RNA virus of the order Mononegavirales. In a specific embodiment, the single-stranded RNA virus is a virus selected from the group consisting of Rhabdoviridae, Paramyxoviridae, Filoviridae, Nyamiviridae, Pneumoviridae, and Bornaviridae. Preferably, the single-stranded RNA virus is a virus of the family Paramyxoviridae, preferably measles virus (MeV) or a virus of the family Rhabdoviridae, preferably a virus of the genus Vesiculovirus, and most preferably vesicular stomatitis virus (VSV). In one embodiment, the virus is an oncolytic virus, preferably the oncolytic virus is VSV. In an even more preferred embodiment, the vesiculovirus is a vesicular stomatitis virus having the glycoprotein GP of lymphocytic choriomeningitis virus (LCMV), preferably having the WE-HPI strain. Such a VSV is described, for example, in International Patent Publication WO2010 / 040526 and is designated VSV-GP.

[0011] In one embodiment, the single-stranded RNA virus is a negative-sense single-stranded RNA virus of the order Mononegavirales, and at least one protein essential for viral transcription and / or replication is selected from the group consisting of a polymerase cofactor, a polymerase, and a nucleocapsid, and preferably, the at least one protein essential for viral transcription and / or replication is (a) a polymerase cofactor, preferably a P protein or a functional equivalent thereof; (b) a polymerase, preferably an L protein; and / or (c) a combination thereof.

[0012] The protease used in accordance with the present invention regulates the activity of at least one protein essential for viral transcription and / or replication. Therefore, the protease also regulates viral transcription and / or replication. In one embodiment, the protease is an autocatalytic protease. In another embodiment, or in addition, the protease is a viral protease, preferably derived from HCV or HIV. In another embodiment, the protease is an HIV-1 protease, preferably a single-chain dimer of HIV-1 protease. Suitable HIV-1 protease inhibitors include, but are not limited to, indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, amprenavir, fosamprenevir, atazanavir, tipranavir, or darunavir.

[0013] In embodiments having an insertion at an intramolecular insertion site (ON switch), the insertion at the intramolecular insertion site of at least one protein essential for viral transcription and / or replication does not affect or does not substantially affect the activity of at least one protein essential for viral transcription and / or replication.

[0014] In certain embodiments, the cleavage sites for at least the protease and optionally the additional protease are located within the intramolecular insertion site of at least one protein essential for viral transcription and / or replication, and proteolytic cleavage of the protein cleaves at the protease cleavage site within the intramolecular insertion site at least one protein essential for viral transcription and / or replication. Cleavage within the intramolecular insertion site inactivates at least one protein essential for viral transcription and / or replication. Further cleavage within the intramolecular insertion site of at least one protein essential for viral transcription and / or replication inhibits viral transcription and / or replication. Thus, the virus is active in the presence of a specific inhibitor of the protease and inactive in the absence of the specific inhibitor of the protease. The virus may further encode at least one heterologous protein, which is expressed when the virus is active in the presence of the specific inhibitor of the protease and is not expressed when the virus is inactive in the absence of the specific inhibitor of the protease.

[0015] In another embodiment, the single-stranded RNA virus is vesicular stomatitis virus (VSV), and the at least one protein essential for viral transcription and / or replication is the P protein and / or the L protein. A non-limiting example of a suitable intramolecular insertion site in the P protein is the flexible hinge region of the VSV P protein, preferably located at amino acid positions 193-199, more preferably at amino acid position 196, of the VSVi P protein (such as the amino acid sequence of SEQ ID NO: 27). In one embodiment, the VSV P protein is derived from the VSV Indiana strain (VSVi), and the intramolecular insertion site in the P protein is the flexible hinge region of the VSV P protein, preferably located at amino acid positions 193-199, more preferably at amino acid position 196, of the VSVi P protein (such as the amino acid sequence of SEQ ID NO: 27). Non-limiting examples of suitable intramolecular insertion sites in the L protein include those within the loop of the methyltransferase (MT) domain of the L protein corresponding to amino acids 1614-1634, preferably amino acids 1614-1629, more preferably amino acids 1616-1625, and even more preferably amino acid 1620, of the VSVi L protein (such as the amino acid sequence of SEQ ID NO: 28). In one embodiment, the VSV L protein is derived from the VSV Indiana strain (VSVi), and the intramolecular insertion site in the L protein is within the loop of the methyltransferase (MT) domain of the L protein derived from amino acids 1614-1634, preferably amino acids 1614-1629, more preferably amino acids 1616-1625, and even more preferably amino acid 1620, of the VSVi L protein (such as the amino acid sequence of SEQ ID NO: 28). In one embodiment, the vesicular stomatitis virus (VSV) and at least one protein essential for viral transcription and / or replication is a P protein and an L protein having an insertion at the intramolecular insertion site as described above.

[0016] In the option with an insertion at the intermolecular insertion site (OFF switch), at least one protein essential for viral transcription and / or replication is encoded as a fusion protein comprising a protease fused to the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication, separated by a cleavage site for the protease. In certain embodiments, proteolytic cleavage of the fusion protein releases the at least one protein essential for viral transcription and / or replication in its active form. At least one protein essential for viral transcription and / or replication in the fusion protein comprising a protease fused to the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication, separated by a cleavage site for the protease, is inactive in the absence of proteolytic cleavage. Proteolytic cleavage of the fusion protein can be inhibited using a specific inhibitor of the protease. Thus, the virus is inactive in the presence of a specific protease inhibitor of the protease and active in the absence of the specific inhibitor of the protease. The virus may further encode at least one heterologous protein, which is not expressed when the virus is inactive in the presence of a specific inhibitor of the protease, but is expressed when the virus is active in the absence of the specific inhibitor of the protease. The fusion protein may also contain an additional viral protein fused to the opposite end of the protease fused to the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication, the additional viral protein and the protease also being separated by a cleavage site of the protease. In one embodiment, the protease replaces the intergenic region flanked on either side by the cleavage site of the protease and linking at least one protein essential for viral transcription and / or replication with the additional viral protein.Loss of the protease thus results in an additional inactive fusion protein comprising a protein essential for viral transcription and / or replication and an additional viral protein. The fusion protein may also comprise a heterologous protein fused to the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication, opposite the protease fused to the N-terminus or C-terminus of the protein, wherein the heterologous protein and the protease are separated by a cleavage site for the protease. In one embodiment, the protease replaces the intergenic region flanking the cleavage site for the protease on either side and linking at least one protein essential for viral transcription and / or replication with the heterologous protein. Loss of the protease thus results in an additional inactive fusion protein comprising a protein essential for viral transcription and / or replication and a heterologous protein. The fusion protein may further comprise a linker between the protease and the at least one protein essential for viral transcription and / or replication, or, if applicable, between the protease and the additional viral or heterologous protein. The linker may separate the protease from the cleavage site, or may separate the cleavage site from at least one protein essential for viral transcription and / or replication, and / or may separate the protease from a further viral or heterologous protein.

[0017] In a preferred embodiment of this alternative (OFF switch), the single-stranded RNA virus is a negative-sense single-stranded RNA virus of the order Mononegavirales, and at least one protein essential for viral transcription and / or replication is the L protein. In another preferred embodiment of this alternative (OFF switch), the fusion protein comprises a protease fused to the N-terminus of at least one protein essential for viral transcription and / or replication, separated by a cleavage site for said protease. In yet another embodiment of this alternative (OFF switch), the single-stranded RNA virus is a negative-sense single-stranded RNA virus of the order Mononegavirales, at least one protein essential for viral transcription and / or replication is the L protein, and the fusion protein comprises a protease fused to the N-terminus of the L protein, separated by a cleavage site for said protease.

[0018] In another aspect, the present invention relates to an RNA virus comprising a modified viral genome comprising a polynucleotide sequence encoding at least one heterologous protein, a protease, and a cleavage site for said protease, wherein the at least one heterologous protein comprises an insertion at an intramolecular insertion site comprising at least the cleavage site for said protease, and optionally further comprising a protease. The virus may be an oncolytic virus, preferably VSV.

[0019] The heterologous protein may be a therapeutic protein, a reporter, or a tumor antigen.

[0020] In a further aspect, the present invention relates to an RNA virus according to the invention for use in therapy, in particular for use in the treatment of cancer. The cancer may be a solid tumor, preferably selected from the group consisting of colon cancer, prostate cancer, breast cancer, lung cancer, NSCLC (non-small cell lung cancer), skin cancer, liver cancer, bone cancer, ovarian cancer, pancreatic cancer, brain tumor, head and neck cancer, HNSCC (head and neck squamous cell carcinoma), lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma), brain cancer, neuroblastoma, mesothelioma, Wilms' tumor, retinoblastoma, and sarcoma.

[0021] In yet another aspect, the present invention relates to a recombinant VSV L protein comprising an insertion within the loop of the methyltransferase domain of the L protein corresponding to amino acids 1614 to 1634, preferably amino acids 1614 to 1629, more preferably amino acids 1616 to 1625, and even more preferably amino acid 1620 of the VSV L protein (SEQ ID NO: 28). The insertion may comprise a reporter protein (such as luciferase or a fluorescent protein) or a protease cleavage site, or a protease and a cleavage site for the protease. When the insertion comprises a protease cleavage site, or a protease and a cleavage site for the protease, the protease may be a viral protease and / or an autocatalytic protease. Preferably, the protease is derived from HCV or HIV. In one embodiment, the protease is HIV-1 protease, preferably a single-chain dimer of HIV-1 protease. Suitable HIV-1 protease inhibitors include, but are not limited to, indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, amprenavir, fosamprenevir, atazanavir, tipranavir, or darunavir. In certain embodiments, the L protein may comprise a secondary mutation. Also provided in accordance with the present invention is a vesicular stomatitis virus (VSV) comprising a recombinant VSV L protein.

[0022] The present invention further provides a method for controlling RNA virus replication, comprising (a) transducing or transducing a host cell with an RNA virus with an option (ON switch) of the present invention having an insertion at an intramolecular insertion site, and (b) maintaining the host cell in the presence or absence of a protease inhibitor specific for said protease, wherein addition of said protease inhibitor allows viral transcription and / or replication, and absence of said protease inhibitor inhibits viral transcription and replication.

[0023] The present invention also provides a method for controlling RNA virus replication, comprising (a) transducing or transducing a host cell with an RNA virus according to an option (OFF switch) of the present invention having an insertion at the intermolecular insertion site, and (b) maintaining the host cell in the presence or absence of a protease inhibitor specific for said protease, wherein addition of said protease inhibitor inhibits viral transcription and / or replication, and absence of said protease inhibitor allows viral transcription and replication.

[0024] The present invention also provides a method for controlling expression of heterologous proteins from RNA viruses, comprising: (a) transducing or transducing a host cell with an RNA virus according to an alternative embodiment of the present invention, wherein at least one heterologous protein comprises an insertion at an intramolecular insertion site containing at least the cleavage site of the protease, and optionally further comprising a protease; and (b) maintaining the host cell in the presence or absence of a protease inhibitor specific for the protease, wherein addition of the protease inhibitor allows expression of the heterologous protein, and absence of the protease inhibitor inhibits heterologous protein expression. The protein in the method according to the present invention may be an autocatalytic protease, preferably an HIV-1 protease, more preferably a single-chain dimer of HIV-1 protease. Suitable HIV-1 protease inhibitors include, but are not limited to, indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, amprenavir, fosamprenevir, atazanavir, tipranavir, or darunavir. [Brief explanation of the drawings]

[0025] [Figure 1]Figure 1 illustrates the principle of the VSV-prot-ON system. Figure 1A: The HIV protease dimer construct was inserted into two VSV proteins, the P protein and the L protein, which form the polymerase complex. The HIV protease functions as a dimer. To ensure the function of the HIV protease as part of the open reading frame for the VSV P protein and polymerase (L protein), we used a pre-bound protease dimer. In this way, the P protein already contains a functional protease dimer that becomes autocatalytically active during translation and cleaves the P protein unless a protease inhibitor is present. Figure 1B: The protein ribbon structure of the HIV protease dimer is shown. The HIV protease functions as a dimer. To ensure the function of the HIV protease as part of the open reading frame for the VSV phosphoprotein and polymerase, we used a pre-bound protease dimer (the dark gray loop at the bottom of the protein ribbon structure).

[0026] [Figure 2] Figure 2 shows A) the structure of the linked dimeric protease in the expression plasmid at aa 196 (P-196PR2) and B) the construct structure of P-196PR2. The cDNA sequence of the P protein (P-196PR2), in which the linked dimeric protease is at aa 196, and the flanking sequences of the VSV nucleoprotein (N protein) and matrix protein (M protein) were synthesized by GeneArt. The linked protease dimer is flanked by a flexible linker consisting of the amino acid sequence (GGSG)3. This separation minimizes disruption of the intramolecularly inserted protein by the tertiary structure of the P and L proteins. The protease cleavage sequences are located before the first protease and after the second protease. The two proteases are connected via the dimeric linker sequence.

[0027] [Figure 3]Figure 3 shows the protease-linked regulation of trans-supplied VSV P protein to complement VSV-ΔP virus. The functionality of the phosphoprotein-protease construct was first tested using a P expression plasmid (P-prot) into which P-196PR2 was cloned. BHK cells were transfected with this P-prot construct and then infected with the VSV-ΔP mutant. VSV-ΔP contained red fluorescent protein as a reporter gene. VSV-ΔP function requires the working P protein transduced by cells expressing P-Prot. Representative photographs are shown for transfected cells treated with the positive control at the indicated times after transfection: A (1–3): no amprenavir added; B (1–3): 1 μM amprenavir added; and C (1–3): normal P expression plasmid.

[0028] [Figure 4] Figure 4 shows the construct of a plasmid carrying the full-length VSV-P-prot sequence and the phosphoprotein (P protein) with a dimeric protease (P196PR2) linked to aa position 196. The P protein gene in VSV Indiana GFP was replaced by P-196PR2. Enhanced GFP (eGFP), located at position 5 of the VSV genome (between the G and L proteins), was used as a marker gene.

[0029] [Figure 5] 5 shows a schematic diagram of an HIV protease dimer insert construct containing a protease cleavage sequence and a flexible linker, and a VSV P protein having the amino acid sequence of the HIV protease dimer insert (SEQ ID NO: 29). The linked protease dimers are adjacent to a flexible linker consisting of the amino acid sequence (GGSG)3. The protease cleavage sequence is located before the first protease and after the second protease. The two proteases are connected via a linker sequence.

[0030] [Figure 6] Figure 6 shows that the protease inhibitor amprenavir regulates the activity of VSV-P-prot, which expresses a protease switch. Figure 6A: To test the genomic integrity of VSV-P-prot, viral genomic RNA was purified and reverse transcribed, and PCR was performed with P196PR2. A VSV mutant lacking the protease insertion was used as a negative control. Markers are used in lanes 1 and 4 of the gel, while lanes 2 and 3 show the PCR products of the VSV control and VSV-P-Prot, respectively. We found that the PCR fragments of P196PR2 and the protease-negative P protein were of the expected sizes (the expected size of the protease-containing P protein is 1490 bp; the expected size of the protease-free P protein is 773 bp; the standard titer of VSV-P-Prot is 1.8 × 10). The PCR products were then sequenced. Figure 6B: After generating full-length VSV-P-Prot, its functionality was tested by infection of BHK cells in the presence and absence of 10 μM amprenavir (APV). In the presence of APV, viral activity was observed by eGFP expression (left column) and cytopathic effect in standard cell culture dishes (right column). Figure 6C: Plaque assays of VSV-P-Prot in the presence and absence of APV are shown. In the presence of APV, cytopathic effect was confirmed by plaque assay. In the absence of APV, neither eGFP signal nor cytopathic effect was observed. Sequencing of the insertion site by two Sanger sequencing reactions to assess whether mutations had occurred within the protease dimer sequence with the original DNA sequence of SEQ ID NO:4 revealed no mutations in the protease dimer sequence.

[0031] [Figure 7]Figure 7 shows that VSV-P-prot activity can be modulated by various HIV protease inhibitors. We tested whether VSV-P-prot could replicate in the presence of second-generation protease inhibitors such as saquinavir and indinavir. The functionality of VSV-P-prot was confirmed by the fluorescent signals of the reporter gene eGFP (Figure 7A, left panel), cytopathic effect (Figure 7A, right panel), and plaque formation (Figure 7B).

[0032] [Figure 8] Figure 8 shows that the protease inhibitor amprenavir dose-dependently modulates VSV-P-prot activity. This figure shows the dose response of amprenavir, an HIV protease inhibitor, on VSV-P-prot viral activity. BHK cells were infected at an MOI of 1, and viral spread was assessed 24 hours later. Figure 8A: Viral eGFP expression and cytopathic effect increased with increasing APV dose. Figure 8B: VSV-P-prot replication initiated at a dose of 100 nM amprenavir, reached a plateau of maximum activity in the dose range of 3–100 μM, and worsened at higher doses. The replication curves showed a slight decrease in VSV-P-prot activity compared to VSV.

[0033] [Figure 9-1] Figure 9 shows the abrogation of VSV-P-prot neurotoxicity. Figure 9A: Intracranial injection of wild-type VSV-dsRed (2 x 10 TCID in 2 μl) resulted in severe signs of neurotoxicity. No neurotoxicity was observed with VSV-P-prot, with or without amprenavir. Figure 9B: Survival graph showing mice injected with VSV-dsRed that had to be sacrificed within 4 days for humane reasons. Figure 9C: Body weight graph showing a significant loss of body weight in mice injected with VSV-dsRed. [Figure 9-2]Figure 9 shows the abrogation of VSV-P-prot neurotoxicity. (Figure 9D) Histological fluorescence analysis of coronal brain sections revealed widespread diffusion of red-fluorescent VSV-dsRed. Viral infection was found throughout the striatum, subcortical regions, and hypothalamus (bilaterally). In contrast, with or without amprenavir, GFP expression from VSV-P-Prot was restricted to the area immediately medial to the injection needle track, with no signs of intracranial spread.

[0034] [Figure 10] Figure 10 shows that the protease-regulating activity of VSV-P-Prot remains stable even after multiple virus passages. The virus was passaged 20 times at a suboptimal APV concentration, and all passages were transferred to cells without protease inhibitors to detect escape mutants. Figure 10A shows BHK cells inoculated with passaged VSV-P-Prot with or without APV. Figure 10B shows viral genomic RNA isolated and reverse transcribed after passage. PCR was performed on the region of the insert, followed by PCR sequencing. We found that the P196PR2 and protease-negative P protein PCR fragments were each the expected size (1490 bp with P-Prot; 773 bp without P-Prot).

[0035] [Figure 11-1] Figure 11 shows the domain organization, structure, and insertion site in the VSV L protein. Figure 11A shows a schematic VSV genome organization showing the genes in the 3' to 5' direction, and the VSV L protein domain organization (Liang et al., Cell, 2015, 162(2): 314-327) with corresponding domain boundaries labeled with numbers above. CD1506, CD1537, MT1603, MT1620, and MT1889 represent the candidate insertion sites tested here. Figure 11B shows the VSV L protein structure determined by structural information. The right diagram zooms in and visualizes the complementary domain (CD), methyltransferase domain (MT), and C-terminal domain (CTD). [Figure 11-2] Figure 11 shows the domain organization, structure, and insertion site in the VSV L protein. Figure 11C: Zoom in on the CD, MT, and CTD with the loop selected as the insertion site. Figure 11D: Molecular model of the VSV L protein with mCherry inserted at MT1620.

[0036] [Figure 12-1] Figure 12 shows that insertion of mCherry at MT1620 results in replication-competent virus. Figure 12A, top: VSV L protein domain organization with insertion sites is shown. Micrographs show 293T cells transfected with five different L-mCherry expression plasmids. Corresponding insertion sites are labeled with the domain abbreviation followed by the amino acid number. Red (top row) indicates L-mCherry expression. Figure 12A, bottom: Similar transfected 293T cells are shown after infection with VSV-GFP-ΔL at an MOI of 10. Green fluorescence indicates functional L-mCherry fusion protein and polymerase activity. Figure 12B: Fluorescence and phase-contrast images of VSV-L-mCherry, VSV-GFP-L-mCherry, and VSV-L-mWasabi 24 hours after infection of BHK-21 cells are shown. The viral genome sequence is displayed above the fluorescence images. [Figure 12-2] Figure 12 shows that insertion of mCherry at MT1620 results in replication-competent virus. Figure 12C: Immunoblot for mCherry under reducing conditions on a 12% polyacrylamide gel. β-actin was used as a loading control. BHK-21 cells infected with VSV, VSV-GFP, VSV-L-mCherry, and VSV-GFP-L-mCherry were used to prepare lysates 8 hours postinfection.

[0037] [Figure 13-1]Figure 13 shows that insertion of mCherry at the MT1620 position results in a moderate reduction. Figure 13A: Evaluation of viral replication fitness by crystal violet plaque assay. Representative photographs from a 6-well dish are shown with the corresponding microscope inset showing a single plaque. BHK-21 monolayers were inoculated with virus for 1 h, washed, and then cultured for 24 h. Figure 13B: Viral replication kinetics of different VSV strains, showing the single-step growth kinetics of VSV (filled circles) and VSV-L-mCherry (open triangles) in BHK-21 cells. Titers were quantified using TCID50 measurements. [Figure 13-2] Figure 13 shows that insertion of mCherry at the MT1620 position results in a moderate reduction. Figure 13C: Comparison of virus-induced cytotoxic activity in an IFN-responsive MTT viability assay. IFN-responsive BHK-21 cells were treated with increasing amounts of IFN (0, 10, 100, 500, and 1000 U / ml) and infected at an MOI of 0.1, 1, and 10. Viability is shown normalized to the untreated control. Bars represent mean ± SEM (n=4). In the absence of IFN treatment, both viruses reduce viability in infected cells to a similar extent.

[0038] [Figure 14] Figure 14 shows the insertion of a protease switch into the VSV L protein, generating an alternative regulatable virus, VSV-L-prot. Figure 14A: BHK cells were inoculated with VSV-L-prot with or without APV. Figure 14B: After plaque purification, viral genomic RNA was isolated and reverse transcribed. PCR was performed on the region of the VSV-L-prot insert and on a control virus with no insert (the expected size of the L protein with the insert is 1830 bp, and the expected size of the L protein without the insert is 1114 bp), followed by PCR sequencing, but no mutations were detected.

[0039] [Figure 15]Figure 15 shows that the protease inhibitor amprenavir dose-dependently modulates VSV-L-prot activity. The dose response of VSV-L-prot to the HIV protease inhibitor amprenavir was tested. BHK cells were infected at an MOI of 1, and viral spread was assessed 24 hours later. Figure 15A: Viral GFP expression increases with increasing doses of amprenavir. Figure 15B: VSV-L-prot activity began at an amprenavir dose of 100 nM and reached maximum activity at 30 μM. Higher amprenavir concentrations were not tested for L-prot due to toxic effects on the cells. Replication curves showed a slight decrease for VSV-L-prot compared to VSV.

[0040] [Figure 16] Figure 16 shows the generation of a VSV with a functional double intramolecular insertion at P and L, generating VSV-P-mWasabi-L-mCherry. As a test of VSV-P-prot-L-prot, a VSV with a functional double intramolecular insertion at P and L, VSV-P-mWasabi-L-mCherry, was generated. Double insert functionality was confirmed by dual fluorescent readout and cytopathic effect in plaque assays (Figure 16A, left: mWasabi, center: mCherry, right: plaques, bottom: schematic diagram of constructs) and testing of genome integrity by cDNA synthesis and PCR (Figure 16B, 1: VSV P site, 2: VSV-P-mWasabi-L-mCherry P site, 3: VSV L site, 4: VSV-P-mWasabi-L-mCherry L site).

[0041] [Figure 17]Figure 17 shows the principle of the VSV-Prot-OFF system and the protein ribbon structure of the HIV protease dimer. Figure 17A: The intergenic region between the GFP and L protein was replaced with the HIV protease construct. Figure 17B: The HIV protease functions as a dimer. A pre-bound protease dimer was used to ensure the function of the HIV protease as part of the open reading frame of the GFP-Prot-L fusion protein.

[0042] [Figure 18] Figure 18 shows the generation of VSV in which the intergenic region has been functionally replaced with an HIV protease dimer. Figure 18A shows BHK cells inoculated with VSV-GFP-Prot-L without the (GGSG)3 linker (the construct containing the linker is not shown). Addition of 10 μM amprenavir abolishes viral activity. Figure 18B shows plaque purification followed by isolation and reverse transcription of viral genomic RNA from VSV-GFP-Prot-L with and without the (GGSG)3 linker. PCR was performed on the insert regions of both the Prot-Off virus and the control virus, followed by PCR sequencing. Shown in the figure are: 1: GFP-L fragment without the protease (959 bp); 2: GFP-Prot-L fragment with the (GGSG)3 linker (1559 bp); and 3: GFP-Prot-L fragment without the (GGSG)3 linker (1487 bp). Sequence alignment of the rescued VSV-Prot-Off virus (without the (GGSG)3 linker) with the construct plasmid and the consensus sequence of the plasmid sequence in the region of the HIV protease insert did not reveal any mutations.

[0043] [Figure 19-1]Figure 19 shows that the protease inhibitor amprenavir dose-dependently modulates VSV-Prot-OFF activity. The dose response of HIV protease inhibitors on VSV-Prot-OFF viral activity is shown. Figure 19A: BHK cells were infected at an MOI of 1, and viral infection was assessed 24 hours later. Viral GFP expression decreased with increasing doses of amprenavir (APV). Figure 19B: Viral replication measured at 24 hpi. VSV-Prot-OFF activity began to decrease at a dose of 30 nM APV. The highest dose of APV tested was 30 μM. APV concentrations higher than 30 μM were not tested with VSV-Prot-OFF due to toxic effects on the cells. 10 μM saquinavir (SQV, open symbols) showed the strongest inhibition of viral replication. [Figure 19-2] Figure 19 shows that the protease inhibitor amprenavir dose-dependently modulates VSV-Prot-OFF activity. The dose response of HIV protease inhibitors on the viral activity of VSV-Prot-OFF is shown. Figure 19C: Viral replication measured at 24 hpi using the indicated concentrations of saquinavir is shown. Figure 19D: BHK cells were infected with the indicated VSV mutants, VSV-GFP or VSV-Prot-Off, at an MOI of 3 during single-step replication kinetics. Virus titers were measured in harvested supernatants and are shown as Log10 TCID50 / ml.

[0044] [Figure 20]Figure 20 shows that VSV-P-prot expression can be regulated in vivo by administration of protease inhibitors. Nude mice were subcutaneously xenografted with U87 glioblastoma cells and injected intratumorally with a single dose of the indicated virus, VSV-P-prot-Luc, or control buffer at a median volume of 0.1 cm3. A protease inhibitor (PI) mixture containing 0.8 mM amprenavir (APV) and 0.2 mM ritonavir (RTV) was administered intraperitoneally at 50 μL every 12 h. Figure 20A: Representative bioluminescence images are shown from 8 days after virus inoculation. Figure 20B: Quantitation of bioluminescence images (BLI) of luciferase signals from VSV-P-prot-Luc-treated tumors in mice receiving PI (black squares) or drug vehicle (gray circles) (n = 5; SD of mean; * p < 0.05).

[0045] [Figure 21] Figure 21 shows that VSV-L-prot can be regulated in vivo by administration of protease inhibitors. Nude mice were xenografted subcutaneously with U87 glioblastoma cells and injected intratumorally with a single dose of the indicated virus, VSV-L-prot, VSV control, or control buffer (placebo), at a median volume of 0.1 cm3. A protease inhibitor (PI) mixture containing 0.8 mM amprenavir (APV) and 0.2 mM ritonavir (RTV) was administered intraperitoneally at 50 μL every 12 hours. Figure 21A: Tumors were measured with calipers, and volumes were calculated using the formula length × width × 0.4. Intratumoral treatment of subcutaneous U87 tumors with VSV-L-prot resulted in attenuation of tumor growth. Figure 21B: Survival plot showing increased survival in animals treated with VSV-L-prot + PI (thin solid line) compared with tumors in mice treated with VSV-L-prot in the absence of PI (thick solid line) (L-prot virus treatment ± PI, n = 5; VSV, n = 3; PBS, n = 6; SD of mean, * p < 0.0X, ** p < 0.01).

[0046] [Figure 22]Figure 22 shows that protease inhibitors modulate VSV-Prot-Off activity in vivo, as indicated by tumor volume and viability. NOD-SCID mice were subcutaneously xenografted with 100 μL of a suspension of G62 glioma cells and injected intratumorally with a single dose of the indicated virus, VSV-Prot-Off, VSV-GFP, or control buffer (placebo), at a median volume of 0.07 cm3, followed by another intratumor injection 7 days later, as indicated by the vertical black dotted lines in A and B. A protease inhibitor (PI) mixture containing 0.8 mM saquinavir (SQV) and 0.2 mM ritonavir (RTV) was administered intraperitoneally at 50 μL every 8 hours. PI treatment was initiated 8 days after the second viral injection, when tumor regression was observed. Figure 22A: Tumors were measured with calipers, and volume was calculated using the formula: length × width × 0.4. FIG. 22B: Survival plot reflects survival of viral neurotoxicity and / or tumor development over the observation period (VSV-Prot-Off virus treatment ± PI, n=8; VSV-GFP, n=8; SD of mean).

[0047] [Figure 23] Figure 23 shows that protease inhibitors modulate VSV-Prot-Off activity in vivo, as demonstrated by immunofluorescence. G62 xenografts with a median volume of 0.07 cm3 were injected intratumorally with the indicated VSV mutants, VSV-Prot-Off or VSV-GFP, or control buffer (placebo). PI treatment (SQV and RTV) was initiated 3 days after the single virus treatment for histological examination. Representative images of immunofluorescence staining from three mice per group are shown. The top panel shows DAPI staining; the middle panel shows anti-VSV-N antibody staining; and the bottom panel shows a magnified area of ​​anti-VSV-N antibody staining. PI treatment restricted the diffusion of VSV-Prot-Off-GFP primarily to the injection site.

[0048] [Figure 24]Figure 24 shows the dose response of saquinavir on the in vitro activity of VSV-Prot-Off encoding soluble IL12. BHK cells were infected with the indicated VSV isomers, VSV-GP, VSV-GP-IL12, VSV-GP-GFP-IL12-Prot-Off-wl, or VSV-GP-GFP-IL12-Prot-Off-w / ol, at an MOI of 0.1 and then washed and cultured in the absence (-ctrl) or presence of 10, 100, 300, 1,000, or 10,000 nmol of the protease inhibitor (PI), saquinavir. Supernatants were collected 30 hours postinfection. Figure 24A: Schematic diagram of the VSV-GP-GFP-IL12-Prot-Off genome, showing the 3' to 5' orientation of the genes (top panel). Viral titers were measured in the supernatants by TCID50. The viral titers of VSV-GP-IL12-Prot-Off with or without the linker (wl, w / ol) were inversely correlated with the saquinavir concentration. (Figure 24B) Enzyme-linked immunosorbent assay (ELISA) was performed to measure the transgene IL12 expressed in the supernatant. IL12 expression was inversely correlated with the saquinavir concentration. As a control, a VSV-GP-IL12 sample without saquinavir (-ctrl) was diluted and measured.

[0049] [Figure 25]Figure 25 shows the dose response of atazanavir on the in vitro activity of VSV-Prot-Off encoding soluble IL12. BHK cells were infected with the indicated VSV mutants, VSV-GP-IL12, VSV-GP-Luc-IL12-Prot-Off-w / ol, or VSV-GP-Luc-IL12-Prot-Off-wl, at an MOI of 1 and then cultured in the absence (-ctrl) or presence of 10, 100, 300, 1,000, or 10,000 nmol of atazanavir. Supernatants were collected 30 hours postinfection. Figure 25A: Schematic diagram of the VSV-GP-Luc-IL12-Prot-Off genome, showing the 3' to 5' orientation of the genes (top panel). Viral titers were measured in the supernatants by TCID50. The viral titers of VSV-GP-Luc-IL12-Prot-Off with or without the linker (wl, w / ol) were inversely correlated with the atazanavir concentration. Figure 25B: Enzyme-linked immunosorbent assay (ELISA) was performed to measure the transgene IL12 expressed in the supernatant. IL12 expression was inversely correlated with the atazanavir concentration. As a control, a VSV-GP-IL12 sample (-ctrl) without atazanavir was diluted and measured.

[0050] [Figure 26]Figure 26 shows the replication kinetics of two VSV-Prot-Off constructs encoding both soluble IL12 and different reporter proteins. Figure 26A: Schematic diagram of the VSV-Prot-Off genome organization of VSV mutants encoding IL12 and either GFP (VSV-GP-Prot-Off-w / ol GFP IL12) or luciferase (VSV-GP-Prot-Off-w / ol Luc IL12). These show the genes encoding soluble IL12 and a fusion protein containing a protease dimer and either GFP or luciferase (Luc) fused (N- to C-terminally) to the L protein, in a 3' to 5' orientation. Figure 26B: BHK cells were infected with the indicated VSV mutants at an MOI of 3 using single-step replication kinetics. After infection, cells were washed and cultured in GMEM for the indicated times. Virus titers were measured in harvested supernatants and are shown as Log10TCID50 / ml.

[0051] [Figure 27] Figure 27 shows a schematic diagram of the VSV-Prot-off construct encoding membrane-anchored IL12. Figure 27A: Schematic diagram of the VSV-Prot-off genome organization, showing the 3' to 5' orientation of the genes encoding the fusion protein containing IL12 fused to the CD4 transmembrane domain, a protease, and the L protein. Figure 27B: Schematic diagram of the fusion protein containing IL12, a CD4 transmembrane domain (TM), a protease (prot dimer), and the L protein (L) located in the transmembrane domain.

[0052] [Figure 28-1]Figure 28 shows proof-of-principle for the expression of membrane-bound therapeutic proteins using the VSV-Prot-off construct. IL12 with the transmembrane domain of CD4 was directly fused to the polymerase, allowing for the reduction of both viral replication and transgene expression in the presence of a protease inhibitor (PI). BHK cells were infected with the indicated VSV mutants at an MOI of 1. After washing, the cells were cultured in the absence (-ctrl) or presence of 10, 100, 300, 1,000, or 10,000 nmol of atazanavir (ATV). Supernatants were harvested 30 hours post-infection. Figure 28A: Viral titers in the supernatants were measured by TCID50. The viral titers of VSV-GP-TM-IL12-Prot-Off(-w / ol) containing no linker or VSV-GP-TM-IL12-Prot-Off(-fl) containing the exact forward linker between the IL12 transmembrane domain and the HIV protease dimer were inversely correlated with AZV concentration, whereas VSV-GP-IL12 was unaffected (Figure 28B). Unfiltered supernatants from cultures infected with VSV-GP-TM-IL12-Prot-Off(-w / ol) containing no linker or VSV-GP-TM-IL12-Prot-Off(-fl) containing the exact forward linker were tested for IL12 by enzyme-linked immunosorbent assay (ELISA). [Figure 28-2]Figure 28 shows proof-of-principle for membrane-bound therapeutic protein expression using the VSV-Prot-Off construct. IL12, with the transmembrane domain of CD4, was directly fused to a polymerase, allowing for reduced viral replication and transgene expression in the presence of a protease inhibitor (PI). BHK cells were infected with the indicated VSV mutants at an MOI of 1. After washing, the cells were cultured in the absence (-ctrl) or presence of 10, 100, 300, 1,000, or 10,000 nmol of atazanavir (ATV). Supernatants were harvested 30 hours postinfection. Figure 28C: Cells infected with VSV-GP-TM-IL12-Prot-Off-fl were diluted in cell lysis buffer, and IL12 concentrations were measured by ELISA on lysed samples (supernatant + lysed cells) compared with cell-containing supernatants (unlysed) or filtered supernatants alone (n=2). Figure 28D: BHK cells were infected with the indicated VSV mutants at an MOI of 3 and cultured for the indicated time periods. The transmembrane IL12 mutants of VSV-Prot-Off without (-w / ol) or with (-fl) the forward linker showed a moderate reduction in expression compared to the parent virus VSV-GP-IL12 only at early time points. Detailed Description of the Invention

[0053] The general embodiments "comprising" or "comprised" encompass the more specific embodiment "consisting of." Furthermore, the singular and plural forms are not used in a limiting sense. As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless expressly stated to specify only the singular form.

[0054] As used herein, the term "homologue" or "homologous" refers to a polypeptide molecule or nucleic acid molecule that is at least 80% identical in sequence to an original sequence or its complementary sequence. Preferably, the polypeptide molecule or nucleic acid molecule is at least 90% identical in sequence to a reference sequence or its complementary sequence. More preferably, the polypeptide molecule or nucleic acid molecule is at least 95% identical in sequence to a reference sequence or its complementary sequence. Most preferably, the polypeptide molecule or nucleic acid molecule is at least 98% identical in sequence to a reference sequence or its complementary sequence. A homologous protein further exhibits the same or similar protein activity as the original sequence.

[0055] As used herein, the terms "corresponding to an amino acid position" or "corresponding to an amino acid position" include defined sequences of VSV, such as the amino acid sequence of the P protein having the sequence of SEQ ID NO:27 or the amino acid sequence of the L protein having the sequence of SEQ ID NO:28, as well as natural variants thereof and sequences from other VSV serotypes. Those skilled in the art also recognize that the genomic sequences of RNA viruses, such as VSV, may vary and therefore not be identical to the sequences provided in SEQ ID NO:27 or SEQ ID NO:28, even if they are from the same serotype. However, using sequence alignment, those skilled in the art know how to identify the position of a sequence in a particular VSV sequence that corresponds to a defined position, i.e., a homologous position, in the sequence of SEQ ID NO:27 or SEQ ID NO:28 of the P protein or L protein, respectively. Such a sequence containing a position corresponding to a defined position in the P protein having the sequence of SEQ ID NO:27 or the L protein having the sequence of SEQ ID NO:28 will have at least 80% sequence identity to the sequence of SEQ ID NO:27 or the sequence of SEQ ID NO:28, and preferably will have at least 90% identity to the sequence of SEQ ID NO:27 or the sequence of SEQ ID NO:28. The corresponding sequences may also contain recombinant insertions such as proteolytic enzymes and / or cleavage sites for said proteolytic enzymes, although these insertions should not be considered to determine the corresponding sequences.

[0056] The term "protein" is used interchangeably with "amino acid residue sequence" or "polypeptide" and refers to a polymer of amino acids of any length. These terms also include proteins that are post-translationally modified by reactions including, but not limited to, glycosylation, acetylation, phosphorylation, glycation, or protein processing. Modifications and changes, such as substitutions, deletions, or insertions of amino acid sequences, can be made in the structure of a polypeptide while the molecule maintains its biologically functional activity. For example, substitutions of specific amino acid sequences can be made in a polypeptide or its underlying nucleic acid coding sequence to obtain a protein with identical properties. The term "polypeptide" typically refers to a sequence having more than 10 amino acids, and the term "peptide" refers to a sequence up to 10 amino acids in length. However, these terms may be used interchangeably.

[0057] The term "fusion protein" refers to a chimeric protein composed of portions from different sources, particularly produced by the joining of two or more genes or gene portions encoding originally separate proteins or fragments thereof. Recombinant fusion proteins are artificially produced by recombinant DNA technology. Fusion proteins may contain a full-length protein (i.e., including all functional domains) or a fragment thereof, such as one or more functional domains, consensus motifs, or cleavage sites, fused to another full-length protein (i.e., including all functional domains) or fragment thereof. "Fused" means that the nucleotide sequence encoding one polypeptide is in-frame with the nucleotide sequence encoding a second polypeptide, such that the nucleotide sequences are expressed as a single protein. Polyproteins are a subtype of fusion protein, typically occurring in RNA viruses. Polyproteins are proteins produced by translation of a single mRNA encoding several proteins in a single open reading frame, i.e., proteins fused to each other (multicistronic mRNA). Polyproteins are typically processed post-translationally or co-translationally by proteolytic enzymes to form single proteins.

[0058] The term "genomic RNA" as used herein refers to the hereditary genetic information of an RNA virus. However, in the context of the present invention, the term "genome" also typically refers to the genome of an RNA virus, thus an RNA genome having a ribonucleic acid sequence. Those skilled in the art will recognize that the genome of an RNA virus may also be provided as a DNA sequence in a vector such as a plasmid. The RNA genome is then produced in the host cell following gene transfer in the host cell by transcription.

[0059] As used herein, the term "gene" refers to a DNA or RNA locus of an inherited genomic sequence that is expressed as a functional product or that influences the traits of an organism by regulating gene expression. Genes and polynucleotides may contain introns and exons, as in a genomic sequence, or may contain only coding sequences, as in a cDNA, such as an open reading frame (ORF) that contains a start codon (methionine codon) and a translation stop codon. Genes and polynucleotides may also contain regions that regulate their expression, such as transcription initiation, translation, and transcription termination. Thus, regulatory elements such as promoters are also included.

[0060] As used herein, the terms "nucleic acid," "nucleotide," and "polynucleotide" are used interchangeably and refer to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to 3' end, and include double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA, negative-sense and positive-sense), double-stranded RNA (dsRNA), genomic DNA, cDNA, cRNA, recombinant DNA or RNA, and derivatives thereof, such as those containing modified backbones.

[0061] As used herein, the terms "ribonucleic acid," "RNA," or "RNA oligonucleotide" refer to a molecule consisting of a sequence of nucleotides constructed from nucleic acid bases, ribose sugars, and phosphate groups. RNA is typically a single-stranded molecule and can perform a variety of functions. The term "ribonucleic acid" specifically includes messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA), each of which plays a specific role in biological cells. Ribonucleic acids include small non-coding RNAs such as microRNA (miRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), and Piwi-interacting RNA (piRNA). The term "non-coding" means that the RNA molecule is not translated into an amino acid sequence.

[0062] The terms "upstream" and "downstream" refer to relative positions within DNA or RNA. Each strand of DNA or RNA has a 5'-end and a 3'-end that are related to the terminal carbon positions of the deoxyribose or ribose units. By convention, "upstream" refers to the direction toward the 5'-end of a polynucleotide, and "downstream" refers to the direction toward the 3'-end of a polynucleotide. In the case of double-stranded DNA, such as genomic DNA, the term "upstream" refers to the direction toward the 5'-end of the coding strand, and the term "downstream" refers to the direction toward the 3'-end of the coding strand.

[0063] The term "coding strand" or "positive-sense strand" refers to the RNA strand that encodes the protein.

[0064] The terms "non-coding strand," "antisense strand," or "negative-sense strand" or "negative strand" refer to the RNA strand that must be transcribed into positive-strand RNA by RNA-dependent RNA polymerase before translation.

[0065] A "vector" is a nucleic acid that can be used to introduce a heterologous polynucleotide into a cell. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule to which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-deficient or active forms of retroviruses, adenoviruses, adeno-associated viruses, VSV, and MeV), which can introduce additional DNA or RNA segments into the viral genome.

[0066] The terms "encode" and "code" broadly refer to any process that uses information from a polymeric macromolecule to induce the production of a second molecule that differs from the first. The second molecule may have a chemical structure that differs from the chemical nature of the first molecule. For example, the term "encode" refers to the semiconservative DNA replication process in which one strand of a double-stranded DNA molecule is used as a template to encode a newly synthesized complementary sister strand by DNA-dependent DNA polymerase. Furthermore, a DNA molecule can encode an RNA molecule (e.g., using DNA-dependent RNA polymerase), or a (negative-strand) RNA molecule can encode a (positive-strand) RNA molecule (e.g., using RNA-dependent RNA polymerase). A (positive-strand) RNA molecule can also encode a polypeptide, as in the process of translation. The term "encode," used to describe the process of translation, also extends to triplet codons that code for amino acids. An RNA molecule can also encode a DNA molecule, for example, by the process of reverse transcription using RNA-dependent DNA polymerase. When referring to a DNA molecule that encodes a polypeptide, the processes of transcription and translation are referred to.

[0067] The term "heterologous polypeptide" or "heterologous protein" as used herein refers to a protein derived from a different organism or species than the receptor, i.e., the RNA virus. In the context of the present invention, those skilled in the art will understand that it refers to a protein that is not naturally expressed by the virus. When used as a portion of a protein, the term "heterologous" may also indicate that the protein contains two or more amino acid sequences that are not distinguished as essentially identical to each other. In the context of the present invention, it is typically a therapeutic protein, an antigen such as a tumor-specific or tumor-associated antigen, or a reporter (such as luciferase or a fluorescent protein).

[0068] The term "therapeutic protein" refers to proteins that can be used in human and / or veterinary medicine, including but not limited to antibodies, growth factors, blood clotting factors, cytokines such as interferons and interleukins, chemokines, and hormones, preferably growth factors, cytokines, chemokines, and antibodies.

[0069] The term "cytokine" refers to a small protein released by cells and acting as an intercellular mediator, for example, influencing the behavior of cells surrounding the secreting cell. Cytokines may be secreted by immune cells such as T cells, B cells, NK cells, and macrophages, or other cells. Cytokines may be involved in intercellular signaling events, such as autocrine signaling, paracrine signaling, and endocrine signaling. They may mediate a range of biological processes, including, but not limited to, immunity, inflammation, and hematopoiesis. Cytokines may be chemokines, interferons, interleukins, lymphokines, or tumor necrosis factors.

[0070] As used herein, "growth factor" refers to a protein or polypeptide that can stimulate cell proliferation.

[0071] As used herein, the term "expression" refers to the transcription and / or translation of a heterologous nucleic acid sequence in a host cell. The expression level of a gene product of interest in a host cell can be determined based on either the amount of corresponding mRNA (or positive-strand RNA) present in the cell or the amount of a polypeptide encoded by a selected sequence. For example, RNA transcribed from a selected sequence can be quantified by Northern blot hybridization, ribonuclease RNA protection, in situ hybridization to cellular RNA, or PCR such as qPCR. The protein encoded by a selected sequence can be quantified by various methods, such as ELISA, Western blot, radioimmunoassay, immunoprecipitation, protein biological activity measurement, protein immunostaining followed by FACS analysis, or homogeneous time-resolved fluorescence (HTRF) measurement. The expression level of non-coding RNA, such as miRNA or shRNA, may also be quantified by PCR, such as qPCR.

[0072] The term "gene product" refers to both mRNA polynucleotides and polypeptides encoded by a gene or DNA polynucleotide.

[0073] As used herein, a "reporter gene" is a polynucleotide that encodes a reporter protein or "reporter" that can be easily detected and quantified. Thus, measurements of the expression level of the reporter typically indicate the level of transcription and / or translation. A gene that encodes a reporter is a reporter gene. For example, a reporter gene may encode a reporter such as an enzyme whose activity can be quantified, e.g., alkaline phosphatase (AP), chloramphenicol acetyltransferase (CAT), Renilla luciferase, or firefly luciferase protein. Reporters also include fluorescent proteins, such as green fluorescent protein (GFP) or recombinant variants of any GFP, including enhanced GFP (EGFP), blue fluorescent protein (BFP and other derivatives), cyan fluorescent protein (CFP and other derivatives), yellow fluorescent protein (YFP and other derivatives), and red fluorescent protein (RFP and other derivatives), or other fluorescent proteins such as mCherry or mWasabi.

[0074] The terms "protease" and "proteinase" are used synonymously herein and refer to enzymes that aid in the catabolism of proteins through proteolysis, i.e., hydrolysis of peptide bonds. Proteases can be classified into seven broad groups: serine proteases, cysteine ​​proteases, threonine proteases, aspartic proteases, glutamic proteases, metalloproteases, and asparagine peptide-lysing enzymes. Proteases function in all organisms, including prokaryotes, eukaryotes, and viruses. In principle, all proteases are suitable in the context of the present invention, as long as they are highly specific, i.e., have a restricted set of substrate sequences, and specific inhibitors are available. Protease inhibitors must be specific for the protease and suitable for in vivo use, i.e., known to be safe, bioavailable, and active in vivo, such as after oral or parenteral administration to a subject, preferably a human subject. Viral proteases are advantageous because they are common targets of antiviral drugs. Therefore, many protease inhibitors that inhibit viral proteases have been approved and evaluated as safe in humans. For example, in the case of human immunodeficiency virus (HIV) proteases, a variety of well-characterized protease inhibitors are available, allowing for the modulation of the system with desired kinetics. Examples of suitable HIV protease inhibitors include, but are not limited to, indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, amprenavir, fosamprenevir, atazanavir, tipranavir, and darunavir. In therapy, protease inhibitors, particularly HIV protease inhibitors, may be administered in combination with ritonavir. Ritonavir increases the plasma concentration of other protease inhibitors. Human proteases are advantageous because they are endogenous proteins in human patients and therefore do not induce an immune response.The protease used in the RNA virus according to the present invention is a heterologous protease, i.e., a protease that is not endogenous to the virus. As used herein, the term "Prot" or "prot" is an abbreviation for protease, so for example, L-Prot refers to an L protein containing an intramolecular protease disclosed herein, P-Prot refers to a P protein containing an intramolecular protease disclosed herein, or Prot-L refers to a protease fused to an L protein, and Prot-P refers to a protease fused to a P protein.

[0075] The protease may be a monomer or a dimer. Preferably, the dimer is used in the form of a single-chain dimer, with the monomers linked via a flexible linker. An example of a protease that is only active as a dimer is the HIV protease used in the Examples. As described for HIV-1 protease, the single-chain dimer is preferably codon-optimized to avoid homology between the first and second proteases. A codon-optimized single-chain dimer of HIV-1 protease may have the DNA sequence of SEQ ID NO: 5, for example. This reduces the risk of "copy-selection" recombination events, as previously described for VSV (Simon-Loriere and Holmes 2011), in which the viral polymerase, the L protein, can switch between templates, skipping sequence extension. "Copy-selection" occurs when the polymerase is guided by sequence homology of the nascent RNA strand with the newly selected template. Preferably, the protease is autocatalytically active, i.e., it mediates cis-cleavage. This may be an inherent property of the protease, or it may be generated by cloning the respective cleavage sites adjacent to the protease, i.e., the protease has an N-terminal cleavage site and / or a C-terminal cleavage site. Preferably, the protease is assembled with the cleavage sites of the protease on either side. Thus, the protease has two cleavage sites of the protease, one at the N-terminus and the other at the C-terminus of the protease. Preferably, the two cleavage sites are not identical. A protease having a cleavage site may further have a linker on one or both sides, which may be adjacent to one or both cleavage sites or between the protease and one or more cleavage sites.

[0076] Thus, a regulatory element or "switch" according to the present invention comprises a protease, at least one cleavage site of said protease, and a protease inhibitor specific for said protease.

[0077] As used herein, the term "RNA virus" refers to a virus that has ribonucleic acid (RNA) as its genetic material. RNA viruses can be single-stranded (ssRNA) or double-stranded (dsRNA). Single-stranded RNA viruses include the taxonomic division (phylum) of "negative-sense ssRNA viruses (NegaRNAviricota)," which specifically includes the Mononegavirales and Articularis (which includes the Orthomyxoviridae, including influenza viruses), as well as the taxonomic division of "positive-sense ssRNA viruses," such as the Coronaviridae, Flaviviridae, and Enteroviridae families. In particular, negative-sense ssRNA viruses of the Mononegavirales order include the Bornaviridae (e.g., Borna disease virus (BDV)), Nyamiviridae (Nyamanini virus (NYMV)), Rhabdoviridae (rabies virus, vesicular stomatitis virus (VSV), Maraba virus), Filoviridae (Ebola virus, including EBOV), Paramyxoviridae (including measles virus (MeV) and Newcastle disease virus (NDV)), and Pneumoviridae (e.g., human respiratory syncytial virus (HRSV)). In the context of the present invention, Rhabdoviridae and Paramyxoviridae are preferred, and more preferably, RNA viruses of the genus Vesiculovirus.

[0078] Negative-sense viral RNA is complementary to mRNA and must be converted to positive-sense RNA by RNA-dependent RNA polymerase before translation. Therefore, purified negative-sense RNA is not infectious because it must first be transcribed, which requires RNA-dependent RNA polymerase contained in the virus particle (virion). Because the RNA sequence is reverse transcribed for sequencing, the sequences of recombinant RNA viruses are generally provided as cDNA sequences.

[0079] The term "linker" refers to a sequence encoding a separating peptide of variable length, approximately 6 to 30 amino acids, preferably 7 to 15 amino acids, that separates different portions of a protein without affecting the function of the different portions or having any function of its own. The linker may be flexible or rigid, and preferably the linker is a flexible linker. Preferably, no linker is used between the protease cleavage site and at least one protein essential for viral transcription and / or replication. Conditional regulation of RNA viruses

[0080] In one aspect, the present invention relates to a single-stranded RNA virus comprising a modified viral genome comprising a polynucleotide sequence encoding at least one protein essential for viral transcription and / or replication, a protease, and a cleavage site for said protease, wherein the at least one protein essential for viral transcription and / or replication comprises an insertion at an intramolecular insertion site comprising the protease cleavage site, and optionally further comprising a protease. The protease cleaves the at least one protein essential for viral transcription and / or replication at the protease cleavage site at the intramolecular insertion site. Cleavage at the intramolecular insertion site inactivates the protein essential for viral transcription and / or replication. Because addition of a protease inhibitor "switches on" the at least one protein essential for viral transcription and / or replication, this aspect may also be referred to as an "ON switch" in the context of the present invention. Preferably, the insertion comprises a flexible linker, such as a glycine-serine linker, on either side.

[0081] In another aspect, the present invention relates to a single-stranded RNA virus comprising a modified viral genome including a polynucleotide sequence encoding at least one protein essential for viral transcription and / or replication, a protease, and a cleavage site for the protease, wherein the at least one protein essential for viral transcription and / or replication is encoded as a fusion protein comprising the protease fused to its N-terminus or C-terminus, separated by the cleavage site for the protease. The fusion protein may or may not include a linker, such as a glycine-serine linker, between the cleavage site and the protein essential for viral transcription and / or replication. More generally, the fusion protein may or may not include a linker between the protease and the protein essential for viral transcription and / or replication, i.e., between the protease and the cleavage site, or between the cleavage site and the protein essential for viral transcription and / or replication. Preferably, the fusion protein does not include a linker between the cleavage site and the protein essential for viral transcription and / or replication. Alternatively, the fusion protein may or may not include a linker between the protease and the cleavage site. The protease cleaves at the protease cleavage site located at the N-terminus or C-terminus of the at least one protein essential for viral transcription and / or replication encoded by the fusion protein, releasing the at least one protein essential for viral transcription and / or replication. Thus, the protease and the protease cleavage site of the protease are intermolecularly located. The proteolytic release of the at least one protein essential for viral transcription and / or replication activates the protein essential for viral transcription and / or replication. That is, the proteolytic cleavage releases the at least one active protein essential for viral transcription and / or replication. As used herein, the term "release" or "proteolytic release" refers to the removal of the sequence fused to the at least one protein essential for viral transcription and / or replication, rendering the protein inactive.This embodiment may also be referred to as an OFF switch in the context of the present invention, since the addition of a protease inhibitor "switches off" at least one protein essential for viral transcription and / or replication.

[0082] Thus, a fusion protein may consist of a protease fused to the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication, separated by a cleavage site for the protease. The fusion protein may optionally further comprise a linker, such as a glycine-serine linker, between the protease and the at least one protein essential for viral transcription and / or replication. Thus, the fusion protein may or may not comprise a linker between the cleavage site and the protein essential for viral transcription and / or replication, or the fusion protein may or may not comprise a linker between the protease and the cleavage site. Preferably, the fusion protein does not comprise a linker between the cleavage site and the protein essential for viral transcription and / or replication. However, no additional elements are required for the fusion protein to inactivate the at least one protein essential for viral transcription and / or replication. Thus, a fusion protein may consist of a protease fused to the N- or C-terminus of at least one protein essential for viral transcription and / or replication, separated by a protease cleavage site, and optionally a linker. The fusion protein may also comprise or consist of (a) a protease fused to the N- or C-terminus of a protein essential for viral transcription and / or replication, separated by a protease cleavage site, and (b) an additional viral or heterologous protein fused to the opposite end of the protease fused to the N- or C-terminus of a protein essential for viral transcription and / or replication, wherein the additional viral or heterologous protein and the protease are also separated by the protease cleavage site. The fusion protein may optionally further comprise a linker between the protease and the protein essential for viral transcription and / or replication and / or a linker between the protease and the additional viral or heterologous protein.In this context, it is important that the protease contains cleavage sites on either side of the protease (assembled by the cleavage sites of the protease on either side) to release both the protein essential for viral transcription and / or replication and the additional viral or heterologous protein. Preferably, the protease is fused to the N-terminus of the protein essential for viral transcription and / or replication, or at least one protein essential for viral transcription and / or replication is the L protein, or the protease is fused to the N-terminus of the L protein. The two cleavage sites of the protease on either side (and optionally the linker) are preferably different from each other. A protease having cleavage sites on either side may further have a linker on one or both sides, which may be adjacent to one or both cleavage sites or between the protease and one or more cleavage sites. Preferably, the fusion protein does not contain a linker between the cleavage site and at least one protein essential for viral transcription and / or replication.

[0083] In the context of the present invention, suitable RNA viruses are particularly single-stranded RNA viruses. The term "single-stranded RNA virus" includes positive-sense single-stranded RNA viruses or negative-sense single-stranded RNA viruses. Preferably, the RNA virus is a negative-sense single-stranded RNA virus. In one embodiment, the RNA virus is a virus of the order Mononegavirales. More specifically, the single-stranded RNA virus of the order Mononegavirales may be a virus of a family selected from the group consisting of Rhabdoviridae, Paramyxoviridae, Filoviridae, Nyamiviridae, Pneumoviridae, and Bornaviridae, preferably a virus of the family Rhabdoviridae or Paramyxoviridae, preferably a virus of the genus Vesiculovirus, more preferably vesicular stomatitis virus (VSV) or measles virus (MeV), even more preferably VSV.

[0084] In one embodiment, the at least one protein essential for viral transcription and / or replication is a protein of a polymerase complex comprising an RNA-dependent RNA polymerase (RdRp) and / or an RNA-dependent RNA polymerase and / or a nucleocapsid protein. Preferably, the at least one protein essential for viral transcription and / or replication is selected from the group consisting of a polymerase cofactor, a polymerase, and a nucleocapsid protein. The term "polymerase cofactor" refers to an essential component of an RNA polymerase transcription and replication complex. In VSV, the RdRp complex comprises a large protein (L protein) that acts as an RdRp and a phosphorylation protein (P protein). The P protein has two domains, one involved in transcription and the other involved in replication. Typically, the P protein binds to the viral ribonucleocapsid and positions the RNA-dependent RNA polymerase on the template surface.

[0085] In certain embodiments, the RNA virus is a Mononegavirales virus, and the at least one protein essential for viral transcription and / or replication is a polymerase cofactor, such as a phosphoprotein (P protein) or a functional equivalent thereof, a polymerase, such as a large protein (L protein), and / or a nucleocapsid, such as a nucleoprotein (N protein). The at least one protein essential for viral transcription and / or replication may be one, two, or three proteins essential for viral transcription and / or replication, preferably one or two proteins essential for viral transcription and / or replication. The term "functional equivalent" of the P protein refers to an essential component of the RdRp complex other than the RNA-dependent RNA polymerase itself.

[0086] The order Mononegavirales includes, but is not limited to, Bornoviridae, Nyamiviridae, Rhabdoviridae, Filoviridae, Paramyxoviridae, and Pneumoviridae. Polymerase refers to the L protein in Bornoviridae (e.g., BDV), Nyamiviridae (e.g., NYMV), Rhabdoviridae (e.g., VSV, Maraba virus), Filoviridae (e.g., EBOV), Paramyxoviridae (e.g., MeV), and Pneumoviridae (e.g., HRSV). Nucleocapsid refers to the N protein in Bornoviridae (e.g., BDV), Nyamiviridae (e.g., NYMV), Rhabdoviridae (e.g., VSV), Paramyxoviridae (e.g., MeV), and Pneumoviridae (e.g., HRSV), and refers to the NP protein in Filoviridae (e.g., EBOV). Thus, an example of a functional equivalent of N protein is the NP protein of Filoviridae. Polymerase cofactor refers to the P protein in Nyamiviridae (e.g., NYMV), Rhabdoviridae (e.g., VSV), and Pneumoviridae (e.g., HRSV), the X / P protein in Bornoviridae (e.g., BDV), the VP35 protein in Filoviridae (e.g., EBOV), and the P / V / C protein in Paramyxoviridae (e.g., MeV). In addition to the P protein, Nyamiviridae contains an additional polymerase cofactor, the X protein. Thus, examples of functional equivalents of the P protein are the X / P protein in Bornoviridae, the VP35 protein in Filoviridae, the P / V / C protein in Paramyxoviridae, and the X protein in Nyamiviridae.

[0087] In one embodiment, the RNA virus is a Mononegavirales virus, and at least one protein essential for viral transcription and / or replication is a polymerase cofactor, such as a P protein or a functional equivalent thereof, and / or a polymerase, such as an L protein, or a combination thereof. Preferably, at least one protein essential for viral transcription and / or replication is a polymerase, such as an L protein. Without being bound by theory, transcription of viral genes occurs sequentially from a single promoter at the 3' end of the genome, resulting in a reduced amount of each transcript, and therefore less modification of the L protein at the end of the genome compared to modification of other proteins.

[0088] Proteases regulate the activity of at least one protein essential for viral transcription and / or replication. At intramolecular locations, proteases inactivate proteins essential for viral transcription and / or replication through at least the cleavage site of the protease, whereas at intermolecular locations, proteases activate proteins essential for viral transcription and / or replication. This protease refers to a protease in its active state and in the absence of its protein inhibitor. Thus, proteases also regulate viral transcription and / or replication. The term "cleavage site of the protease" refers to a consensus amino acid sequence that serves as a substrate for proteolytic cleavage of a polypeptide. Cleavage sites for each protease are known in the art.

[0089] In one embodiment, the protease is an autocatalytic protease, or the protease acts as an autocatalytic protease. A protease acting as an autocatalytic protease means that the protease is autocatalytically active, i.e., the protease mediates cis-cleavage. Typically, an autocatalytically active protease also mediates trans-cleavage in addition to cis-cleavage in different polypeptides containing the respective cleavage sites. Mediating cis-cleavage may be an inherent property of the protease (autocatalytic protease). For example, the protease is flanked by one or more cleavage sites. Thus, the protease mediates cleavage of the polypeptide containing the protease. In certain cases, the protease may be released from the polyprotein by autocatalytic cleavage. A protease may also become autocatalytically active by incorporating one or two cleavage sites at the N- or C-terminus of the protease. Thus, proteases that act as autocatalytic proteases or catalytically active proteases can also be generated by cloning the respective cleavage sites adjacent to the proteases (without naturally expressing the cleavage sites on the same polypeptide surface), resulting in a recombinant protease with cleavage sites at the N-terminus and / or C-terminus. Preferably, the proteases are assembled with the cleavage sites of the proteases on either side. Thus, the protease has two cleavage sites of the proteases, one at the N-terminus and the other at the C-terminus of the protease. Autocatalytic proteases or proteases that act as autocatalytic proteases favor aspects related to an ON switch (addition of a protease inhibitor "switches on" at least one protein essential for viral transcription and / or replication) and an OFF switch (addition of a protease inhibitor "switches off" at least one protein essential for viral transcription and / or replication).However, in aspects related to ON switches, the insertion at the intramolecular insertion site may contain only the cleavage site for the protease, but the protease may be provided in trans, i.e., preferably as a separate polypeptide encoded by the RNA virus. Thus, at least one protein essential for viral transcription and / or replication comprises an insertion at the intramolecular insertion site containing at least the cleavage site for the protease, preferably the protease and the cleavage site for the protease. Thus, in the case of an insertion at the intramolecular insertion site containing only the cleavage site for the protease, the protease is provided in trans.

[0090] Furthermore, the protease or autocatalytic protease may be any protease, particularly a viral protease, a prokaryotic protease, or a eukaryotic protease, particularly a viral or eukaryotic protease. The protease used in the RNA virus of the present invention is a heterologous protease, i.e., a protease that is not endogenous to the virus. In one embodiment, the protease is a viral protease, such as one derived from HCV or HIV. Those skilled in the art will recognize that proteases suitable for the context of the present invention are highly specific, i.e., have a limited set of unique and rare substrate sequences, and further have specific inhibitors available. The availability of specific inhibitors allows for the conditional regulation of RNA viruses. A protease inhibitor inhibits the proteolytic activity of a protease, where "inhibition" means that the protease exhibits at least 80% inhibition, at least 90% inhibition, at least 95% inhibition, at least 99% inhibition, and preferably 100% inhibition, compared to the protease without the inhibitor. Preferably, the inhibitor is used in a dose that corresponds to a therapeutic serum concentration.

[0091] Protease inhibitors must be specific for the protease and suitable for in vivo use, i.e., safe, bioavailable, and active in vivo after oral or parenteral administration to a subject, preferably a human subject. Viral proteases are advantageous because they are common targets for antiviral drugs, and therefore, several protease inhibitors that inhibit viral proteases have been approved and evaluated as safe for humans. For example, in the case of human immunodeficiency virus (HIV) proteases, a variety of well-characterized protease inhibitors are available, allowing for modulation of the system with desired kinetics. Examples of suitable HIV protease inhibitors include, but are not limited to, indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, amprenavir, fosamprenavir, atazanavir, tipranavir, and darunavir. Human proteases are advantageous because they are endogenous proteins to human patients and therefore do not induce an immune response. Examples of suitable human proteases include, but are not limited to, caspases and metalloproteinases. Examples of suitable human protease inhibitors include, but are not limited to, the caspase inhibitors emricasan and nivocasan; and the matrix metalloproteinase inhibitors batimastat, tanomastat, and ecaliximab.

[0092] The protease may be monomeric or dimeric. Preferably, the dimer is used in the form of a single-chain dimer, with the monomers linked via a flexible linker. An example of a protease that is only active as a dimer is the HIV protease used in the Examples. As described for HIV-1 protease, the single-chain dimer is preferably codon-optimized to avoid homology between the first and second proteases. This reduces the risk of "copy selection" recombination events, in which the viral polymerase L protein can switch between templates and skip sequence extension, as previously described for VSV (Simon-Loriere and Holmes 2011). "Copy selection" occurs when the polymerase is guided by sequence homology of the nascent RNA strand with the newly selected template. Preferably, the protease is autocatalytically active, i.e., it mediates cis-cleavage. In one embodiment, the protease is HIV-1 protease, preferably a single-chain dimer of HIV-1 protease (e.g., a single-chain dimer of HIV-1 protease having the DNA sequence of SEQ ID NO: 5). Suitable HIV-1 protease inhibitors include, but are not limited to, indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, amprenavir, fosamprenavir, atazanavir, tipranavir, or darunavir, preferably amprenavir, saquinavir, or indinavir. In another embodiment, the protease is HCV protease NS3, and suitable NS3 inhibitors include, but are not limited to, boceprevir, telaprevir, asunaprevir, cilprevir, faldaprevir, vaniprevir, narlaprevir, simeprevir, or danoprevir, preferably vaniprevir, narlaprevir, simeprevir, or danoprevir.

[0093] The single-stranded RNA virus according to the present invention may further encode a heterologous protein, preferably a therapeutic protein, a reporter, or a tumor antigen, more preferably a therapeutic protein or a tumor antigen, the production of which depends on the intrinsic activity of the viral transcription complex.

[0094] In certain embodiments, the virus is an oncolytic virus. An oncolytic virus is a virus that preferentially infects and kills cancer cells. The killed cancer cells release new infectious virus particles that infect additional cancer cells and release cell fragments that stimulate the host's anti-tumor immune response. Clinically tested oncolytic RNA viruses include, but are not limited to, reovirus, measles virus, Newcastle disease virus, influenza virus, Semliki Forest virus, Sindbis virus, poliovirus, coxsackievirus, Seneca Valley virus, Maraba virus, and VSV. Preferably, the oncolytic virus is VSV. This virus includes derivatives thereof, such as VSV-GP, pseudotyped with the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), as described in International Patent Publication WO 2010 / 040526. ON switch

[0095] Provided is a single-stranded RNA virus comprising a modified viral genome comprising a polynucleotide sequence encoding at least one protein essential for viral transcription and / or replication, a protease, and a cleavage site for said protease, wherein the at least one protein essential for viral transcription and / or replication comprises an insertion at an intramolecular insertion site comprising at least the cleavage site for said protease, and optionally further comprising a protease, preferably comprising a protease and a cleavage site for said protease.

[0096] The insertion at the intramolecular insertion site of at least one protein essential for viral transcription and / or replication does not affect the activity of at least one protein essential for viral transcription and / or replication. The activity of at least one protein essential for viral transcription and / or replication is measured by a TCID that is a viral reporter gene expression. 50 Replication assay or MTT killing assay (see Figure 13C) is detected, preferably by TCID 50 Replication assays may be used to assess the activity of the virus, i.e., the TCID of at least one protein essential for viral transcription or replication, wherein the modified protein essential for viral transcription and / or replication may be expressed by a single-stranded RNA virus having an insertion at an intramolecular site, or may be expressed in trans on the surface of a plasmid with a single-stranded RNA virus lacking said protein essential for viral transcription and / or replication. 50 A virus is considered to have no effect on the activity of at least one protein essential for viral transcription or replication if it provides a titer of log 2 or less, preferably a titer of log 1.5 or less, more preferably a titer of log 1 or less, and even more preferably an equivalent titer, as compared to a recombinant single-stranded RNA virus (control) that does not contain an insert at the intramolecular insertion site, and if there is a protease at the intramolecular insertion site in the presence of a protease inhibitor in the control sample and the test sample.

[0097] In single-stranded RNA viruses, at least the protease cleavage site and optionally further proteases are located within the intramolecular insertion site of at least one protein essential for viral transcription and / or replication, and proteolytic cleavage of the protein cleaves at least one protein essential for viral transcription and / or replication at the protease cleavage site within the intramolecular insertion site. Cleavage within the intramolecular insertion site inactivates at least one protein essential for viral transcription and / or replication. Thus, cleavage within the intramolecular insertion site of at least one protein essential for viral transcription and / or replication further inhibits viral transcription and / or replication. As a result, the virus is active in the presence of a specific inhibitor of the protease and inactive in the absence of the specific inhibitor of the protease. The single-stranded RNA virus may further encode at least one heterologous protein, which is expressed when the virus is active in the presence of the specific inhibitor of the protease and is not expressed when the virus is inactive in the absence of the specific inhibitor of the protease. The production of such heterologous proteins is dependent on the native activity of the viral transcription complex. Suitable heterologous proteins are proteins such as therapeutic proteins, reporters, or tumor antigens.

[0098] In a specific embodiment, the protease is an autocatalytic HIV protease dimer inserted into the intramolecular insertion site of one or two proteins (P protein and / or L protein, individually and in combination) of the vesicular stomatitis virus (VSV) that constitute the polymerase complex. In the presence of a protease inhibitor, the integrity of the viral proteins is maintained, allowing viral replication. In the absence of a protease inhibitor, the HIV protease dimer is autocatalytically active and cleaves essential viral proteins during translation. Similar to regulatory modules of DNA viruses (e.g., Tet-On), this mechanism is referred to in the examples as "prot-ON."

[0099] In one embodiment, the insert at the intramolecular insertion site comprises a protease and at least one cleavage site of said protease. At the intramolecular location, the protease has two cleavage sites. Thus, the protease has cleavage sites on either side and, optionally, a linker on one or both sides. The linker may be adjacent to one or both cleavage sites, or may be located between the protease and one or more cleavage sites. The two cleavage sites of the protease (and optionally the linker) on either side of the protease are preferably different from each other.

[0100] In one embodiment, the RNA virus is a Mononegavirales virus, and the at least one protein essential for viral transcription and / or replication is a polymerase cofactor, such as a P protein or a functional equivalent thereof; a polymerase, such as an L protein; and / or a nucleocapsid, such as an N protein. The at least one protein essential for viral transcription and / or replication may be one, two, or three proteins essential for viral transcription and / or replication, preferably one or two proteins essential for viral transcription and / or replication. In a specific embodiment, the at least one protein essential for viral transcription and / or replication is a P protein or a functional equivalent thereof, or an L protein, or a combination thereof.

[0101] The flexible linker and codon usage for the protease dimer are optimized to avoid homology between the first and second proteases. Although so-called "copy-selection" recombination events in VSV have been previously described (Simon-Loriere and Holmes 2011), this precaution was adopted because the viral polymerase, the L protein, could potentially switch templates and skip sequence extension. "Copy-selection" occurs preferentially when the polymerase is guided by sequence homology of the nascent RNA strand with the newly selected template. Furthermore, point mutations occur frequently in RNA viruses, with a nucleotide mutation rate of approximately 1 in 10,000 in VSV. Theoretically, every genome contains one mutation, leading virologists to refer to the VSV genome (and other RNA virus genomes) not as a single sequence but as a mixture of so-called "quasi-species." Therefore, the occurrence of a mutation within the HIV protease sequence that inactivates the proteolytic switch is a realistic possibility. To avoid escape mutant or revertant viruses that may overwhelm the conditional ON switch, the protease module (ON switch) may be duplicated by introducing a protease dimer into the first and second essential VSV proteins, such as the P and L proteins. The proteases and their respective cleavage sites may be the same or different in the two proteins essential for viral transcription and / or replication and may therefore be regulated by the same or different protease inhibitors.

[0102] A suitable insertion site at amino acid 196 of the P protein of VSV has been previously described (Das et al., J Virol, 2006, 80(13):6368-6377 and Das and Pattnaik, J Virol, 2005, 79(13):8101-8112), where the numbering corresponds to the insertion site of serotype VSV strain Indiana (VSV) (e.g., having the nucleotide sequence of SEQ ID NO: 1 and / or the amino acid sequence of SEQ ID NO: 27). I ) refers to the P protein.

[0103] Although L protein insertion sites have been described, the resulting viruses were temperature-sensitive and unstable after passaging (Ruedas and Perrault 2009, Ruedas and Perrault 2014). Based on the recently published complete structural information for the VSVL protein (Liang, Li et al. 2015), potential permissive sites were identified and evaluated, first with fluorescent proteins and then with HIV protease dimers.

[0104] Thus, in one embodiment, the single-stranded RNA virus is vesicular stomatitis virus (VSV). While multiple VSV serotypes exist, the most well-characterized and commonly used VSV serotype for treatment is the VSV Indiana strain (VSVi). All sequences disclosed and used herein are derived from VSVi. Also encompassed are derivatives of VSVi, such as VSV-GP, which is described in detail in International Patent Publication WO 2010 / 040526. Because the VSV Indiana strain is an RNA virus, several complete genome nucleotide sequences are available, including the cDNA sequence of SEQ ID NO: 22 (GenBank accession number: MH919398.1). The virus generated in the Examples is derived from the DNA sequence of SEQ ID NO: 20. Accordingly, the positions designated below are provided as corresponding amino acid positions in the P or L protein of VSVi, or in representations of the P or L protein of VSVi having the amino acid sequences of SEQ ID NOs: 27 or 28, respectively. A person skilled in the art knows how to identify corresponding amino acid sequences in the sequences of the P or L proteins of further VSVi by sequence alignment.

[0105] In one embodiment, the single-stranded RNA virus is VSV, and the at least one protein essential for viral transcription and / or replication is the VSV P protein and / or L protein. A suitable intramolecular insertion site for the P protein is within the flexible hinge region of the VSV P protein, preferably at a position corresponding to amino acids 193-199, more preferably amino acid 196, of the VSVi P protein. In a specific embodiment, the intramolecular insertion site for the P protein is at amino acids 193-199, preferably amino acid 196, of the VSVi P protein. These numbers refer to the VSVi P protein, and one exemplary sequence for the VSVi P protein has the amino acid sequence of SEQ ID NO: 27. In one embodiment, the intramolecular insertion site for the P protein is located at amino acids 193 to 199 of the P protein of VSVi having the sequence of SEQ ID NO: 27 or a homolog thereof, preferably at amino acid 196 of the P protein of VSVi having the sequence of SEQ ID NO: 27 or a homolog thereof, which homolog has at least 80% sequence identity to SEQ ID NO: 27, preferably at least 90% sequence identity to SEQ ID NO: 27. A suitable intramolecular insertion site for the L protein is located within the methyltransferase domain (MT) of the L protein, in particular within the loop of the methyltransferase domain of the L protein corresponding to amino acids 1614 to 1634, preferably amino acids 1614 to 1629, more preferably amino acids 1616 to 1625, more preferably amino acid 1620 of the VSVi L protein. In a particular embodiment, the intramolecular insertion site for the L protein is located at amino acids 1614 to 1634, preferably amino acids 1614 to 1629, more preferably amino acids 1616 to 1625, more preferably amino acid 1620 of the VSVi L protein. Here, these numbers refer to the L protein of VSVi, and one exemplary sequence of the L protein of VSVi has the amino acid sequence of SEQ ID NO:28.In one embodiment, the intramolecular insertion site of the L protein is located at amino acids 1614 to 1634, preferably amino acids 1614 to 1629, more preferably amino acids 1616 to 1625, even more preferably amino acid 1620 of the L protein of VSVi having the sequence of SEQ ID NO: 28 or a homolog thereof, which homolog has at least 80% sequence identity to SEQ ID NO: 28, preferably at least 90% sequence identity to SEQ ID NO: 28. In another embodiment, the single-stranded RNA virus is VSV, and the P protein comprises an insertion at an intramolecular insertion site comprising at least a cleavage site for the protease and, optionally, an additional protease, within the flexible hinge region of the VSV P protein at a position corresponding to amino acids 193-199 of the VSVi P protein, preferably at amino acids 193-199 of the VSVi P protein; or the L protein comprises an insertion at an intracellular insertion site comprising at least a cleavage site for the protease and, optionally, an additional protease, within a loop of the methyltransferase domain (MT) of the L protein at a position corresponding to amino acids 1614-1634 of the VSVi L protein, preferably at amino acids 1614-1634 of the VSVi L protein. In yet another embodiment, the single-stranded RNA virus is VSV, and the P protein comprises an insertion at an intramolecular insertion site comprising at least a cleavage site for the protease and, optionally, an additional protease, within the flexible hinge region of the VSV P protein at a position corresponding to amino acids 193-199 of the VSVi P protein, preferably amino acids 193-199 of the VSVi P protein, and the L protein comprises an insertion at an intracellular insertion site comprising at least a cleavage site for the protease and, optionally, an additional protease, within a loop of the methyltransferase domain (MT) of the L protein at a position corresponding to amino acids 1614-1634 of the VSVi L protein, preferably amino acids 1614-1634 of the VSVi L protein. Placing ON switches in multiple, preferably two, proteins essential for viral transcription and / or replication reduces the risk of escape mutants.Despite repeated passaging, no escape mutants of the ON switch have been observed, but insertions into two proteins essential for viral transcription and / or replication result in greater stability. As used herein, the term "amino acid position" refers to the following: amino acid position 196 of the VSVi P protein having the sequence of SEQ ID NO:27 means between amino acids 196 and 197 of the VSVi P protein having the sequence of SEQ ID NO:27, and amino acid 1620 of the VSVi L protein having the sequence of SEQ ID NO:28 means between amino acids 1620 and 1621 of the VSVi L protein having the sequence of SEQ ID NO:28.

[0106] The ON switch system inherently embodies an element of environmental safety: because viral progeny depend on the presence of a protease inhibitor, any virus that is potentially released is not active for productive infection. This could be important if the therapeutic RNA virus is likely to cause disease in animals.

[0107] VSV is typically associated with neurotoxicity and intracranial spread. In vivo data demonstrated that the ON switch system completely inhibited neurotoxicity and intracranial spread. Because the protease inhibitor amprenavir does not cross the blood-brain barrier, systemic administration of the compound did not confer viral activity in the brain. Despite the systemic presence of the ON switch system, which allows viral replication in the presence of amprenavir throughout the body, no neurotoxicity was observed.

[0108] In one embodiment, the single-stranded RNA virus according to the invention is a virus for therapeutic use, in particular for cancer therapy, especially for human use. OFF switch

[0109] Further provided is a single-stranded RNA virus comprising a modified viral genome comprising a polynucleotide sequence encoding at least one protein essential for viral transcription and / or replication, a protease, and a cleavage site for the protease, wherein the at least one protein essential for viral transcription and / or replication is encoded as a fusion protein comprising the protease fused to the N-terminus or C-terminus, separated by the cleavage site for the protease. In one embodiment, the fusion protein does not comprise the amino acid sequence of SEQ ID NO: 30. Preferably, the protease is fused to the N-terminus of the at least one protein essential for viral transcription and / or replication. Thus, the at least one protein essential for viral transcription and / or replication is encoded as a fusion protein comprising the protease fused directly to the N-terminus of the at least one protein essential for viral transcription and / or replication, separated by the cleavage site for the protease and, optionally, a linker. The fusion protein may or may not comprise a linker, such as a glycine-serine linker, between the protease and the protein essential for viral transcription and / or replication. Thus, the fusion protein may or may not contain a linker between the cleavage site and the protein essential for viral transcription and / or replication, or the fusion protein may or may not contain a linker between the protease and the cleavage site. Preferably, the fusion protein does not contain a linker between the cleavage site and the protein essential for viral transcription and / or replication.

[0110] At least one protein essential for viral transcription and / or replication is encoded as a fusion protein comprising a protease fused to the N-terminus or C-terminus of the at least one protein essential for viral transcription and / or replication, separated by a cleavage site for the protease, and proteolytic cleavage of the fusion protein releases the at least one protein essential for viral transcription and / or replication in its active form. Thus, the at least one protein essential for viral transcription and / or replication and the protease are expressed as a fusion protein separated by a cleavage site for the protease. Upon proteolytic cleavage, the protease and the at least one protein essential for viral transcription and / or replication are separated. The at least one protein essential for viral transcription and / or replication is inactive in the fusion protein and becomes active upon release by proteolytic cleavage (also referred to as proteolytic release). According to the present invention, the fusion protein does not comprise the amino acid sequence of SEQ ID NO: 30. This sequence functions as a degron within the SMASh tag described by Chung et al. (Nature Chemical Biology (2015), 11: 713-722), resulting in protein degradation. In contrast, according to the present invention, fusing a protease to at least one protein essential for viral transcription and / or replication separated by a protease cleavage site inactivates the at least one protein essential for viral transcription and / or replication. The fusion protein is therefore expressed and detectable, i.e., not degraded. Thus, within the fusion protein, at least one protein essential for viral transcription and / or replication is functionally inactive. Functionally inactivating at least one protein essential for viral transcription and / or replication, such as the L protein, within the fusion protein is sufficient to efficiently switch off virus production without the need for protein degradation.The single-stranded RNA virus may further encode at least one heterologous protein, and the heterologous protein is expressed when the virus is active in the presence of a specific inhibitor of protease, and is not expressed when the virus is inactive in the absence of a specific inhibitor of protease.The production of such heterologous protein depends on the original activity of the viral transcription complex.Suitable heterologous proteins include therapeutic proteins, reporters, or tumor antigens.

[0111] Therefore, at least one protein essential for viral transcription and / or replication is inactive in a fusion protein comprising a protease fused to the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication, separated by the cleavage site of the protease without involving proteolytic cleavage.The proteolytic cleavage of the fusion protein is inhibited by a specific inhibitor of the protease.Therefore, the single-stranded RNA virus is inactive in the presence of a specific inhibitor of the protease, and is active in the absence of a specific inhibitor of the protease.

[0112] In one embodiment, the protease and the cleavage site for the protease replace the intergenic region linking the protein essential for viral transcription and / or replication with the additional viral protein. Thus, in one embodiment, the fusion protein comprises the protein essential for viral transcription and / or replication and the additional viral protein separated by the protease and the cleavage site for the protease, with the protease preferably adjacent to either side of the cleavage site for the protease (i.e., at the N-terminus and C-terminus of the protease). Thus, loss of the protease results in an additional, inactive fusion protein comprising the protein essential for viral transcription and / or replication and the additional viral protein. In other words, deletion of the protease and the cleavage site for the protease in a revertant or escape mutant results in a new, non-functional fusion protein comprising the protein essential for viral transcription and / or replication in its inactive state fused to the additional viral protein. Therefore, replacing the entire intergenic region makes the virus safer because the insertion containing the protease and the deletion of the protease cleavage site result in an additional fusion protein, which contains a protein essential for viral transcription and / or replication and an additional viral protein. The additional viral protein may be a second protein essential for viral transcription and / or replication. This function provides protection from escape mutants, as the deletion of the protease insertion does not provide any advantage to the virus.

[0113] If the viral genome does not contain two proteins essential for viral transcription and / or replication adjacent to each other, this may be achieved by gene shuffling. It is known that genes can be shuffled in VSV. However, the order of genes in the genome correlates with the frequency of translation. Therefore, gene shuffling usually involves some degree of attenuation. Alternatively, the additional viral protein is a viral protein that is not essential for viral transcription and / or replication.

[0114] In another embodiment, the protease and the cleavage site of the protease replace the intergenic region linking proteins essential for viral transcription and / or replication with a heterologous protein, as shown in Figure 17. In other words, the protease is fused at one end to a protein essential for viral transcription and / or replication and at the other end to a heterologous protein, with the respective ends separated by the cleavage site of the protease. Thus, in one embodiment, the fusion protein may also comprise a heterologous protein fused to the opposite end of the protease fused to the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication, with the heterologous protein and the protease also separated by the cleavage site of the protease. In one embodiment, the protease replaces the intergenic region linking at least one protein essential for viral transcription and / or replication, flanked on either side by the cleavage site of the protease, with the heterologous protein. Thus, loss of the proteolytic enzyme results in an additional inactive fusion protein comprising a protein essential for viral transcription and / or replication and a heterologous protein.

[0115] When the intergenic region is replaced by a protease, the protease should be an autocatalytic protease flanked on either side by cleavage sites for said protease, i.e., comprising two cleavage sites for said protease, one at the N-terminus and the other at the C-terminus of the protease (or single-chain dimeric protease). Thus, in one embodiment, the fusion protein further comprises an additional viral or heterologous protein fused to the end opposite the protease fused to the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication, wherein said additional viral or heterologous protein and said protease are also separated by cleavage sites for said protease. In a specific embodiment, the protease flanking the cleavage site of the protease on either side replaces the intergenic region linking at least one protein essential for viral transcription and / or replication with an additional viral or heterologous protein, preferably forming a fusion protein with at least one additional viral or heterologous protein due to the deletion of the protease in the revertant virus or escape mutant, resulting in the inactivation of at least one protein essential for viral transcription and / or replication. Replacing the intergenic region with the protease has the advantage of reducing (in the case of an additional viral protein) or not increasing (in the case of a heterologous protein) the number of intergenic regions, thus reducing the risk of viral attenuation. The two cleavage sites of the protease (and optionally the anchor) on either side of the protease are preferably different from each other.

[0116] In one embodiment, the RNA virus is a Mononegavirales virus, and the at least one protein essential for viral transcription and / or replication is a polymerase cofactor, such as the P protein or a functional equivalent thereof; a polymerase, such as the L protein; and / or a nucleocapsid, such as the N protein. The at least one protein essential for viral transcription and / or replication may be one or two proteins essential for viral transcription and / or replication, preferably one protein essential for viral transcription and / or replication. Preferably, the at least one protein essential for viral transcription and / or replication is the P protein or a functional equivalent thereof or the L protein, preferably the L protein. Furthermore, the protease is preferably fused to the N-terminus of the P protein (or a functional equivalent thereof) or the L protein, more preferably to the N-terminus of the L protein.

[0117] In a preferred embodiment, the at least one protein essential for viral transcription and / or replication is the L protein; or a protease is fused to the N-terminus of the at least one protein essential for viral transcription and / or replication, separated by a cleavage site for the protease; or the at least one protein essential for viral transcription and / or replication is the L protein and the protease is fused to the N-terminus of the L protein, separated by a cleavage site for the protease. According to the present invention, the at least one protein essential for viral transcription and / or replication, preferably the L protein, is inactive in the fusion protein, preferably a fusion protein comprising the protease fused to the N-terminus of the at least one protein essential for viral transcription and / or replication, and becomes active upon release by proteolytic cleavage.

[0118] In one embodiment, a single-stranded RNA virus comprises a modified viral genome comprising a polynucleotide sequence encoding at least one protein essential for viral transcription and / or replication, a protease, and a cleavage site for the protease, wherein the at least one protein essential for viral transcription and / or replication is encoded as a fusion protein consisting of the protease fused to the N-terminus or C-terminus of the protein essential for viral transcription and / or replication, separated by the cleavage site for the protease, and the fusion protein optionally further comprising a linker, such as a glycine-serine linker, between the protease and the protein essential for viral transcription and / or replication. Thus, the fusion protein may or may not comprise a linker between the cleavage site and the protein essential for viral transcription and / or replication; alternatively, the fusion protein may or may not comprise a linker between the protease and the cleavage site. Preferably, the fusion protein does not comprise a linker between the cleavage site and the protein essential for viral transcription and / or replication. Preferably, the protease is fused to the N-terminus of a protein essential for viral transcription and / or replication, or at least one protein essential for viral transcription and / or replication is the L protein, or the protease is fused to the N-terminus of the L protein.

[0119] In another embodiment, the single-stranded RNA virus comprises a modified viral genome comprising a polynucleotide sequence encoding at least one protein essential for viral transcription and / or replication, a protease, and a cleavage site for the protease, wherein the at least one protein essential for viral transcription and / or replication is encoded as a fusion protein comprising or consisting of: (a) the protease fused to the N-terminus or C-terminus of the at least one protein essential for viral transcription and / or replication, separated by the cleavage site for the protease; and (b) an additional viral or heterologous protein fused to the opposite end of the protease fused to the N-terminus or C-terminus of the at least one protein essential for viral transcription and / or replication, wherein the additional viral or heterologous protein and the protease are also separated by the cleavage site for the protease, and the fusion protein optionally further comprises a linker between the protease and the protein essential for viral transcription and / or replication and / or a linker between the protease and the additional viral or heterologous protein. Thus, the fusion protein may or may not contain a linker between the cleavage site and the protein essential for viral transcription and / or replication; alternatively, the fusion protein may or may not contain a linker between the protease and the cleavage site; and / or the fusion protein may or may not contain a linker between the cleavage site and the additional viral or heterologous protein; alternatively, the fusion protein may or may not contain a linker between the other side of the protease and the cleavage site. Preferably, the fusion protein does not contain a linker between the cleavage site and the protein essential for viral transcription and / or replication.

[0120] Preferably, the protease is fused to the N-terminus of a protein essential for viral transcription and / or replication, or at least one protein essential for viral transcription and / or replication is an L protein, or the protease is fused to the N-terminus of the L protein. The two cleavage sites of the protease (and optionally a linker) on either side of the protease are preferably different from each other. A protease having a cleavage site on either side may be adjacent to one or both cleavage sites, or may have a linker on one or both sides between the protease and one or more cleavage sites. Preferably, the fusion protein does not contain a linker between the cleavage site and at least one protein essential for viral transcription and / or replication.

[0121] In one embodiment, the single-stranded RNA virus according to the invention is a virus for therapeutic use, in particular for cancer therapy, especially for human use. Conditional expression of heterologous proteins

[0122] The single-stranded RNA virus of the present invention comprises a modified viral genome comprising a polynucleotide sequence encoding at least one protein essential for viral transcription and / or replication, a protease, and a cleavage site for the protease, wherein (a) the at least one protein essential for viral transcription and / or replication comprises an insertion in an intramolecular insertion site comprising the protease and, optionally, at least a cleavage site for an additional protease, or (b) the at least one protein essential for viral transcription and / or replication is encoded as a fusion protein comprising a protease fused to its N-terminus or C-terminus, separated by a cleavage site for the protease, and optionally further encoding at least one heterologous protein. Production of such a heterologous protein depends on native viral transcription and / or replication, and therefore requires that at least one protein essential for viral transcription and / or replication be active. Thus, in option (a) (ON switch), the heterologous protein is expressed when the virus is active in the presence of a specific inhibitor of the protease, but not when the virus is inactive in the absence of the specific inhibitor of the protease; and in option (b) (OFF switch), the heterologous protein is not expressed when the virus is inactive in the presence of a specific inhibitor of the protease, but is expressed when the virus is active in the absence of the specific inhibitor of the protease. Suitable heterologous proteins are proteins such as therapeutic proteins, reporters, or tumor antigens. Particularly for therapeutic purposes, the heterologous protein is preferably a therapeutic protein with immunoregulatory or cell death-regulatory function, or a tumor antigen. The therapeutic protein may also be a protein encoded by a suicide gene.

[0123] Further provided herein is an RNA virus comprising a modified viral genome comprising a polynucleotide sequence encoding at least one heterologous protein, a protease, and a cleavage site for said protease, wherein the at least one heterologous protein comprises an insertion at an intramolecular insertion site comprising said protease and, optionally, at least a cleavage site for a further protease. In one embodiment, the RNA virus is a single-stranded RNA virus.

[0124] The term "single-stranded RNA virus" includes positive-sense single-stranded RNA viruses or negative-sense single-stranded RNA viruses. Preferably, the RNA virus is a negative-sense single-stranded RNA virus. In one embodiment, the RNA virus is a virus of the order Mononegavirales. More specifically, the single-stranded RNA virus of the order Mononegavirales can be a virus of a family selected from the group consisting of Rhabdoviridae, Paramyxoviridae, Filoviridae, Nyamiviridae, Pneumoviridae, and Bornaviridae, preferably a virus of the family Rhabdoviridae or Paramyxoviridae, preferably a virus of the genus Vesiculovirus, more preferably vesicular stomatitis virus (VSV) or measles virus (MeV), even more preferably VSV.

[0125] In certain embodiments, the virus is an oncolytic virus. An oncolytic virus is a virus that preferentially infects and kills cancer cells. The dead cancer cells release new infectious virus particles that infect additional cancer cells and release cell fragments that stimulate the host's anti-tumor immune response. Clinically tested oncolytic RNA viruses include, but are not limited to, reovirus, measles virus, Newcastle disease virus, influenza virus, Semliki Forest virus, Sindbis virus, poliovirus, Coxsackievirus, Seneca Valley virus, Maraba virus, and VSV. Preferably, the oncolytic virus is VSV.

[0126] The term "heterologous" refers to an RNA virus, not a host or patient infected with the virus, and thus explicitly encompasses eukaryotic, particularly human, proteins. A heterologous protein is a protein derived from a different organism or species than the recipient, i.e., an RNA virus. At least one heterologous protein encoded by an RNA virus according to the present invention may be a therapeutic protein, a reporter, or a tumor antigen. Preferably, the at least one heterologous protein is a therapeutic protein with immunomodulatory or cell death-regulating function, preferably selected from the group consisting of cytokines, chemokines, growth factors, and antibodies. The therapeutic protein may also be a membrane-associated protein, or may be membrane-bound by fusing a transmembrane domain, such as the transmembrane domain of CD4, to the heterologous protein, preferably via a linker. The therapeutic protein may also be an encoded suicide gene. Alternatively, or in addition, the at least one heterologous protein is a tumor antigen (including tumor-specific and / or tumor-associated antigens), such as a lineage antigen, neoantigen, testis antigen, or tumor virus antigen. The term "tumor-specific antigen" refers to an antigen that is expressed only in tumor cells and not in any other tissue of an organism. The term "tumor-associated antigen" refers to an antigen that is overexpressed, i.e., expressed at a higher concentration, in tumor cells compared to other tissues in an organism. A tumor antigen may also be one or more neoantigens, where neoantigens are newly formed antigens resulting from somatic mutations in tumors. Those skilled in the art know how to detect and determine neoantigens from patients. In another embodiment, the heterologous protein is a reporter protein such as green fluorescent protein, red fluorescent protein, mCherry, or mWasabi. For therapeutic purposes, the heterologous protein is preferably a therapeutic protein or tumor antigen with immunomodulatory or cell death-regulating functions.

[0127] Those skilled in the art will appreciate that an insert comprising at its intramolecular insertion site at least the cleavage site for said protease and optionally at least the cleavage site for the further protease corresponds to an insert comprising at its intramolecular insertion site for at least one protein essential for viral transcription and / or replication, i.e., the ON switch, comprising at its intramolecular insertion site for said protease and optionally at least the cleavage site for the further protease. Thus, the above disclosure and embodiments relating to ON switches equally apply to RNA viruses comprising a modified viral genome comprising at least one heterologous protein, a protease, and a cleavage site for said protease, wherein the at least one heterologous protein comprises an insert comprising at its intramolecular insertion site at least the cleavage site for said protease and optionally at least the cleavage site for the further protease. In one embodiment, the RNA virus is a single-stranded RNA virus.

[0128] Those skilled in the art will further appreciate that aspects relating to the ON switch of a heterologous protein may be combined with the ON switch of at least one protein essential for viral transcription and / or replication. Thus, armed viruses with two independent switches, one controlling viral activity and the other controlling the activity of a virally encoded therapeutic protein, are also contemplated. Both switches are preferably controlled by two independent compounds.

[0129] In one embodiment, the RNA virus according to the invention is a virus for therapeutic use, in particular for cancer therapy, especially for human use.

[0130] Further provided are polynucleotide sequences encoding at least one recombinant protein, a protease, and a cleavage site for said protease, wherein the at least one recombinant protein comprises an insert at an intramolecular insertion site comprising the protease and the cleavage site for said protease, or the at least one recombinant protein comprises an intramolecular insertion site comprising the protease and the cleavage site for said protease. Thus, the ON switches described herein can also be used for therapeutic proteins, particularly therapeutic proteins with a small therapeutic window. Examples of such therapeutic proteins are cytokines.

[0131] In one embodiment, the polynucleotide or recombinant protein according to the invention is a virus for therapeutic use, in particular for use in human therapy, and therefore the proteolytic enzymes are preferably of human origin to prevent immune reactions.

[0132] Thus, in one embodiment, the protease is a human protease, such as a metalloproteinase or a caspase. Examples of suitable human protease inhibitors include, but are not limited to, emericasan, nivocasan, batimastat, tanomastat, and ecaliximab. In a further embodiment, the protease is an autocatalytic protease or a protease that acts as an autocatalytic protease. Thus, the protease may be adjacent to the cleavage site of the protease, and preferably, the protease is adjacent to the cleavage site of the protease on either side. The two cleavage sites of the protease (and optionally the linker) on either side of the protease are preferably different from each other. Preferably, the insert includes a flexible linker, such as a glycine-serine linker, on either side. Therapeutic Use

[0133] Further provided herein is a single-stranded RNA virus according to the present invention, comprising a modified viral genome comprising a polynucleotide sequence encoding at least one protein essential for viral transcription and / or replication, a protease, and a cleavage site for said protease, wherein (a) at least one protein essential for viral transcription and / or replication comprises an insertion in an intramolecular insertion site comprising at least said protease and optionally a cleavage site for an additional protease, or (b) at least one protein essential for viral transcription and / or replication is encoded as a fusion protein comprising a protease fused to its N-terminus or C-terminus, separated by a cleavage site for said therapeutic protease, and the single-stranded RNA virus optionally further encodes at least one heterologous protein, such as a therapeutic protein or tumor antigen. Suitable therapies include cancer therapy, gene therapy, and / or prophylactic and therapeutic vaccination.

[0134] In a preferred embodiment, the single-stranded RNA virus is a virus used to treat cancer.

[0135] Also provided herein is an RNA virus according to the present invention, comprising a modified viral genome comprising a polynucleotide sequence encoding at least one heterologous protein, a protease, and a cleavage site for said protease, wherein the at least one heterologous protein comprises an insertion in an intramolecular insertion site comprising at least the cleavage site for said protease, and optionally further comprises a protease for therapeutic use. Suitable therapeutic methods include cancer therapy, gene therapy, and / or prophylactic and therapeutic vaccination.

[0136] In a preferred embodiment, the RNA virus is a virus used to treat cancer.

[0137] The virus may be administered intravenously, intratumorally, subcutaneously, intramuscularly, intradermally, intranasally, or intraperitoneally, preferably intravenously or intratumorally. The virus may be administered using a physiological buffer or related formulation. The protease inhibitor used is specific for the protease. Examples of suitable HIV protease inhibitors include, but are not limited to, indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, amprenavir, fosamprenavir, atazanavir, tipranavir, and darunavir. Other suitable protease inhibitors are well known in the art. In therapy, protease inhibitors, particularly HIV protease inhibitors, may be administered in combination with blockers of protease enzymes such as Cyp family members, such as ritonavir. Ritonavir or other protease inhibitors increase the plasma concentration of other protease inhibitors.

[0138] The protease inhibitor may be administered by any suitable route, preferably subcutaneously, orally, or intravenously, more preferably orally.

[0139] The cancer may be a solid tumor, preferably selected from the group consisting of colon cancer, prostate cancer, breast cancer, lung cancer, skin cancer, liver cancer, bone cancer, ovarian cancer, pancreatic cancer, brain cancer, head and neck cancer, lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma), brain cancer, neuroblastoma, mesothelioma, Wilms' tumor, retinoblastoma, and sarcoma (such as rhabdomyosarcoma). L protein insertion site

[0140] Further provided is a recombinant VSV L protein comprising an insertion within the methyltransferase (MT) domain of the L protein or within a loop of the methyltransferase domain of the L protein corresponding to amino acids 1614-1634, preferably amino acids 1614-1629, more preferably amino acids 1616-1625, and even more preferably amino acid 1620, of a VSViL protein, particularly a VSViL protein having the amino acid sequence of SEQ ID NO: 28. In certain embodiments, the intramolecular insertion site of the L protein is located at amino acids 1614-1634, preferably amino acids 1614-1629, more preferably amino acids 1616-1625, and even more preferably amino acid 1620, of the VSVi L protein, where these numbers refer to the VSVi L protein, and one exemplary sequence of a VSVi L protein has the amino acid sequence of SEQ ID NO: 28. In one embodiment, the intramolecular insertion site of the L protein is located at amino acids 1614 to 1634, preferably amino acids 1614 to 1629, more preferably amino acids 1616 to 1625, even more preferably amino acid 1620 of the L protein of VSVi having the sequence of SEQ ID NO: 28 or a homolog thereof, wherein the homolog has at least 80% sequence identity to SEQ ID NO: 28, preferably at least 90% sequence identity to SEQ ID NO: 28.

[0141] As used herein, the term "insert" refers to an amino acid sequence of variable length, ranging from a few amino acids (at least 3, at least 5, at least 10, and preferably at least 15 amino acids) to several hundred amino acids, such as up to 500, 300, and 250 amino acids. An insert is introduced into another sequence, in this case the L protein sequence, resulting in a net addition of amino acids. Thus, an insert may contain, for example, at least a cleavage site for a protease (e.g., at least about 15 amino acids, such as SEQ ID NOS: 6 and 7); a protease and at least a cleavage site for the protein; or a reporter protein. Preferably, an insert contains a flexible linker, such as a glycine-serine linker, on either side. In certain embodiments, an insert is 15 to 500 amino acids, preferably 15 to 300 amino acids, and more preferably 15 to 250 amino acids. An insert results in a net addition of amino acids and does not include amino acid substitutions, i.e., simply replacing one or more amino acids with the same number of different amino acids.

[0142] Insertions at the intramolecular insertion site of the L protein do not affect the activity of the L protein. The activity of the L protein can be determined by detecting the expression of a viral reporter gene, i.e., TCID 50 Preferably, TCID 50 Replication assays may be used to assess the activity of the protein, e.g., TCID, where a modified L protein with an insertion at an intramolecular site may be expressed by a single-stranded RNA virus or may be expressed in trans on a plasmid with a single-stranded RNA virus lacking the L protein. Insertion at the intramolecular insertion site of the L protein may also affect the activity of the protein, e.g., TCID, 50It is considered that the activity of the L protein is not affected if viral replication, as measured by the method described above, provides a titer of 2 or less in logarithm, preferably 1.5 or less in logarithm, more preferably 1 or less in logarithm, and even more preferably an equal titer, compared to a recombinant single-stranded RNA virus (control) that has no insert at the intramolecular insertion site, and if there is a protease at the intramolecular insertion site in the control sample and the test sample where a protease inhibitor is present.

[0143] In one embodiment, the insert comprises a fluorescent protein. In another embodiment, the insert comprises a protease cleavage site, or alternatively, a protease and said protease cleavage site. The protease may be a single-chain dimer flanked by two protease cleavage sites, or the protease may be a monomer flanked by N-terminal and / or C-terminal protease cleavage sites. In one embodiment, the protease is a viral protease, such as from HCV or HIV. Preferably, the protease is or acts as an autocatalytic protease.

[0144] The autocatalytic protease may be an HIV-1 protease, preferably a single-chain dimer of HIV-1 protease, and the protease may be inhibited by a protease inhibitor selected from the group consisting of indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, amprenavir, fosamprenavir, atazanavir, tipranavir, and darunavir.

[0145] The L protein may further comprise a secondary mutation, preferably in the methyltransferase domain of the L protein, which, in one embodiment, restores activity of the L protein.

[0146] Further provided herein is a vesicular stomatitis virus (VSV) comprising a recombinant VSV L protein according to the invention. In vitro methods

[0147] Further provided is a method for controlling the replication of an RNA virus, the method comprising transducing or transducing a host cell with a single-stranded RNA virus according to the present invention, the virus comprising a modified genome comprising a polynucleotide sequence encoding at least one protein essential for viral transcription and / or replication, a protease, and a cleavage site for said protease; and maintaining the host cell in the presence or absence of a protease inhibitor specific for said protease, wherein at least one protein essential for viral transcription and / or replication comprises an insertion in an intramolecular insertion site comprising at least said protease and optionally a cleavage site for a further protease, and wherein addition of said protease inhibitor allows viral transcription and / or replication, and absence of said protease inhibitor inhibits viral transcription and replication.

[0148] Also provided is a method for controlling the replication of an RNA virus, the method comprising transducing or transducing a host cell with a single-stranded RNA virus according to the present invention, the virus comprising a modified viral genome comprising a polynucleotide sequence encoding at least one protein essential for viral transcription and / or replication, a protease, and a cleavage site for said protease; and maintaining the host cell in the presence or absence of a protease inhibitor specific for said protease, wherein at least one protein essential for viral transcription and / or replication is encoded as a fusion protein comprising the protease fused to its N-terminus or C-terminus, separated by the cleavage site for said protease, and wherein addition of the protease inhibitor inhibits viral transcription and / or replication, and absence of the protease inhibitor allows viral transcription and replication.

[0149] Further provided is a method for controlling expression of a heterologous protein by an RNA virus, the method comprising transducing or transducing a host cell with an RNA virus according to the present invention; and maintaining the host cell in the presence or absence of a protease inhibitor specific for said protease, wherein the protease is located within an intramolecular insertion site of at least one heterologous protein, and addition of said protease inhibitor enables expression of the heterologous protein, and absence of said protease inhibitor inhibits heterologous protein expression.

[0150] Preferably, the method according to the invention is an in vitro method, so that the transduction or gene transfer and maintenance steps are carried out in vitro in cell culture.

[0151] The protease is preferably an autocatalytic protease, more preferably an HIV-1 protease, and even more preferably a single-chain dimer of HIV-1 protease. This protease is particularly advantageous because multiple protease inhibitors are available. Suitable protease inhibitors include, for example, indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, amprenavir, fosamprenavir, atazanavir, tipranavir, or darunavir. In view of the above, it can be seen that the present invention also encompasses the following items. Item 1: A single-stranded RNA virus comprising a modified viral genome comprising a polynucleotide sequence encoding at least one protein essential for viral transcription and / or replication, a protease, and a cleavage site for said protease, (a) at least one protein essential for viral transcription and / or replication contains an insertion in an intramolecular insertion site that includes at least the cleavage sites for said protease and optionally further proteases, or (b) At least one protein essential for viral transcription and / or replication is encoded as a fusion protein comprising a protease fused to the N-terminus or C-terminus, separated by a cleavage site for the protease. Item 2: The single-stranded RNA virus according to item 1, (a) the protease cleaves at least one protein essential for viral transcription and / or replication at a cleavage site for said protease located at the intramolecular insertion site; or (b) The protease cleaves at its cleavage site located at the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication encoded as the fusion protein, thereby releasing at least one protein essential for viral transcription and / or replication. Item 3: 3. The single-stranded RNA virus according to item 1 or 2, Proteases can be inhibited using protease inhibitors. Item 4: The single-stranded RNA virus according to any one of items 1 to 3, The at least one protein essential for viral transcription and / or replication is an RNA-dependent RNA polymerase or a protein of a polymerase complex comprising an RNA-dependent RNA polymerase or a nucleocapsid, and preferably, the at least one protein essential for viral transcription and / or replication is selected from the group consisting of a polymerase cofactor, a polymerase, and a nucleocapsid. Item 5: 5. The single-stranded RNA virus according to any one of items 1 to 4, The single-stranded RNA virus is a negative-sense single-stranded RNA virus, preferably a negative-sense single-stranded RNA virus of the Mononegaviridae family. Item 6: 6. The single-stranded RNA virus according to any one of items 1 to 5, Single-stranded RNA viruses (a) a virus of a family selected from the group consisting of Rhabdoviridae, Paramyxoviridae, Filoviridae, Nyamiviridae, Pneumoviridae, and Bornaviridae; and / or (b) a virus of the Paramyxoviridae family, preferably measles virus (MeV), or a virus of the Rhabdoviridae family, preferably vesicular stomatitis virus (VSV); Item 7: Item 7. The single-stranded RNA virus according to Item 5 or 6, The at least one protein essential for viral transcription and / or replication is selected from the group consisting of a polymerase cofactor, a polymerase, and a nucleocapsid, and preferably the at least one protein essential for viral transcription and / or replication is selected from the group consisting of: (a) a polymerase cofactor, preferably a P protein or a functional equivalent thereof; (b) a polymerase, preferably an L protein; and / or (c) A combination thereof. Item 8 8. The single-stranded RNA virus according to any one of items 1 to 7, (a) The protease regulates the activity of at least one protein essential for viral transcription and / or replication; (b) proteolytic enzymes regulate viral transcription and / or replication; (c) the protease is an autocatalytic protease; (d) the protease is a viral protease; and / or (e) The protease is derived from HCV or HIV. Item 9 9. The single-stranded RNA virus according to any one of items 1 to 8, The protease is an HIV-1 protease, preferably a single-chain dimer of HIV-1 protease, which can be inhibited by a protease inhibitor selected from the group consisting of indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, amprenavir, fosamprenavir, atazanavir, tipranavir, and darunavir. Item 10: 10. The single-stranded RNA virus according to any one of items 1 to 9, The insertion at the intramolecular insertion site of at least one protein essential for viral transcription and / or replication does not affect the activity of the at least one protein essential for viral transcription and / or replication. Item 11: The single-stranded RNA virus according to any one of items 1 to 10, at least the cleavage site for said protease and optionally for further proteases is located within an intramolecular insertion site of at least one protein essential for viral transcription and / or replication, (a) proteolytic cleavage of the protein cleaves at least one protein essential for viral transcription and / or replication at a cleavage site for said protease within the intramolecular insertion site; (b) cleavage within the intramolecular insertion site renders inactive at least one protein essential for viral transcription and / or replication; (c) cleavage within the intramolecular insertion site of at least one protein essential for viral transcription and / or replication inhibits viral transcription and / or replication; (d) the virus is active in the presence of a specific inhibitor of the protease and is inactive in the absence of a specific inhibitor of the protease; and / or (e) The virus further encodes at least one heterologous protein, which is expressed when the virus is active in the presence of a specific inhibitor of the protease and is not expressed when the virus is inactive in the absence of the specific inhibitor of the protease. Item 12: 12. The single-stranded RNA virus according to any one of items 1 to 11, The single-stranded RNA virus is vesicular stomatitis virus (VSV), and at least one protein essential for viral transcription and / or replication is the P protein and / or the L protein, and the intramolecular insertion site is (a) within the flexible hinge region of the VSV P protein, preferably at a position corresponding to amino acids 193-199, more preferably amino acid 196, of the VSV P protein (e.g., the sequence of SEQ ID NO: 27); (b) within a loop of the methyltransferase domain of a VSVi L protein corresponding to amino acids 1614 to 1634, preferably amino acids 1614 to 1629, more preferably amino acids 1616 to 1625, more preferably amino acid 1620 of the L protein having the sequence of SEQ ID NO: 28; or (c) A combination of (a) and (b). Item 13: 10. The single-stranded RNA virus according to any one of items 1 to 9, at least one protein essential for viral transcription and / or replication is encoded as a fusion protein comprising a protease fused to the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication, separated by a cleavage site for said protease; (a) Proteolytic cleavage of the fusion protein releases at least one protein essential for viral transcription and / or replication in an active form; (b) at least one protein essential for viral transcription and / or replication in a fusion protein comprising a protease fused to the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication, separated by a cleavage site for said protease, is inactive in the absence of proteolytic cleavage; (c) proteolytic cleavage of the fusion protein is inhibited using specific inhibitors of proteases; (d) the virus is inactive in the presence of a specific protease inhibitor of the protease and active in the absence of a specific inhibitor of the protease; (e) the virus further encodes at least one heterologous protein that is not expressed when the virus is inactive in the presence of a specific inhibitor of the protease and that is expressed when the virus is active in the absence of the specific inhibitor of the protease; (d) the fusion protein further comprises an additional viral or heterologous protein fused to the end opposite the protease fused to the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication, wherein the additional viral or heterologous protein and the protease are also separated by a cleavage site for the protease; (e) the protease flanking the protease cleavage site on either side replaces an intergenic region linking at least one protein essential for viral transcription and / or replication with an additional viral or heterologous protein; and / or (f) The protease flanking the protease cleavage site on either side replaces the intergenic region linking at least one protein essential for viral transcription and / or replication with an additional viral or heterologous protein, and loss of the protease results in an additional inactive fusion protein comprising the protein essential for viral transcription and / or replication and the additional viral or heterologous protein. Item 14: Item 14. The single-stranded RNA virus according to Item 1 or 13, The single-stranded RNA virus is a negative-sense single-stranded RNA virus of the Mononegaviridae family, and at least one protein essential for viral transcription and / or replication is encoded as a fusion protein comprising a protease fused to the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication, separated by a cleavage site for the protease; (a) at least one protein essential for viral transcription and / or replication is the L protein; and / or (b) The fusion protein comprises a protease fused to the N-terminus of at least one protein essential for viral transcription and / or replication, separated by a cleavage site for said protease. Item 15: Item 14. The single-stranded RNA virus according to Item 1 or 13, At least one protein essential for viral transcription and / or replication is encoded as a fusion protein, which fusion protein comprises: (a) consisting of a protease fused to the N-terminus or C-terminus of a protein essential for viral transcription and / or replication, separated by a cleavage site for said protease, wherein the fusion protein optionally contains a linker between the protease and the protein essential for viral transcription and / or replication; (b) comprising a protease fused to the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication, separated by a cleavage site for said protease and a further viral protein, or a heterologous protein fused opposite a protease fused to the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication, wherein said further viral or heterologous protein and said protease are also separated by a cleavage site for said protease, and the fusion protein optionally further comprises a linker between the protease and the at least one protein essential for viral transcription and / or replication and / or a linker between the protease and the further viral or heterologous protein; or (c) does not contain the amino acid sequence of SEQ ID NO: 30. Item 16: An RNA virus comprising a modified viral genome comprising a polynucleotide sequence encoding at least one heterologous protein, a protease, and a cleavage site for said protease, wherein the at least one heterologous protein comprises an insertion in an intramolecular insertion site comprising at least the cleavage site for said protease and optionally a further protease. Item 17: 17. The RNA virus according to item 16, wherein the heterologous protein is a therapeutic protein, a reporter, or a tumor antigen. Item 18: 18. The single-stranded RNA virus according to any one of items 1 to 15, or the RNA virus according to item 16 or 17, for use in therapy. Item 19: 18. The single-stranded RNA virus according to any one of items 1 to 15, or the RNA virus according to item 16 or 17, for use in the treatment of cancer. Item 20: Item 19. A single-stranded RNA virus or an RNA virus for use according to Item 19, wherein the cancer is a solid tumor, preferably selected from the group consisting of colon cancer, prostate cancer, breast cancer, lung cancer, skin cancer, liver cancer, bone cancer, ovarian cancer, pancreatic cancer, brain tumor, head and neck cancer, lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma), brain cancer, neuroblastoma, mesothelioma, Wilms' tumor, retinoblastoma, and sarcoma. Item 21: A recombinant VSV L protein comprising an insertion within a loop of the methyltransferase domain of the L protein corresponding to amino acids 1614 to 1634, preferably amino acids 1614 to 1629, more preferably amino acids 1616 to 1625, and even more preferably amino acid 1620, of a VSV L protein having the sequence of SEQ ID NO:28. Item 22: 22. The recombinant VSV L protein of item 21, wherein the L protein further comprises a secondary mutation. Item 23: 23. A vesicular stomatitis virus (VSV) comprising a recombinant VSV L protein according to item 21 or 22. Item 24: 1. A method for controlling RNA virus replication, the method comprising: (a) transducing or transducing a host cell with an RNA virus according to any one of items 10 to 12, and (b) maintaining the host cell in the presence or absence of a protease inhibitor specific for the protease; The addition of the protease inhibitor allows viral transcription and / or replication, and the absence of the protease inhibitor inhibits viral transcription and replication. Item 25: 1. A method for controlling RNA virus replication, the method comprising: (a) transducing or transducing a host cell with an RNA virus according to any one of items 13 to 15, and (b) maintaining the host cell in the presence or absence of a protease inhibitor specific for the protease; The addition of said protease inhibitor inhibits viral transcription and / or replication, and the absence of said protease inhibitor allows viral transcription and replication. Item 26: 1. A method for controlling heterologous protein expression by an RNA virus, the method comprising: (a) transducing or transducing a host cell with an RNA virus according to item 16 or 17; wherein the proteolytic enzyme is located within an intramolecular insertion site of at least one heterologous protein; and (b) maintaining the host cell in the presence or absence of a protease inhibitor specific for the protease; The addition of the protease inhibitor allows expression of the heterologous protein, and the absence of the protease inhibitor inhibits expression of the heterologous protein. [Example]

[0152] material and method P protein

[0153] The DNA sequence (P196PR2, DNA sequence SEQ ID NO: 3) of a phosphoprotein (P protein with cDNA sequence SEQ ID NO: 1; corresponding to the amino acid sequence SEQ ID NO: 27) with a dimeric protease linked at aa position 196, and flanking sequences of VSV nucleoprotein and matrix protein were synthesized by GeneArt. The P protein gene in the VSV Indiana strain was replaced by P196PR2. GFP at position 5 was used as a marker gene. P196PR2, with flanking VSV-N and VSV-M sequences, was amplified by PCR using a 30-bp sequence spanning two restriction enzyme sites (XbaI and Bst1107I) and digested with these enzymes into a full-length VSV vector. The construct was subsequently generated by cloning using Gibson assembly (Figure 4).

[0154] The protease-linked dimer construct (DNA sequence of SEQ ID NO: 5) was flanked by a (GGSG)3 linker sequence (DNA sequence of SEQ ID NO: 4; amino acid sequence of SEQ ID NO: 29) to spatially separate the P protein and the protease dimer. The 5' GGSG linker had the DNA sequence of SEQ ID NO: 8, and the 3' GGSG linker had the DNA sequence of SEQ ID NO: 9. The linker codons were manually designed to avoid homology between the upstream and downstream linkers. Cleavage sites were located between each protease domain and the linker and had the DNA sequences of SEQ ID NO: 6 and SEQ ID NO: 7. The protease dimer codons were selected through a combination of an optimization algorithm and manual adjustment. The optimization process was a compromise between using human codons and avoiding homology between the first and second proteases. Overhangs were introduced by Gibson assembly cloning.

[0155] The functionality of the phosphoprotein-protease construct was first tested using a P expression plasmid (Figure 2), in which P196PR2 DNA, produced by GeneArt, was cloned by digestion of the vector with XbaI and Bst1107I and then ligated with T4 ligase. BHK cells were transfected with this P-Prot (P protein and protease) construct and infected with the VSV-ΔP mutant. VSV-ΔP contained red fluorescent protein (RFP) as a reporter gene. VSV-ΔP function required a functional P protein provided in trans by cells expressing P-Prot. In this construct, we were able to show that the activity of VSV-ΔP-RFP was directly correlated with the presence of a protease inhibitor (amprenavir, at concentrations of 0.1, 1, and 10 μM). In the absence of the protease inhibitor, the P protein was cleaved and no RFP signal was detected. L protein

[0156] The VSV L viral insert was introduced into the entire VSV genome by Gibson assembly of four fragments. The majority of the vector (fragment 4) was provided by restriction enzyme digestion of pVSV-GFP with the enzymes SfoI and FseI. The HIV protease dimer insert (fragment 1) was amplified with primer sequences specific for the flexible (GGSG)3 linkers at both ends of the construct (DNA sequence of SEQ ID NO:4; amino acid sequence of SEQ ID NO:29). The L protein sequence (fragments 2 and 3) surrounding fragment 1 was amplified from pVSV by introducing overhangs at the 5' end of fragment 3 with primer MT1620-insertGGSGfor (SEQ ID NO:25) and at the (GGSG)3 linker at the 3' end of fragment 2 with primer MT1620-insertGGSGrev (SEQ ID NO:26). The L protein has the cDNA sequence of SEQ ID NO:2 and the amino acid sequence of SEQ ID NO:28, with the insert introduced at amino acid position 1620 of SEQ ID NO:28. The resulting L protein with the protease insert had the DNA sequence of SEQ ID NO: 10, which was confirmed by sequencing. Further overhangs to fragment 4 were introduced at the 5' end of fragment 2 using a forward primer, 49 bp-before-Fsel [5'-GCT GCC AAG TAA TAC ACC GG-3'] (SEQ ID NO: 23), which binds 49 nucleotides upstream of the nearest restriction enzyme cleavage site, Fsel, and at the 3' end of fragment 3 using a reverse primer, 50 bp-after-Sfol [5'-TTT ATC TCC TCC TAA AGT TTC-3'] (SEQ ID NO: 24), which binds 50 nucleotides downstream of the nearest restriction enzyme cleavage site, SfoI. VSV vector

[0157] The WT VSV vector (Indiana strain) and the VSV-GFP vector (Indiana strain) have the DNA sequences of SEQ ID NO: 20 and SEQ ID NO: 21, respectively (for details, see Schnell et al., J. Virol. 1996, 70(4): 2318-2323, Boritz et al., J. Virol. 1999, 73(8): 6937-6945, and Muik et al., Cancer Res. 2014, 74(13): 3567-3578). VSV-GFP-ΔP (a recombinant VSV Indiana strain lacking the viral envelope protein P) was generated as described elsewhere (Muik et al., J. Mol. Med. (Berl), 2012, 90(8): 959-970). Infectious virus was recovered using standard helper virus-free calcium phosphate rescue techniques in 293T cells in the presence of 10 μM amprenavir for Prot-On virus and in the absence of amprenavir for protease-free Prot-Off virus (Witko et al., J Virol, 2006, 135(1):91-101). BHK21 cells were used for amplification of replication-competent VSV mutants. cell line

[0158] BHK-21 cells (American Type Culture Collection, Manassas, VA) were cultured in Glasgow minimum essential medium (GMEM) supplemented with 10% fetal bovine serum, 5% tryptose phosphate broth, 100 units / mL penicillin, and 0.1 mg / mL streptomycin.

[0159] 293T cells (293tsA1609neo) and 293-VSV (293 cells expressing VSV N, P-GFP, and L) (Panda et al., J Virol, 2010, 84(9): 4826-4831) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, 1% P / S, 2% glutamine, 1x sodium pyruvate, and 1x non-essential amino acids. In silico experiments

[0160] Structure visualization and molecular modeling: All structures were solved using Coot 0.8.7.1 (Emsley et al., Acta Crystallogr D Biol Crystallogr, 2010, 66(Pt4):486-501) and UCSF Chimera 1.12 (Pettersen et al., J Comp Chem, 2004, 25(13):1605-1612). Images of molecular structures were generated using UCSF Chimera 1.12. The VSV-L-MT1620-mCherry model was generated as follows. The VSV L protein (Protein Data Bank (PDB) accession code 5a22) with the amino acid sequence of SEQ ID NO: 28 and mCherry (PDB accession code 2h5q) with the DNA sequence of SEQ ID NO: 11 (including the linker) were docked using the ZDock server (Pierce et al., Bioinformatics, 2014, 30(12):1771-1773). The VSV L protein was defined as a control structure docked with unconstrained mCherry in rigid-body mode. One of the top hits was selected because the N- and C-termini of mCherry are located near MT1620 (amino acid 1620 of SEQ ID NO: 28 within the methyltransferase domain (MT) insertion site). The rigid-body protein docking solution was then flexibly refined using FiberDock (Mashiach et al., Poteins, 2010, 78(6):1503-1519). The (GGSG)3 linker was manually introduced into Coot 0.8.7.1 and modeled using ModLoop (Pieper et al., Nucleic Acids Res, 2014, 42(Database issue): D336-346). RNA extraction, cDNA synthesis, and PCR

[0161] Viral RNA was first purified using the viral DNA / RNA kit peqGOLD (Peqlab) according to the manufacturer's instructions. cDNA synthesis was then performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. PCR was then performed using Q5® HotStart High-Fidelity DNA Polymerase (NEB). The annealing temperature was selected according to the recommendations of the NEB Annealing Temperature Calculator. The extension time was 1 min / 1000 nucleotides. Replication dynamics

[0162] 1 × 10 BHK-21 cells 5 Cells / well were seeded into 24-well plates and cultured overnight at 37°C. The next day, the medium was removed and the cells were infected with the corresponding VSV mutant at a multiplicity of infection (MOI) of 0.1. The cells were incubated with the inoculum for 1 hour and then washed twice with PBS. 1 mL of fresh medium was added to the cells and the cells were cultured at 37°C. The time immediately after washing was set as the 0-hour value. Further supernatants were collected after 4, 8, 12, 16, 24, and 36 hours. Samples were analyzed using the TCID in BHK21 cells. 50 The solution was stored at −80°C until the virus titer was determined by assay. Dose response

[0163] 1 × 10 BHK-21 cells 5 Cells were seeded into 24-well plates at 100 cells / well and cultured overnight at 37°C. The next day, the medium was removed, and the cells were infected with the corresponding VSV mutant at a multiplicity of infection (MOI) of 1. Amprenavir concentrations of 0 nM, 30 nM, 100 nM, 300 nM, 1 μM, 3 μM, 10 μM, 30 μM, and 100 μM were added. Viral progeny were harvested at 24 hpi. TCID 50 measurement

[0164] Viral titers were determined using the Spearman-Kärber method as previously described (Kaerber, Archiv fur Experimentalle Pathologie und Pharmakologie, 1931, 162:480-483) to determine the 50% tissue culture infectious dose (TCID 50 ) assay. Briefly, 10-fold serial dilutions of virus were prepared. 100 μL of each dilution was added in quadruplicate to confluent BHK-21 cells in a 96-well plate and incubated at 37°C for 24–48 h until cytopathic effects were observed. The number of infected wells was counted and the TCID 50 values ​​were calculated. In vitro cytotoxicity assay

[0165] Plaques were obtained by crystal violet staining and fixation of BHK-21 cells infected at 60% confluency (15 g crystal violet from Fluka, 85 mL EtOH, 250 mL 37% formaldehyde, and 1000 mL HO). The final confluency before fixation was approximately 80%. Five-fold serial dilutions of the virus stock were prepared and diluted 1:10. 6.5 , 1:10 7 , 1:10 7.5 , 1:10 8 , 1:10 8.5 , and 1:10 9 Cells in 6-well plates were infected with PBS. One hour post-infection, cells were washed with PBS and overlaid with a 2.5% plaque agarose / GMEM mixture. The agarose / medium mixture was carefully removed from the wells before fixation with crystal violet, which was performed 24 hours post-infection. IFN killing assay

[0166] Viral cell killing was assessed in an interferon response assay, in which IFN-competent BHK-21 cells were treated with increasing amounts (10, 100, 500, and 1000 U / mL) of recombinant pan type I IFN (PBL assay science, Piscataway Township, NJ) and infected at MOIs of 0.1, 1, and 10. Cells were infected at 10 1 day before IFN treatment. 4 Cells were seeded at 100°C for 1 hour. INF treatment was performed 16 hours prior to infection. Infection was allowed to proceed for 72 hours. After this infection period, thiazolyl blue was added for 4 hours. Cells were then lysed in 0.1 M NaCl containing 1000 mg SDS for an additional 4 hours. MTT-formazan was measured at 540 nm. Lytic plaque measurement

[0167] Monolayers of BHK-21 cells were infected with serial dilutions of virus stock. One hour after infection, cells were washed twice with PBS and overlaid with a 1:1 dilution of 2.5% plaque agarose and whole GMEM medium. The next day, the plaque agarose was removed and the cells were stained with crystal violet. Immunoblotting

[0168] BHK-21 cells were infected with VSV, VSV-GFP, VSV-L-mCherry, or VSV-GPF-L-mCherry at an MOI of 5, and cell lysates were prepared 4, 8, 12, and 24 hours later. Uninfected BHK-21 cells served as controls. Cells were lysed for 30 minutes in ice-cold cell lysis buffer (50 mmol / L HEPES, pH 7.5; 150 mmol / L NaCl; 1% Triton X-100; 2% aprotinin; 2 mmol / L EDTA, pH 8.0; 50 mmol / L sodium fluoride; 10 mmol / L sodium pyrophosphate; 10% glycerol; 1 mmol / L sodium vanadate; and 2 mmol / L Pefabloc SC). To remove cell debris, the cell lysate was centrifuged at 13,000 rpm for 10 minutes. The protein-containing supernatant was stored at -80°C.

[0169] SDS-PAGE of protein lysates was performed under reducing conditions on a 12% polyacrylamide gel. Lysates from the 8-hour time point were used for comparison of VSV, VSV-GFP, VSV-L-mCherry, and VSV-GFP-L-mCherry. Proteins were transferred to a 0.45 μm nitrocellulose membrane (Whatman, Dassel, Germany) using a tank blot apparatus. The blot time was 90 minutes. The membrane was blocked overnight in 1x PBS containing 5% nonfat milk and 0.1% Tween 20 (PBS™) and then incubated for 3 hours at room temperature with a rabbit monoclonal antibody specific for mCherry diluted 1:1,000 in PBS™. The antibody was raised against recombinant mCherry and purified in-house (as published later). After washing, a goat antibody specific for peroxidase-conjugated rabbit IgG (Invitrogen, Carlsbad, CA) diluted 1:5,000 in PBS™ was added, and the blot was incubated for an additional hour. After further washing, the blot was developed by enhanced chemiluminescence (ECL). After the first detection, the same blot was washed extensively and reused for staining as a loading control. Actin was stained with a mouse β-actin-specific monoclonal antibody (A2228; Sigma, Munich, Germany) diluted 1:5,000 in PBS™, followed by a secondary goat antibody specific for horseradish peroxidase-conjugated mouse IgG. Washing, incubation, and blot development were performed as described for the first detection. Gene transfer

[0170] Transfection of L-mCherry expression plasmid was performed in 293T cells using the TransIT®-LT1 transfection kit from Mirus. The amount of plasmid DNA and transfection reagent was 2.7 × 10 per well one day before transfection. 5293T cells were seeded in 24-well plates and selected according to the manufacturer's recommendations. The P expression plasmid was co-transfected with the L-mCherry expression plasmid. 24 hours after transfection, 293T cells were infected with VSV-GFP-ΔL at an MOI of 10. Images were acquired 48 hours after infection. Animal experiments

[0171] Animal experiments were conducted in accordance with the national animal experimentation law. Permission for animal experiments was granted by the national authorities.

[0172] Stereotaxic method: Stereotaxic mouse brain injections of virus were performed using a mouse stereotaxic construct (Harvard Apparatus, Hollistion, MA). Anesthesia was induced with a mixture of 100 mg / kg ketamine and 10 mg / kg xylazine. Postoperatively, analgesia was administered with 5 mg / kg ketoprofen and antibiotic therapy with 5 mg / kg enrofloxacin. Analgesia was continued with oral ibuprofen solution (0.1 mg / mL) in drinking water. During stereotaxic surgery, mice were immobilized in the stereotaxic construct. The mouse head was shaved and washed twice with betadine and twice with ethanol. The scalp was opened with a scalpel. The injection hole site was positioned oriented toward the bregma. A 1 mm diameter injection hole was drilled with an electric drill (FST, Foster City, CA). The virus injection volume was 10 μL at an injection rate of 1 μL / min. During surgery, mice were placed on a heating pad and their eyes were protected with petrolatum.

[0173] Image analysis: Virus-infected cells in mouse brain and tumor cultures and histological sections were analyzed using a fluorescence microscope (Nikon, Japan).

[0174] Statistical analysis: Statistical significance was determined by Student's t-test and analysis of variance (ANOVA). A P value of less than 0.05 was considered statistically significant. GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA) was used for statistical analysis and data presentation. Adobe Photoshop software was used to compose multicolor photographic panels and overlaid images. Example 1: Generation of the protease-regulated ON switch, VSV-P-prot

[0175] To generate a tunable switch that controls the activity of an RNA virus, we developed a system to integrate an autocatalytically active protease sequence into a gene essential for VSV gene expression and replication (Fig. 1a). In the first ON switch construct, an HIV protease dimer was introduced into the P protein (SEQ ID NO: 1), a cofactor for VSV polymerase, to generate VSV-P-prot. It has previously been shown that the intramolecular insertion site does not affect the function of the P protein (Das et al., J Virol., 2006, 80(13):6368-6377). The function of the HIV protease requires dimerization. To immediately promote posttranslational proteolytic activity, the gene insertion construct was designed to contain two copies of the HIV protease (PR) linked by a flexible linker (Fig. 1b, Fig. 2) (Krausslich, PNAS, 1991, 88(8):3213-3217). Flexible linkers (SEQ ID NOs: 8 and 9) were also applied upstream and downstream of the protease construct (SEQ ID NO: 5), resulting in a protease dimer with a cleavage site and linker with the coding sequence of SEQ ID NO: 4, ensuring independent function of the protease from the rest of the fusion protein (Chen et al., Adv Drug Deliv Rev, 2013, 65 (10:1357-1369). The codon usage of both protease sequences was a compromise to allow for human codon usage and adjustments between the two sequences to avoid sequence homology so as to minimize the risk of copy selection events during replication (Simon-Loriere and Holmes, Nat Rev Microbiol, 2011, 9(8):617-626). The resulting sequence (SEQ ID NO: 29) is shown in Figure 5.The single-stranded binding dimer (PR2) is further flanked by the corresponding cleavage sites (SEQ ID NOs: 6 and 7) as shown in Figures 4 and 5 (de Oliveira et al., J Virol, 2003, 77(17):9422-9430) and was cloned into the flexible hinge region of the VSV P protein at aa position 196 (P196), which has previously been described as a region that allows functional intramolecular insertions (Das et al. 2006).

[0176] To confirm native VSV phosphoprotein function in the integrated autoproteolytic ON switch, we transfected BHK cells with a plasmid encoding the isolated P196PR2 construct and subsequently infected them with VSV lacking its P protein and expressing a red fluorescent protein (RFP) reporter gene in situ (VSV-ΔP-RFP) (Muik et al., J Mol Med (Berl), 2012, 90(8):959-970). The RFP signal in VSV-ΔP-RFP-infected cells was detectable only in the presence of P196PR2 and a specific HIV protease inhibitor (here, amprenavir) (Figure 3, panels B1-B3). The absence of the protease inhibitor resulted in a lack of viral gene expression and viral replication (Figure 3, panels A1-A3), indicating that P196PR2 maintains essential viral P protein function and can be controlled by inhibition of the proteolytic ON switch.

[0177] Next, we generated a recombinant VSV (VSV-P-prot) expressing P196PR2 instead of its native P protein, which also contained an eGFP reporter gene at the fifth gene position (Fig. 4). Rescue and propagation of VSV-P-prot were performed in media containing 10 μM amprenavir. PCR amplification and sequencing confirmed that the P196PR2 construct was correctly integrated into the VSV genome (Fig. 6A). Infection of BHK cells with VSV-P-prot resulted in a strong GFP signal within 24 h in the presence, but not in the absence, of amprenavir (10 μM), indicating that the supplied protease inhibitor regulated VSV-P-prot gene expression (Fig. 6B). Conversely, viral replication, as observed by viral plaque formation of VSV-P-prot, was also found to be dependent on the protease inhibitor (Fig. 6C). Viral RNA was reverse transcribed and subjected to sequence confirmation. The sequence of the insert from the viral genome sequence aligned perfectly with the viral construction plasmid. Example 2: VSV-P-prot is regulated in a dose-dependent manner and by various HIV protease inhibitors

[0178] To test whether the amprenavir-dependent activity of VSV-P-prot is generalizable to other HIV protease inhibitors, BHK cells were cultured with the second-generation compounds saquinavir (10 μM) and indinavir (10 μM) and subsequently infected with VSV-P-prot at an MOI of 0.01. Consistent with the effects of amprenavir, both inhibitors promoted viral gene expression (GFP signal) and viral replication (plaque formation) (Figure 7), confirming the universal targeting ability of the HIV protease-based VSV ON switch system. Furthermore, lopinavir (10 μM) and other HIV protease inhibitors were also shown to modulate VSV-P-prot (data not shown).

[0179] The amprenavir dose used for virus production and initial studies was chosen according to previously described APV plasma concentrations in patients treated orally with APV (Sadler et al., Antimicrob Agents Chemother, 1999, 43(7):1686-1692). Furthermore, dose-response studies were performed to determine whether amprenavir's inhibitory activity on VSV-P-prot was dose-dependent. Viral gene expression (GFP) and viral replication (TCID ) were measured as previously described. 50 We assessed the effect of amprenavir on both VSV-P-prot and VSV-P-prot replication assays. BHK cells were infected at an MOI of 1, and virus infection was assessed 24 hours later (Fig. 8A). Single-step growth curves revealed that VSV-P-prot activity began at an amprenavir dose of 100 nM, reached a plateau of maximal activity in the dose range of 3–100 μM, and declined at higher doses (Fig. 8B). Virus replication of standard recombinant VSV lacking the P-prot regulatory mechanism yielded titers >1.5 logs higher and was unaffected by amprenavir doses up to 30 μM, indicating that amprenavir is unable to regulate VSV replication in the absence of the P-prot switch. Replication curves showed a slight decline in VSV-P-prot compared to VSV. Example 3: Lack of neurovirulence and intracranial spread of VSV-Pprot

[0180] VSV is known to have significant neurotoxicity in experimental animals when introduced into the CNS space. VSV glycoproteins exhibit a strong affinity for neurons, and both anterograde and retrograde axonal spread has been reported. To examine the extent to which the neurotoxicity of VSV-P-prot is abrogated compared to normal VSV, we used direct stereotaxic injection into the mouse striatum. Intracranial injection of wild-type VSV-dsRed (2 x 10 in 2 μL) was performed. 5 TCID 50) resulted in severe signs of neurotoxicity (Figure 9A) manifested as hindlimb paralysis, lack of coordination, hunched back, and severe weight loss (Figure 9C) beginning within 2 days after injection. For humane reasons, all mice had to be euthanized within 4 days (Figure 9B). In stark contrast, brain injection of the same dose of VSV-P-prot did not result in any signs of neurotoxicity. Furthermore, mice treated simultaneously with amprenavir and ritonavir (100 μM amprenavir and 25 μM ritonavir (which inhibits APV degradation) in 100 μL of PBS, administered intraperitoneally twice daily for 10 days) did not show any brain-related adverse signs after intracranial VSV-P-prot injection (Figure 9A-C). To examine potential intracranial spread after stereotaxic injection, brains were collected on the day of toxicity-related euthanasia (VSV-dsRed) or 10 days after VSV-P-prot inoculation. Histological fluorescence analysis of coronal brain sections demonstrated widespread diffusion of red-fluorescent VSV-dsRed. Viral infection was observed throughout the striatum, subcortical regions, and (bilaterally) the hypothalamus. In contrast, GFP expression from VSV-P-prot was completely restricted to the area immediately behind the needle track, without any signs of intracranial spread of VSV-P-prot, regardless of whether amprenavir was administered systemically (Figure 9D). These data confirm that VSV-P-prot is not associated with the neurotoxicity and intracranial spread typical of VSV.

[0181] Thus, further ligand-dependent viral activity was confirmed in vivo by complete suppression of parental VSV-associated neurotoxicity and intracranial spread. Because amprenavir does not cross the blood-brain barrier, systemic administration of the compound did not confer viral activity and was absent of neurotoxicity despite the systemic presence of the ON system. Example 4: Protease inhibitor-dependent genetic stability of VSV-P-prot

[0182] RNA viruses are prone to frequent mutations, especially in the case of VSV, where the mutation rate is approximately 10,000-fold lower (Steinhauer and Holland 1986, Steinhauer, de la Torre et al. 1989). To test whether the tunable viral-controlled ON switch of VSV-P-prot remained functionally stable over multiple rounds of viral replication, we used in vitro serial virus passages under optimal (10 μM) and suboptimal (1 μM) amprenavir conditions. After each passage, protease inhibitor dependence was assessed by GFP expression after transferring a sample of the supernatant to a parallel dish cultured without amprenavir. After 20 passages (P20), no amprenavir escape virus mutants were observed (Figure 10A). To confirm the genomic integrity of VSV-P-prot, viral genomic RNA from passage P20 from suboptimal amprenavir-treated virus propagation was purified, reverse transcribed, and PCR was performed on the insert P-196PR2. A VSV mutant lacking the protease insert was used as a negative control. The P-196PR2 and protease-negative P protein PCR fragments were found to be of the expected size (Figure 10B). Subsequent sequencing of the P-prot region and alignment comparison with the respective sequence in the parental plasmid construct (SEQ ID NO: 4) revealed one mutation in the construct (protease 2: nucleotide G23A; amino acid R8K), which did not render the construct nonfunctional. It remains to be determined whether this mutation is functionally silent, reflects adaptation to low APV concentrations, or exchanges of rare codons (R: 7%) for frequent codons (K: 74%). Example 5: Identification of protease insertion sites in the VSV L protein

[0183] To test whether the autocatalytically active protease approach to viral control could also function when inserted into another essential VSV protein, we attempted to generate VSV-L-prot. Because stable, non-attenuating intramolecular insertions have not been well documented to date (Ruedas and Perrault, J Virol, 2009, 83(23):12241-12252; Ruedas and Perrault, J Virol, 2014, 88(24):14458-14466), we first investigated the ability of the VSV L protein to tolerate the insertion of a reporter gene (mCherry). Using a structure-guided approach, we identified five distinct mCherry L protein insertion sites (amino acid positions CD1506, CD1537, MT1603, MT1620, and MT1889 of the L protein, which has the amino acid sequence of SEQ ID NO: 28). These insertion sites were plausible because they were located on the surface and in flexible loops, minimizing the possibility of steric clashes (Figure 11A, C). To preserve the structural integrity of the L protein, we avoided insertion sites within α-helices and β-sheets. Figure 11D shows a model of insertion at MT1620, which resulted in replication-competent virus, as described below. To screen the proposed in silico-designed structural predictions for candidate insertion sites, we generated VSV L protein expression vectors inserted at CD1506, CD1537, MT1603, MT1620, and MT1889. These five constructs were transfected into HEK cells, followed by infection with a replication-incompetent VSV-GFP-ΔL virus encoding eGFP as a reporter. In this screen, all sites displayed mCherry signals, but only two sites (CD1506, MT1620) displayed eGFP signals, indicating transcriptional activity of the L-mCherry fusion protein (Figure 12A). Thus, all insertion sites allow correct mCherry folding, albeit with varying efficiency, but only two insertions retain polymerase activity.To test eGFP-positive clones for viral replication competence, we chose to clone these sites (CD1506, MT1620) into the entire VSV genome. Each site was cloned into two VSV backbones: one with eGFP as a reporter at position 5 in the genome and the other without eGFP. VSV mutants with the CD1506 construct could not be rescued even after multiple attempts. In contrast, VSV-L-MT1620 and VSV-GFP-L-MT1620 virus rescue generated replication-competent viruses, as confirmed by cytopathic effects and fluorescent signals (Figure 12B). As expected, VSV-eGFP-L-MT1620-mCherry showed fluorescent signals in both the FITC (green) and TRITC (red) bands, while VSV-L-MT1620-mCherry showed fluorescent signals only in the TRITC band. mCherry, flanked on both sides by linkers, has the DNA sequence of SEQ ID NO: 11. VSV-L-MT1620-mWasabi showed green fluorescence in the FITC band (Figure 12B). Similarly, the mWasabi protein is flanked on both sides by linkers and encoded by the DNA sequence of SEQ ID NO: 12. To verify the presence of mCherry based on protein concentration, immunoblotting was performed using an mCherry-specific antibody. BHK-21 cells were infected with VSV, VSV-GFP, VSV-L-MT1620-mCherry, and VSV-GFP-L-MT1620-mCherry. As a positive control, BHK-21 cells were transfected with a vector containing only mCherry. The mCherry in the L protein showed a signal at a high molecular weight (predicted at 267 kDa), consistent with the production of the L-mCherry fusion protein after viral infection (Figure 12C).

[0184] Taken together, these results demonstrate that insertion of mCherry at the MT1620 site was successful, resulting in replication-competent virus.

[0185] To assess the replication capacity and potential attenuation of the VSV-L insert compared to wild-type base VSV, plaque assays were performed as judged by plaque size (Fig. 13A) and TCID to quantify viral replication. 50 Measurements were performed (Figure 13B). Both studies revealed a reduction in the VSV-L insert compared to wild-type VSV, resulting in approximately a 1-2 log decrease in viral replication titer. Furthermore, MTT viability assays were performed to assess the ability of the VSV-L insert to induce cell killing in BHK cells in the presence or absence of interferon compared to VSV. In the absence of IFN, viral cytotoxicity after infection with the VSV-L insert was comparable to that of VSV infection (Figure 13C). In the presence of IFN, the two L-MT1620-mCherry VSV mutants exhibited stronger IFN dependence compared to VSV and VSV-GFP, resulting in slightly reduced killing (Figure 13C; data for the GFP mutants not shown), confirming the finding that insertion of mCherry at the MT1620 position results in a mild reduction in viral replication or cytolysis compared to wild-type VSV without significantly compromising its ability to replicate or lyse.

[0186] The sequencing results of L-mCherry obtained in this example are provided as SEQ ID NO: 13. Upon sequence verification of all rescued VSV-L insertion mutants, one to three secondary nonsynonymous mutations were observed in most viruses, located near the insertion site. These mutations may be conditional and may favor proper polymerase function. Example 6: Generation of another protease-regulated ON switch, VSV-L-prot

[0187] The discovery of a functional insertion site within the VSV L protein allowed us to generate another tunable VSV-prot variant, VSV-L-Prot. Like VSV-P-prot, VSV-L-Prot contains a protease insertion within the L protein (SEQ ID NO: 10) and therefore replicates to high titers in response to and in the presence of APV (as well as saquinavir and indinavir) but not in the absence of protease inhibitors (Figure 14A).

[0188] To test the genomic integrity of VSV-L-Prot, viral genomic RNA was purified, reverse transcribed, and PCR was performed on L-MT1620PR2. A VSV mutant lacking the protease insert was used as a negative control. We found that the PCR fragments of L-MT1620PR2 and the protease-negative L protein were of the expected size (Figure 14B).

[0189] To assess whether mutations had occurred within the protease dimer sequence, we sequenced the insertion site using two Sanger sequencing reactions. The sequencing results were consistent with the plasmid sequence. No mutations were observed within the protease dimer sequence. Example 7: VSV-L-prot can be regulated in a dose-dependent manner

[0190] Similar to VSV-P-prot, a dose-response study was performed to determine whether the amprenavir-inhibitory activity of VSV-L-prot was dose-dependent. As previously described, viral gene expression (GFP) (Figure 15A) and viral replication (TCID 50 The effect of amprenavir on both VSV-L-prot and VSV-P-prot (replication assay) was assessed (Figure 15B). VSV-L-prot activity began at an amprenavir dose of 100 nM and reached maximum activity at a dose of 30 μM. Higher amprenavir concentrations were not tested with L-prot because previous studies with VSV-P-prot indicated that this was toxic to cells, resulting in decreased titers. Furthermore, the replication curve showed a slower decline with VSV-L-prot than with VSV, corresponding to the curve seen with VSV-P-prot. Example 8: Generation of a tandem protease-regulated ON switch, VSV-PL-prot

[0191] To investigate the possibility of developing a revertant virus that could lose conditional ON switch control, we further investigated the possibility of inserts in the P and L proteins. As a first demonstration of the concept of a VSV mutant with a functional double insert, we then generated a VSV with a functional double intramolecular insert within P and L, i.e., VSV-P-mWasabi-L-mCherry. Functionality of the double insert was confirmed by dual fluorescent readout in the plaque assay (Figure 16A) and cytopathic effect, and by testing genome integrity by cDNA synthesis / PCR and Sanger sequencing.

[0192] To test the genomic integrity of VSV-P-mWasabi-L-mCherry, viral genomic RNA was purified, reverse transcribed, and PCR was performed with P-196-mWasabi (SEQ ID NO: 15) and L-MT1620-mCherry (SEQ ID NO: 14). A VSV mutant lacking the fluorescent protein insert was used as a negative control. We found that PCR fragments for P-196-mWasabi, L-MT1620-mCherry, and the fluorescent protein-negative P and L proteins were of their predicted sizes (Figure 16B).

[0193] To assess whether mutations may have occurred within the fluorescent marker sequence, we performed two Sanger sequencing reactions to confirm the insertion site. The sequencing results were consistent with the plasmid sequence. No mutations were found in the insertion sequence. Based on the sequencing results, the cDNA sequences of P-196-mWasabi and L-MT1620-mCherry from VSV-P-mWasabi-L-mCherry are presented as SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0194] After confirming the feasibility of tandem intramolecular insertions, we next generated a double ON switch-regulated VSV mutant, VSV-PL-prot. The virus was successfully rescued and appeared to behave similarly to both the VSV-P-prot and VSV-L-prot single-switch constructs. This virus also exhibited protease inhibitor dependence, producing plaques only in the presence of amprenavir (data not shown). However, the double-switch virus showed a slightly greater reduction compared to the single-switch virus.

[0195] Overall, therefore, we generated a construct in which an HIV protease dimer was intramolecularly inserted into two proteins (P and L proteins, separately and in combination) of vesicular stomatitis virus (VSV) that constitute the polymerase complex. In the presence of a protease inhibitor, the integrity of the viral proteins was maintained, and the virus could replicate. In the absence of a protease inhibitor, the HIV protease dimer was autocatalytically active, cleaving essential viral proteins upon translation. Similar to regulatory modules in DNA viruses (such as Tet-On), we termed this mechanism "prot-ON."

[0196] We optimized the codon usage of the flexible linker and protease dimer to avoid homology between the first and second proteases. This precaution was taken because the so-called "copy selection" recombination event in VSV has previously been described (Simon-Loriere and Holmes 2011), which could potentially cause the viral polymerase, the L protein, to switch templates and skip sequence extension. "Copy selection" occurs preferentially when the polymerase is guided by sequence homology of the nascent RNA strand with the newly selected template. Furthermore, point mutations occur frequently in RNA viruses, with a nucleotide mutation rate of approximately 1 in 10,000 in VSV. Theoretically, every genome contains a single mutation, which has led virologists to refer to the VSV genome (and other RNA virus genomes) as a mixture of so-called "quasi-species" rather than as a single sequence. Therefore, the occurrence of a mutation within the HIV protease sequence that inactivates the proteolytic switch is a realistic possibility. To avoid such escape mutant or revertant viruses that could usurp the control of the conditional ON switch, we introduced a protease dimer into a second essential VSV protein, such as the P protein and L protein, to duplicate the protease module (ON switch).

[0197] The only other previously published functional intramolecular insertion site in a VSV protein that appears to support viral replication over successive passages was described in the M protein (Soh and Whelan, Virol, 2015, 89(23):117050-11760). However, because controlling the M protein could potentially facilitate viral replication and possibly escape mutations, direct control of the VSV replication machinery is preferable. Insertion sites within the L protein have been described, but the resulting viruses were temperature-sensitive and unstable after passage (Ruedas and Perrault 2009, Ruedas and Perrault 2014). In contrast, we were able to generate functional insertions containing first a fluorescent protein and then an HIV protease dimer. Both P-prot and L-prot replicated in response to all tested HIV protease inhibitors in a compound dose-dependent manner. In the absence of protease inhibitors, viral gene expression was terminated and replication ceased.

[0198] Although not tested in this study, the ON switch system inherently provides an additional environmental safety element: because viral progeny depends on the presence of protease inhibitors, any virus potentially released is not active against productive infection. This is particularly important if the therapeutic RNA virus has the potential to cause or mimic disease in notifiable animals. Example 9: Generation of a protease-regulated OFF switch, VSV-GFP-prot-L

[0199] Following the generation of the protease-based ON switch, we also generated a VSV mutant capable of being turned off, VSV-Prot-off. Using the same HIV protease-mediated autocatalytic switch system, site-changing insertion from an intramolecular site to an intermolecular site reversed the direction of viral promotion to viral arrest. In this OFF switch, a mutant codon-optimized protease dimer was inserted into the VSV genome to form a fusion protein of GFP, a protease dimer, and the viral polymerase L. This large fusion protein, with the protease dimer fused to the N-terminus of the L protein, is predicted to be functionally inactive but is activated by proteolytic release of the L protein in the absence of a protease inhibitor. In this OFF construct, we replaced the intergenic region of VSV with an HIV protease flanked by its cleavage site. The intergenic region plays an important role in generating multiple proteins from a single RNA strand. We selected the intergenic region between the nonessential reporter protein GFP and the L protein in VSV-GFP (Figure 17A) to generate two viruses. One virus has a flexible linker region (SEQ ID NO: 16) surrounding the HIV protease dimer construct, while the other virus lacks the region (SEQ ID NO: 17) surrounding the HIV protease dimer construct. It is advantageous to avoid the flexible linker region because it is likely to remain as a C-terminal tag on GFP and an N-terminal tag on the L protein after the HIV protease dimer cleaves its recognition sequence. However, these two constructs differ slightly in their replication capabilities. Addition of amprenavir (10 μM) resulted in the abolition of viral activity at the level of both viral transgene expression (GFP) and viral replication (plaque assay) (Figure 18A). This system added an additional, unique safety feature. Theoretically, loss of the protease insert could allow viral progeny to escape OFF switch control.Therefore, the inventors placed a protease OFF switch in place of the intergenic region to address the disadvantage of forming a fusion protein of two adjacent VSV proteins, in this case GFP and a VSV protein, which would render the virus dysfunctional.

[0200] To test the genomic integrity of VSV-GFP-Prot-L, viral genomic RNA was purified, reverse transcribed, and PCR was performed on GFP-Prot-L. A VSV mutant lacking the protease insertion was used as a negative control. We found that the VSV-GFP-Prot-L and protease-negative L protein PCR fragments were of their predicted sizes (Figure 18B).

[0201] To assess whether mutations had occurred within the protease dimer sequence, we sequenced the insertion site using two Sanger sequencing reactions. The sequencing results were consistent with the plasmid sequence. One mutation was observed in the protease dimer sequence in each construct. The mutation was at amino acid position 85 in the linker-containing construct, corresponding to nt 623 of the cDNA sequence of the protease dimer with the linker having the sequence of SEQ ID NO: 18 (i.e., nt 254 of the second protease nucleotide sequence), and the mutation was at amino acid position 86 in the linker-free construct, corresponding to nt 589 of the cDNA sequence of the linker-free protease dimer with the sequence of SEQ ID NO: 19 (i.e., nt 256 of the second protease nucleotide sequence). Both mutations were within the second protease. These mutations were not described as typical protease inhibitor-resistant mutations and did not interfere with regulation by protease inhibitors. Presumably, these mutations made the protease more active when fused between GFP and L. Example 10: VSV-GFP-prot-L is regulated by various protease inhibitors in a dose-dependent manner

[0202] Similar to the two prot-ON constructs, a dose-response study was performed to determine whether the amprenavir-regulating activity of VSV-GFP-Prot-L (VSV-Prot-Off) was dose-dependent. As previously described, the amprenavir-regulating activity of VSV-GFP-Prot-L (VSV-Prot-Off) was measured by measuring viral gene expression (GFP) and viral replication (TCID ). 50 The effect of amprenavir on both VSV-GFP-Prot-L and VSV-GFP-Prot-L (Figures 19A and 19B) was assessed. In the absence of protease inhibitors, VSV-GFP-Prot-L activity was not reduced compared to normal VSV. VSV-GFP-Prot-L activity was high at low amprenavir concentrations (0–300 nM) and began to decline at 1 μM. We also tested whether VSV-GFP-Prot-L responds to protease inhibitors other than amprenavir. Treatment with 10 μM saquinavir resulted in even more potent inhibition of viral replication than amprenavir (Figure 19B). This inhibition was further confirmed using saquinavir concentrations ranging from 0 to 30 μM, as shown in Figure 19C. Furthermore, single-step replication kinetics was investigated using 10 μM saquinavir per well in a 12-well plate. 5 BHK cells were seeded and infected with VSV-Prot-Off or VSV-GFP at an MOI of 3 (Figure 19D). One hour after infection, cells were washed twice with PBS and cultured in 500 μL of GMEM until the indicated time points. At the initial value, i.e., time point 0, 500 μL of GMEM was immediately removed. VSV-Prot-Off showed no reduction compared to VSV-GFP (Figure 19D).

[0203] Therefore, using the same autoproteolytic system as the "prot-ON" construct but at a functionally distinct genomic location, we further developed a mechanism for reversing protease-dependent OFF regulation, which works by replacing the intergenic region with an HIV protease dimer. In this construct, as in HIV, the protease must be active to separate the two viral proteins. Addition of a protease inhibitor to this construct results in a nonfunctional fusion protein (polyprotein) that inhibits viral activity. Maintaining similarity to Tet-On / Tet-Off, we named this construct "prot-Off." For optimal viral activity control, both the ON and OFF switches were designed to disrupt the early stages of viral propagation by regulating proteins of the viral replication / transcription machinery. Therefore, our approach can potentially be expanded to surpass current methods for regulating RNA viruses.

[0204] While the replacement of intergenic regions in the Prot-Off virus has been exemplified by the substitution of a non-essential reporter protein, GFP, and the essential L protein, Prot-Off could also be used to evenly replace the intergenic regions linking essential viral proteins with additional viral proteins, thereby rendering the virus safer because elimination of the protease construct could result in a non-functional fusion protein. Classical resistance mutations could theoretically arise within the Prot-Off construct, as these mutations arise in HIV under continuous treatment with protease inhibitors. However, the wide range of available protease inhibitors may allow compensation for such point mutations. Example 11: VSV-P-prot can be regulated in vivo by administration of protease inhibitors

[0205] To validate the ON-switch virus in vivo, we generated a luciferase-expressing mutant, VSV-P-prot-Luc, containing a luciferase reporter gene instead of the eGFP reporter gene for in vivo imaging, as described in Example 1 and shown in Figure 4. Six- to eight-week-old female athymic nude mice (Janvier Labs, Le Genest-Saint-Isle, France) were housed in a BL2 facility with a 12-hour light / dark cycle and free access to food and water. For subcutaneous xenografts, 2 × 10 6 Nude mice were injected with 100 μL of a human U87 glioblastoma cell suspension containing 100 cells into the right flank. After the implantation period, the median volume was 0.1 cm. 3 U87 xenografts were incubated with 10 ml of VSV-Pprot-Luc or control buffer. 7 A single dose of 30 μL containing 1000 TCID of virus 50The tumors were injected intratumorally with a protease inhibitor (dose determined by RITV). Protease inhibitor treatment (50 μL of 0.8 mM APV + 0.2 mM RTV in a drug vehicle containing 10% DMSO, 40% PEG300, 5% Tween 80, and 45% PBS, administered intraperitoneally every 12 hours) was initiated 1 hour before virus administration. Ritonavir acts as a blocker of protease enzymes (Cyp family) in vivo. Ritonavir increases the concentrations of other protease inhibitors. RTV is also used as an additive in HIV treatment for this very purpose. Furthermore, RTV blocks p-glycoprotein, thereby increasing the concentrations of other protease inhibitors in the brain. In vivo imaging of bioluminescence from luciferase-expressing VSV mutants was performed using the IVIS® Lumina II (Perkin Elmer, Waltham, MA) system, as described by Urbiola C et al. (int. J. Cancer, 2018, 148: 1786-1796). Figure 20A shows a representative bioluminescence image taken 8 days after virus injection. At day 8, luminescence was detected only in mice treated with protease inhibitors. This is confirmed by the luciferase signal data quantified by bioluminescence imaging (BLI), shown in Figure 20B. In the absence of protease inhibitors, the luciferase signal peaked between days 2 and 3 and then began to decline. The early bioluminescence signal, independent of protease inhibitor administration, can be explained by the fact that the virus preparation contained amprenavir, which blocks autoproteolysis during virus production and storage. Furthermore, without protease inhibitor injection, the bioluminescence signal significantly decreased after 3 days (Figure 20B), followed by a loss of tumor control (data not shown). In contrast, in the presence of protease inhibitors, the luciferase signal remained constant at a much higher overall level for 17 days (Figure 20B), and tumor size was suppressed (data not shown). These data demonstrate the in vivo functionality of the ON switch construct, which, in the presence of protease inhibitors, allows viral expression and replication of transgenes, including the reporter gene luciferase and therapeutic proteins used in this experiment. Example 12: VSV-L-prot can be regulated in vivo by administration of protease inhibitors

[0206] The in vivo data were further confirmed using VSV-L-prot expressing GFP as a reporter. Nude mice were xenografted subcutaneously with U87 glioblastoma cells as described in Example 11. The median volume was 0.1 cm. 3 Mice were injected intratumorally with a single dose of VSV-L-prot, VSV control, or control buffer (placebo). The generation of VSV-L-prot is described above in Example 6. A protease inhibitor (PI) mixture containing 0.8 mM amprenavir (APV) and 0.2 mM ritonavir (RTV) was administered intraperitoneally in 50 μL every 12 hours. Tumors were measured with calipers and volumes were calculated as length × width. 2 The values ​​were calculated using the formula: × 0.4. Intratumoral treatment of subcutaneous U87 tumors with VSV-L-prot resulted in reduced tumor growth (Figure 21A) and increased survival (Figure 21B) in the presence of the protease inhibitor cocktail compared to treatment without the protease inhibitors. The concentrations of protease inhibitors used in this proof-of-concept study are relatively low and can be further increased. Overall, these data further confirm the in vivo suitability of the viral ON switch. Example 13: Protease inhibitors modulate VSV-Prot-Off activity in vivo

[0207] We further tested the OFF switch in vivo. Six- to eight-week-old female NOD.CB-17-Prkdcscid / Rj mice (Janvier Labs, Le Genest-Saint-Isle, France) were housed in a BL2 facility with a 12-h light / dark cycle and free access to food and water. For subcutaneous xenografts, 2 × 10 6 100 μL of glioblastoma cell suspension containing 100 human G62 glioma cells was injected into the flank of NOD-SCID mice. To test the OFF switch system, a 0.07 cm median volume was used. 3G62 xenografts were injected with 2 × 10 of VSV-Prot-Off (n = 16), VSV-GFP (n = 8), or control buffer (sham; n = 7). 7 Virus (TCID 50 ) was injected intratumorally. Viral treatment was repeated 7 days later. The generation of VSV-Prot-OFF (VSV-GFP-prot-L) is described in Example 6 above and shown in Figure 17A. Treatment with a protease inhibitor cocktail (0.8 mM SQV + 0.2 mM RTV in a drug vehicle containing 10% DMSO, 40% PEG300, 5% Tween80, 45% PBS, injected intraperitoneally in 50 μL every 8 hours) was initiated 8 days after the second viral injection if tumor regression was observed. Tumors were measured with calipers, and the volume was calculated as length × width. 2 The correlation coefficient was calculated using the formula: × 0.4. Starting on day 6, mice treated with VSV-GFP showed signs of neurotoxicity (Figure 22B). After 15 days of treatment, some mice treated with VSV-Prot-Off developed neurological symptoms, and the remaining mice were randomly divided into two groups. The first group (n = 7) was not administered a protease inhibitor, which would allow continued viral replication. Although tumor control was maintained, some mice developed further neurotoxicity. However, neurotoxicity was reduced compared to the parental VSV-GFP-treated mice (3 vs. 6 of 8 mice). Subsequently, the second group (n = 8) was treated with a protease inhibitor mixture (SQV + RTV) three times daily to initiate the OFF switch. No signs of neurotoxicity were observed in this group (Figure 22B). After activation of the OFF switch, tumor control deteriorated and relapse occurred (Figure 22A). Example 14: Protease inhibitors modulate VSV-Prot-Off activity in vivo as shown by immunofluorescence

[0208] Xenografts were implanted as described in Example 13. Median volume was 0.07 cm 3 2 x 10 of VSV-Prot-Off or VSV-GFP into G62 xenografts 6 Virus (TCID 50Tumors were intratumorally injected with 30 μL of a drug containing 10% DMSO, 40% PEG300, 5% Tween 80, and 45% PBS. Protease inhibitor treatment (0.8 mM SQV + 0.2 mM RTV in a drug vehicle containing 10% DMSO, 40% PEG300, 5% Tween 80, and 45% PBS, injected intraperitoneally in 50 μL every 8 h) was initiated 3 days after the first viral treatment. Tumors were harvested 1 week later (day 10), and viral spread was analyzed using anti-VSV-N antibody staining. Representative images (Figure 23) show widespread intratumoral spread of VSV-GFP in the absence of protease inhibitors, while intratumoral spread of VSV-Prot-OFF was slightly reduced, suggesting some reduction in VSV-Prot-OFF in vivo. In contrast, protease inhibitor treatment initiated 3 days after viral inoculation blocked viral spread and restricted it to small, isolated areas. Example 15: The protease inhibitor saquinavir regulates the expression of soluble IL12 using VSV with a protease-regulated OFF switch

[0209] To further investigate whether the expression of a therapeutic protein transgene can be regulated using an OFF switch, VSV-GP-IL12-Prot-Off was tested in cell culture. VSV-GP-IL12-Prot-Off (shown schematically at the top of Figure 24A) was developed based on VSV-GP pseudotyped with the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) to overcome the neurovirulence of VSV, as detailed in International Patent Publication WO2010 / 040526. 10 cells per well were cultured. 5BHK cells were seeded in 12-well plates. Cells were infected with the VSV mutants VSV-GP, VSV-GP-IL12, VSV-GP-GFP-IL12-Prot-Off-wl, or VSV-GP-GFP-IL12-Prot-Off_w / ol at an MOI of 0.1. One hour postinfection, cells were washed with PBS and cultured in standard GMEM (-ctrl) without protease inhibitors or in 10, 100, 300, 1,000, or 10,000 nmol of the protease inhibitor saquinavir. Supernatants were harvested 30 hours postinfection. Viral titers were calculated as TCID 50 The viral titers of VSV-GP-IL12-Prot-Off, with or without the linker (wl, w / ol), were dependent on the presence and concentration of the PI, saquinavir (Figure 24A). Next, enzyme-linked immunosorbent assay (ELISA) was performed to determine whether the expressed transgene IL12 also depended on saquinavir concentration. VSV-GP-IL12-Prot-Off-w / ol had moderately favorable titer characteristics (high titer even without a PI and a strong response to a PI) and was therefore used in subsequent ELISAs. VSV-GP-IL12 and uninhibited Prot-Off viruses yielded IL12 concentrations above the detection limit of the assay. Only the control sample (-ctrl) without a PI was diluted and measured. IL12 concentrations were proportional to the viral titers of Prot-Off viruses treated with various saquinavir doses (Figure 24B). Example 16: The protease inhibitor atazanavir regulates the expression of soluble IL12 using VSV with a protease-regulated OFF switch

[0210] 10 per well 5BHK cells were seeded in 12-well plates. Cells were infected with the VSV mutants VSV-GP-IL12, VSV-GP-Luc-IL12-Prot-Off-wl (Figure 25A), and VSV-GP-Luc-IL12-Prot-Off-w / ol (containing GFP instead of Luc as shown in Figure 25A) at an MOI of 1. One hour post-infection, cells were washed with PBS and cultured in standard GMEM (-ctrl) without PI or in 10, 100, 300, 1,000, or 10,000 nmol of a recently developed protease inhibitor called atazanavir. Supernatants were harvested 30 hours post-infection. Viral titers were measured in TCID 50 Again, the linker-free Prot-Off mutant replicated to higher titers and responded more rapidly to atazanavir (Figure 25A). Therefore, this mutant was used in an IL12 ELISA. VSV-GP-IL12 virus and uninhibited Prot-Off virus yielded IL12 concentrations above the detection limit of the assay. Only a control sample without PI (-ctrl) was diluted and measured (Figure 25B). Example 17: Replication kinetics of VSV with a protease-regulated OFF switch encoding IL12 and a reporter protein

[0211] 10 per well 5 BHK cells were seeded in 12-well plates. For single-step replication kinetics, cells were infected with the VSV mutants VSV-GP-IL12, VSV-GP-Prot-Off-w / ol GFP IL12 (Figure 26A, top), and VSV-GP-Prot-Off-w / ol Luc IL12 (Figure 26A, bottom) at an MOI of 3. One hour postinfection, cells were washed twice with PBS and cultured in 500 μL of GMEM until the indicated time points. At the initial value, i.e., time point 0, 500 μL of GMEM was immediately withdrawn. The VSV-Prot-Off mutants showed a mild reduction in activity at early time points compared to the parent virus, VSV-GP-IL12 (Figure 26B). Example 18: Proof of concept for expression of a membrane-bound therapeutic protein using a protease-regulated OFF switch (Figures 27 and 28)

[0212] Due to the strong toxicity of systemically administered IL12, membrane-anchored mutants have been developed that retain IL12 at the desired site (Poutou, J. et al., Gene Therapy (2015) 22, 696-706). We applied this principle to obtain several advantages over soluble IL12 constructs. First, as explained by Poutou et al., locally produced IL12 reduces systemic toxicity. Nevertheless, toxicity is not completely prevented, and further modulation remains desirable. As shown in Figure 27, by fusing IL12 directly to the VSV polymerase (L protein) along with the CD4 transmembrane domain, both viral replication and transgene expression can be reduced in the presence of protease inhibitors. Furthermore, in the context of viral escape mutants, fusing a potentially toxic transgene to a protease dimer in a regulatory viral OFF switch mutant would force the virus to eliminate both the transgene and the regulatory switch at once. Eliminating the switch alone would result in a non-functional transgene-polymerase fusion protein. Furthermore, combining both a transgene and an OFF switch allows for efficient viral coding. Typically, genes are added to the VSV genome via additional intergenic regions. VSV genes are transcribed in a continuous gradient, whereby every intergenic region reduces the expression of downstream transcripts. Therefore, introducing a transgene without the need for extra intergenic regions can reduce viral attenuation.

[0213] Previous Prot-Off constructs have shown that a flexible linker between the HIV protease dimer and polymerase results in both lower titers and slightly less stringent regulation in the absence of protease inhibitors. A residual linker attached to the N-terminus of the polymerase after protease cleavage could potentially explain the former phenomenon. Furthermore, a flexible linker between the HIV protease and polymerase may allow some activity due to less stringent steric hindrance, potentially resulting in less stringent regulation. Therefore, transmembrane-anchored IL12 viral mutants were designed either with a flexible linker only between the IL12-TM and HIV protease dimer (forward linker-fl) or without any linker adjacent to the protease (no linker-w / ol). The forward linker construct was designed to provide some additional clearance between the CD4 membrane anchor and the protease-polymerase fusion protein. The transmembrane domain has the amino acid sequence of SEQ ID NO: 31 (encoded by the nucleic acid sequence of SEQ ID NO: 32) and is separated from IL12, encoded by the nucleotide sequence of SEQ ID NO: 33, by a linker having the amino acid sequence of SEQ ID NO: 34 (encoded by the nucleic acid sequence of SEQ ID NO: 35).

[0214] Viral titers and IL-12 expression were measured in cell cultures. 10 per well 5 BHK cells were seeded in 12-well plates. Cells were infected with the VSV mutants VSV-GP-TM-IL12-Prot-Off-w / ol, VSV-GP-TM-IL12-Prot-Off-fl, or VSV-GP-IL12 (control) at an MOI of 1. One hour post-infection, cells were washed with PBS and cultured in standard GMEM (-ctrl) without PI or atazanavir at 10, 100, 300, 1,000, or 10,000 nmol. Supernatants were harvested 30 hours post-infection. Viral titers were calculated in TCID 50The concentrations of IL12 in the PI-negative control samples were measured by ELISA (Figure 28A). Furthermore, unfiltered supernatants were tested for IL12 by ELISA. Because IL12 is membrane-bound, in principle, only virally lysed cells would release the protein. Indeed, the maximum IL12 concentration was lower in the PI-negative control samples compared with the secreted IL12 (compare Figure 28B with Figures 24B and 25B). With the exception of the secreted variants, the limits of ELISA measurement did not exceed 2-3 logarithmic scales for undiluted samples.

[0215] Therefore, we compared samples containing lysed cells, cell-containing supernatant, and supernatant only. Lysed cells included cells and unfiltered supernatant with a 1:1 ratio of dead cells and buffer. Cell-containing supernatant refers to unfiltered supernatant containing dead cells, which was centrifuged to remove dead cells. Thus, the supernatant contains only IL12 released by the killing of viral cells. Diluting the sample with cell lysis buffer increased the IL12 concentration 10-fold due to proteins released from the cell membrane. Conversely, centrifugation, and thus removal of the supernatant from the remaining IL12-bearing cells, further reduced the IL12 concentration in the sample.

[0216] We further analyzed the replication kinetics of VSV-encoded transmembrane IL12 in cell culture. 5BHK cells were seeded in 12-well plates. Cells were infected with the VSV mutants VSV-GP-TM-IL12-Prot-Off-fl, VSV-GP-TM-IL12-Prot-Off-w / ol, or VSV-GP-IL12 at an MOI of 3 for single-step replication kinetics. One hour postinfection, cells were washed twice with PBS and cultured in 500 μL of GMEM until the indicated time points. At the initial value, i.e., time point 0, 500 μL of GMEM was immediately removed. The VSV-Prot-Off transmembrane IL12 mutant showed a moderate decrease in activity at early time points compared to the original virus, VSV-GP-IL12 (Figure 28D). This early decrease is likely due to expression of the IL12-protease-polymerase fusion protein in the endoplasmic reticulum. However, VSV replication complexes are formed in the cytoplasm; therefore, the released polymerase must diffuse from the ER to the site of viral replication. However, at later time points, the decline was not evident, and furthermore, no difference was observed between constructs containing the forward linker and those without the linker. [Table 1]

Claims

1. 1. A single-stranded RNA virus comprising a modified viral genome comprising a polynucleotide sequence encoding at least one protein essential for viral transcription and / or replication, a protease, and a cleavage site for said protease, (a) at least one protein essential for transcription and / or replication of said virus contains an insertion in an intramolecular insertion site that includes at least the cleavage sites for said protease and optionally further proteases, or (b) A single-stranded RNA virus, wherein at least one protein essential for transcription and / or replication of the virus is encoded as a fusion protein comprising the protease fused to the N-terminus or C-terminus, separated by a cleavage site for the protease.

2. The single-stranded RNA virus of claim 1 , wherein the protease can be inhibited using a protease inhibitor.

3. The single-stranded RNA virus according to claim 1 or 2, wherein the single-stranded RNA virus is a negative-sense single-stranded RNA virus, preferably a negative-sense single-stranded RNA virus of the Mononegaviridae family.

4. The at least one protein essential for viral transcription and / or replication is selected from the group consisting of a polymerase cofactor, a polymerase, and a nucleocapsid, and preferably the at least one protein essential for viral transcription and / or replication is selected from the group consisting of: (a) a polymerase cofactor, preferably a P protein or a functional equivalent thereof; (b) a polymerase, preferably an L protein; and / or (c) The single-stranded RNA virus of claim 3, which is a combination thereof.

5. The single-stranded RNA virus according to any one of claims 1 to 4, wherein the protease is an HIV-1 protease, preferably a single-chain dimer of HIV-1 protease, and the protease can be inhibited by a protease inhibitor selected from the group consisting of indinavir, saquinavir, ritonavir, nelfinavir, lopinavir, amprenavir, fosamprenavir, atazanavir, tipranavir, and darunavir.

6. at least the cleavage sites for said protease and optionally further proteases are located within the intramolecular insertion site of at least one protein essential for transcription and / or replication of said virus, (a) proteolytic cleavage of the protein cleaves at least one protein essential for viral transcription and / or replication at a cleavage site for the protease within the intramolecular insertion site; (b) cleavage within the intramolecular insertion site inactivates at least one protein essential for transcription and / or replication of the virus; (c) cleavage within said intramolecular insertion site of at least one protein essential for transcription and / or replication of said virus inhibits viral transcription and / or replication; (d) the virus is active in the presence of a specific inhibitor of the protease and is inactive in the absence of a specific inhibitor of the protease; and / or (e) The single-stranded RNA virus according to any one of claims 1 to 5, wherein the virus further encodes at least one heterologous protein, which is expressed when the virus is active in the presence of a specific inhibitor of the protease, and which is not expressed when the virus is inactive in the absence of the specific inhibitor of the protease.

7. The single-stranded RNA virus is a vesicular stomatitis virus (VSV), the at least one protein essential for transcription and / or replication of the virus is a P protein and / or an L protein, and the intramolecular insertion site is (a) within the flexible hinge region of the VSV P protein, preferably at a position corresponding to amino acids 193-199, more preferably amino acid 196, of the VSV P protein; (b) within a loop of the methyltransferase domain of the L protein corresponding to amino acids 1614 to 1634, preferably amino acids 1614 to 1629, more preferably amino acids 1616 to 1625, more preferably amino acid 1620 of the VSVi L protein; or (c) The single-stranded RNA virus according to any one of claims 1 to 6, which is a combination of (a) and (b).

8. the at least one protein essential for viral transcription and / or replication is encoded as a fusion protein comprising the protease fused to the N-terminus or C-terminus of the at least one protein essential for viral transcription and / or replication, separated by a cleavage site for the protease; (a) proteolytic cleavage of the fusion protein releases at least one protein essential for transcription and / or replication of the virus in an active form; (b) at least one protein essential for viral transcription and / or replication in the fusion protein comprising the protease fused to the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication, separated by a cleavage site for the protease, is inactive in the absence of proteolytic cleavage; (c) proteolytic cleavage of the fusion protein is inhibited using a specific inhibitor of the protease; (d) the virus is inactive in the presence of a specific protease inhibitor of the protease and is active in the absence of the specific inhibitor of the protease; (e) the virus further encodes at least one heterologous protein, which is not expressed when the virus is inactive in the presence of a specific inhibitor of the protease and is expressed when the virus is active in the absence of the specific inhibitor of the protease; (d) the fusion protein further comprises an additional viral or heterologous protein fused to the end opposite the protease fused to the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication, wherein the additional viral or heterologous protein and the protease are also separated by a cleavage site for the protease; (e) the protease flanks on either side the cleavage site of the protease, replacing the intergenic region linking at least one protein essential for transcription and / or replication of the virus with an additional viral or heterologous protein; and / or (f) the protease flanking the cleavage site of the protease on either side replaces an intergenic region linking at least one protein essential for viral transcription and / or replication with an additional viral or heterologous protein, and loss of the protease results in an additional inactive fusion protein comprising the protein essential for viral transcription and / or replication and the additional viral or heterologous protein.

9. the single-stranded RNA virus is a negative-sense single-stranded RNA virus of the Mononegaviridae family, and at least one protein essential for transcription and / or replication of the virus is encoded as a fusion protein comprising the protease fused to the N-terminus or C-terminus of the at least one protein essential for transcription and / or replication of the virus, separated by a cleavage site for the protease; (a) at least one protein essential for transcription and / or replication of the virus is the L protein; and / or (b) the fusion protein comprises the protease fused to the N-terminus of at least one protein essential for transcription and / or replication of the virus, separated by a cleavage site for the protease.

10. At least one protein essential for transcription and / or replication of the virus is encoded as a fusion protein, the fusion protein comprising: (a) consisting of the protease fused to the N-terminus or C-terminus of at least one protein essential for transcription and / or replication of the virus, separated by a cleavage site for the protease, the fusion protein optionally comprising a linker between the protease and the protein essential for transcription and / or replication of the virus; or (b) the single-stranded RNA virus according to any one of claims 1 to 8, comprising the protease fused to the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication, separated by a cleavage site for the protease, and an additional viral protein or heterologous protein fused to the opposite side of the protease fused to the N-terminus or C-terminus of at least one protein essential for viral transcription and / or replication, wherein the additional viral protein or heterologous protein and the protease are also separated by a cleavage site for the protease.

11. 1. An RNA virus comprising a modified viral genome comprising a polynucleotide sequence encoding at least one heterologous protein, a protease, and a cleavage site for said protease, wherein said at least one heterologous protein comprises an insertion at an intramolecular insertion site comprising said protease and optionally at least a cleavage site for a further protease, and preferably said heterologous protein is a therapeutic protein, a reporter, or a tumor antigen.

12. A single-stranded RNA virus according to any one of claims 1 to 10 or an RNA virus according to claim 11 for use in therapy.

13. The single-stranded RNA virus according to any one of claims 1 to 10 or the RNA virus according to claim 11, for use in the treatment of cancer.

14. 14. The single-stranded RNA virus or RNA virus for use according to claim 13, wherein the cancer is a solid tumor, preferably selected from the group consisting of colon cancer, prostate cancer, breast cancer, lung cancer, skin cancer, liver cancer, bone cancer, ovarian cancer, pancreatic cancer, brain tumor, head and neck cancer, lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma), brain cancer, neuroblastoma, mesothelioma, Wilms' tumor, retinoblastoma, and sarcoma.

15. A recombinant VSV L protein comprising an insertion within a loop of the methyltransferase domain of the L protein corresponding to amino acids 1614 to 1634, preferably amino acids 1614 to 1629, more preferably amino acids 1616 to 1625, more preferably amino acid 1620, of a VSV L protein having the sequence of SEQ ID NO:

28.

16. A vesicular stomatitis virus (VSV) comprising the recombinant VSV L protein of claim 15.

17. 1. A method for controlling RNA virus replication, comprising: (a) transducing or transducing a host cell with an RNA virus according to claim 6 or 7, and (b) maintaining the host cell in the presence or absence of a protease inhibitor specific for the protease; a method wherein the addition of said protease inhibitor allows viral transcription and / or replication, and the absence of said protease inhibitor inhibits viral transcription and replication; or (a) transducing or transducing a host cell with an RNA virus according to any one of claims 8 to 10, and (b) maintaining the host cell in the presence or absence of a protease inhibitor specific for the protease; a method wherein the addition of said protease inhibitor inhibits viral transcription and / or replication, and the absence of said protease inhibitor allows viral transcription and replication; or (a) transducing or transducing a host cell with the RNA virus of claim 119; wherein said proteolytic enzyme is located within an intramolecular insertion site of at least one heterologous protein; and (b) maintaining the host cell in the presence or absence of a protease inhibitor specific for the protease; The method, wherein the addition of the protease inhibitor allows expression of the heterologous protein, and the absence of the protease inhibitor inhibits expression of the heterologous protein.

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