Abstract
The present invention refers to a vector system, usable as a platform vector and suitable for the production of transgenic viruses of the subfamily Alphaherpesvirinae. Such transgenic viruses can be used as vaccine or as oncolytic virus or in gene therapy. The platform vector of the present invention is a vector system allowing a simplified search for and generation and production of viruses with a modified and increased functionality. The present invention refers also to the use of the platform vector as a vector system for the generation and the production of transgenic viruses, methods for the production of a transgenic virus, using the vector system of the present invention and viruses obtained by such methods.
Claims
1. A vector system comprising a bacterial artificial chromosome (BAC) construct, comprising a viral part and a non-viral part, wherein the viral part is derived of a virus of the subfamily Alphaherpesvirinae, wherein the non-viral part comprises DNA with sequences of a bacterial plasmid, characterized in that the viral part comprises at least two recombination sites, wherein the DNA with sequences of a bacterial plasmid is inserted in between the sequences of a first viral gene and a second viral gene and the first recombination site is inserted in between the sequences of a third viral gene and a fourth viral gene and the second recombination site is inserted in between the sequences of a fifth viral gene and a sixth viral gene.
2. The vector system according to claim 1, wherein the viral part is derived of a virus from the genus Simplexvirus or from the genus Varicellovirus.
3. The vector system according to claim 1, wherein the viral part is derived of a Herpes simplex virus.
4. The vector system according to claim 1, wherein at least one of the at least two recombination sites is inserted in between the sequences of two tail-to-tail oriented genes and/or wherein the DNA with sequences of a bacterial plasmid, and preferably the non-viral part is inserted in between the sequences of two tail-to-tail oriented genes.
5. The vector system according to claim 1, wherein the at least two recombination sites are inserted in between the sequences of two tail-to-tail oriented genes and wherein the DNA with sequences of a bacterial plasmid, and preferably the non-viral part is inserted in between the sequences of two tail-to-tail oriented genes.
6. The vector system according to claim 1, wherein the DNA with sequences of a bacterial plasmid is inserted in between the sequences of the UL3 gene and the UL4 gene and/or wherein one of the at least two recombination sites is inserted in between the sequences of the UL10 gene and the UL11 gene, wherein preferably the second recombination site is inserted in between the sequences of the UL55 gene and the UL56 gene.
7. The vector system according to claim 1, wherein the non-viral part comprises DNA-sequences of the plasmid pBeloBAC11.
8. The vector system according to claim 1, wherein the DNA with sequences of a bacterial plasmid can be removed after transfection in mammalian cells by recombination.
9. (canceled)
10. A method for the production of a transgenic virus comprising the steps: a) introducing a transgene expression cassette into one of the at least two recombination sites of the vector system according to claim 1 to obtain a vector encoding a transgenic virus; b) transfecting the vector obtained in step a) into a mammalian cell; c) cultivating the transfected mammalian cell; and d) isolating the virus produced in the mammalian cell.
11. The method according to claim 10, wherein in step a) a first transgene expression cassette is introduced into the first of the at least two recombination sites and a second transgene expression cassette is introduced into the second of the at least two recombination sites of the vector system according to claim 1.
12. The method according to claim 10, wherein step a) is performed in a prokaryotic system.
13. The method according to claim 10, wherein the transgene cassette encodes at least one protein or at least one peptide or an RNA, preferably a protein or a peptide selected from the group consisting of target control proteins, immunomodulating agents like cytokines, anti-cancer peptides (ACP) or proteins, pro-apoptotic proteins, extracellular matrix degrading proteins, proteins for the tumor-antigene-presentation, pathogen antigens and combinations thereof.
14. The method according to claim 10, for the production of a vaccine or an oncolytic virus or viral vector for gene therapy.
15. A virus obtained by the method according to claim 10.
16. A method for vaccination or gene therapy in a subject, comprising administering to the subject the virus according to claim 15.
Description
[0096] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety. Preferred embodiments of the present invention are described also in the following in the non-limiting examples and the figures.
[0097] FIG. 1 shows schematically a vector system according to the present invention and a virus produced from the vector system;
[0098] FIG. 2 shows the verification of stable insertion of two recombination sites into the HSV-1 genome;
[0099] FIG. 3 shows schematically a further vector system according to the present invention;
[0100] FIG. 4 shows the verification of the stable insertion of two transgene expression cassettes at the recombination sites, also called functionalisation;
[0101] FIG. 5 shows the detailed analysis of the functionalized virus platformNA-ICI-T.
EXAMPLES
[0102] In the examples the advantage of the vector system according to the present invention is shown by insertion of a target control protein and an immune checkpoint inhibitor to obtain an oncolytic virus for treatment of non-small-cell lung carcinoma (NSCLC), more specifically a target control protein for targeting to epidermal growth factor receptor (EGFR) expressing tumor cells and an immune checkpoint inhibitor against programmed cell death protein 1 (PD-1). The immune checkpoint inhibitor corresponds to a single chain variable fragment (scFv) derived of an anti-PD-1 antibody. The target control protein consists of an scFv against EGFR N-terminally fused to HSV-1 glycoprotein H. As a result, a functionalized transgenic virus for a combined virus immune therapy was produced. The main advantage is the simplified, modular and stable insertion of two different transgene expression cassettes to functionalize the vector that allows different transgene combinations.
Example 1
[0103] FIG. 1 shows the schematic representation of the vector system according to the present invention before (basic vector) and after insertion of the recombination sites (platform vector). The genome of HSV-1 consisting of the regions UL (unique long), US (unique short) and the inverted repeats TR (terminal repeat, Terminal repeat long (TRL) and Terminal repeat short (TRS)) and IR (internal repeat, Inverted repeat long (IRL) and Inverted repeat short (IRS)) is depicted. Furthermore, the HSV-1 genome contains three origins of DNA replication of two types: one copy of oriL located at the center of the unique long (UL) region of the genome and two copies of oriS located in the repeats flanking the unique short (US) region of the genome. The non-viral part, here shown as BAC, including sequences of pBeloBAC1 1, a sequence encoding Cre recombinase and a Zeocin selection cassette, is flanked by loxP recombination sites and inserted between UL3 and UL4 together with an additional polyadenylation signal. The two recombination sites to generate the platform vector, FRT and mFRT, are inserted between UL10 and UL11 or UL55 and UL56, respectively. The core plasmid system with and for the recombination sites was kindly provided by Z. Ruzsics.
[0104] In FIG. 1 following symbols are used:
TABLE-US-00001 • polyadenylation signal o additional polyadenylation signal ![custom-character]()
FRT recombination site ![custom-character]()
mFRT recombination site
[0105] FIG. 2 shows the verification of stable insertion of two recombination sites into the HSV-1 genome. A: Schematic depiction of the fragment sizes before (basic vector) and after insertion of the recombination sites (platform vector) starting from oligonucleotides (depicted as arrows) that bind in UL10 and UL11 or UL55 and UL56. Numbers between two oligonucleotides correspond to number of base pairs. B: Fragments of polymerase chain reactions (PCR) using oligonucleotides shown in A and the basic or platform vector as template. The fragments were separated in an agarose gel (2%, Tris acetic acid EDTA buffer) and visualized by Midori Green Advance under UV light. C: Basic and platform virus were reconstituted by transfection of Vero cells with respective BAC DNA and further cultivation. Growth properties of basic and platform virus were determined by infection of Vero cells (MOI of 0,1) in triplicates, harvest of the cell culture supernatant at different hours post infection (hpi) and titration on Vero cells using plaque assay.
Example 2
[0106] FIG. 3 shows a schematic depiction of the vector system according to the present invention after functionalisation with two transgenes based on the schematic depiction of FIG. 1. The two virulence genes UL37 and UL39 contain point mutations and a partial deletion, respectively, to obtain an attenuated transgenic virus that is deficient in neuotoxicity (called platformNA). The functionalized vector platformNA-ICI-T is generated by Flp-mediated insertion of two transgenes at the recombination sites, FRT and mFRT. One transgene expression cassette depicted as ICI encodes an immune checkpoint inhibitor against programmed cell death protein 1 (PD-1), more specifically a single chain variable fragment (scFv) derived of an antibody against PD-1. The other transgene expression cassette depicted as T encodes a target control protein against the epidermal growth factor receptor (EGFR) expressed on tumor cells, more specifically the target control protein consists of an scFv against EGFR (kindly provided by R. Kontermann) N-terminally fused to HSV-1 glycoprotein H.
[0107] In FIG. 3 following symbols are used:
TABLE-US-00002 • polyadenylation signal o additional polyadenylation signal ![custom-character]()
FRT recombination site ![custom-character]()
mFRT recombination site
[0108] FIG. 4 shows the verification of the stable insertion of two transgene expression cassettes at the recombination sites, also called functionalisation. A: Schematic depiction of the fragment sizes before (platform.sub.NA vector) and after insertion of the transgene expression cassettes (platform.sub.NA-ICI-T) starting from oligonucleotides (depicted as arrows) that bind in UL10 and UL11 or UL55 and UL56 or UL55/UL56 and the transgene expression cassettes comprising the target control protein. Numbers between two oligonucleotides correspond to length of fragments/products in base pairs produced by polymerase chain reactions (PCR) separated in B and D. B: Fragments of polymerase chain reactions (PCR) using oligonucleotides shown in A and the platform.sub.NA or the platformna-ICI-T vector as template. The fragments were separated in an agarose gel (1.2%, Tris acetic acid EDTA buffer) and visualized by Midori Green Advance under UV light. C: Isolated BAC DNA was digested with the restriction enzyme NotI, fragments were separated in an agarose gel (0.6%, Tris boric acid EDTA buffer) and visualized by ethidium bromide under UV light. Expected differences regarding the restriction pattern between the platform.sub.NA vector and the functionalized vector platform.sub.NA-ICI-T are highlighted with arrows. D: PCR fragments as described in B but with platform.sub.NA virus (passage 4) and platform.sub.NA-ICI-T virus (passage 8) as template representing the stability of the transgene expression cassette insertion using the two recombination sites. Both viruses were reconstituted by transfection of Vero cells with the respective BAC DNA and further cultivation.
[0109] FIG. 5 shows the detailed analysis of the functionalized virus platformNA-ICI-T including growth properties and expression as well as functional analysis of proteins encoded by inserted transgenes. A: Growth properties of platformNA virus compared to platformNA-ICI-T virus reconstituted as described in FIG. 4D were determined by infection of Vero cells (MOI of 0,1) in triplicates, harvest of the cell culture supernatant at different hours post infection (hpi) and titration on Vero cells using plaque assay. B: Functional analysis of the targeting to EGFR expressing cells was investigated by infection (MOI of 5) of J1.1cl2 cells and J1.1cl2 cells expressing EGFR (suitable for the scFv used), either alone or in presence of Cetuximab (anti-EGFR antibody). 16 hours post infection cell lysates were harvested and analysed by SDS-PAGE followed by Western Blot to detect the HSV-1 glycoprotein B (gB) as an indicator for infection. J1.1cl2, kindly provided by G. Campadelli-Fiume, cannot be infected by HSV-1. C: Expression of the scFv against PD-1 was analysed in Vero cells as well as in the non-small-cell lung carcinoma (NSCLC) cell lines A549 und HCC827. 16 hours post infection (MOI of 0,5) cell lysates were harvested and analysed by SDS-PAGE and Western Blot to detect the Myc-labelled scFv. D: To test the functionality of the virus encoded scFv against PD-1 as an immune checkpoint inhibitor a commercially available PD-1:PD-L1 inhibitor screening assay was applied. Therefore Vero cells were infected (MOI of 0,5), 16 hours post infection cell lysates were harvested and tested in thePD-1:PD-L1 inhibitor screening assay. Pembrolizumab, an anti-PD-1 antibody, was used as inhibition control.
