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WO2025082899A1 - Oncolytic viruses and their uses - Google Patents

Oncolytic viruses and their uses Download PDF

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WO2025082899A1
WO2025082899A1 PCT/EP2024/078852 EP2024078852W WO2025082899A1 WO 2025082899 A1 WO2025082899 A1 WO 2025082899A1 EP 2024078852 W EP2024078852 W EP 2024078852W WO 2025082899 A1 WO2025082899 A1 WO 2025082899A1
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fragment
envelope glycoprotein
irpov
retroviral envelope
genome
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Anne DUPRESSOIR
Ngoc Huong Giang TRAN
Thierry Heidmann
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VIROXIS
Centre National de la Recherche Scientifique CNRS
Institut Gustave Roussy (IGR)
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Saclay
Original Assignee
VIROXIS
Centre National de la Recherche Scientifique CNRS
Institut Gustave Roussy (IGR)
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Saclay
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/766—Rhabdovirus, e.g. vesicular stomatitis virus
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
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    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
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    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20041—Use of virus, viral particle or viral elements as a vector
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    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20041—Use of virus, viral particle or viral elements as a vector
    • C12N2760/20045—Special targeting system for viral vectors

Definitions

  • the invention concerns new oncolytic viruses and their uses.
  • Cancer is a leading cause of death worldwile. Over the past several years, in addition to conventional treatment, new generations of cancer therapy have been developed to deal with the limitations of current treatments.
  • Oncolytic virotherapy has recently emerged as a promising alternative to conventional cancer treatments.
  • the idea is to employ infectious viruses to selectively infect and efficiently kill tumor cells.
  • Oncolytic viruses OVs
  • OVs Oncolytic viruses
  • T-VEC which is an HSV1-derived OV
  • T-VEC demonstrated anticancer potency and has been approved by FDA for the treatment of melanoma (Andtbacka RHI, et al. 2015. Talimogene Laherparepvec Improves Durable Response Rate in Patients With Advanced Melanoma. J Clin Oncol Off J Am Soc Clin Oncol 33:2780-2788).
  • IRPOV Infectious, Replicative and Pseudotyped Oncolytic Virus
  • a first subject matter of the invention concerns the use of said retroviral envelope glycoprotein or a fragment thereof which target a tumor marker for producing an IRPOV specifically directed against said tumor marker.
  • the invention also concerns an IRPOV genome encoding said IRPOV.
  • Another subject matter of the invention relates to a plasmid comprising said IRPOV genome and its use for producing a viral particle, said viral particle being said IRPOV.
  • Another subject matter of the invention thus concerns said IRPOV as such or a pharmaceutical composition comprising it and its use as a drug, in particular for its use in the prevention and/or the treatment of a tumor.
  • a subject matter of the invention concerns a use of a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein, for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus” (IRPOV) specifically directed against said host cell surface protein from an “Infectious and Replicative Oncolytic Virus” (IROV), the native envelope glycoprotein of said IROV being inactivated (resulting in the loss of its natural tropism) in said IRPOV and said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV: ⁇ is capable of allowing the production of an IRPOV;
  • ⁇ has retained its native targeting capacity for said host cell surface protein
  • ⁇ has retained its native infectious activity permitting to said IRPOV to entry into a host cell expressing said host cell surface protein.
  • “Retroviral envelope glycoprotein” means a glycoprotein located on the surface of retroviruses, a type of RNA virus. This protein (Env) is essential for the virus ability to enter and infect host cells. It plays a crucial role in the viral life cycle by mediating the fusion of the viral membrane with the host cell membrane, allowing the virus to enter the host cell.
  • the retroviral Env protein is typically composed of two subunits: Sil (surface glycoprotein) and TM (transmembrane glycoprotein). Sil is responsible for binding to specific receptors on the host cell surface, while TM triggers the fusion process between the viral and host cell membranes together.
  • Sil surface glycoprotein
  • TM transmembrane glycoprotein
  • Virus tropism means the capability of a virus to infect and replicate within specific types of cells, tissues, or organisms. It depends on various factors, including the virus surface proteins or receptors, which determine the host cells or organisms it can target.
  • “Fragment thereof” means a portion or segment of said retroviral envelope glycoprotein. It consists of a sequence of amino acids (or nucleic acids encoding said fragment) and represents only a part of the complete protein structure. These fragments can vary in size and keep the same function as the retroviral envelope glycoprotein from which it comes from.
  • “Host cell surface protein” means a protein molecule located on the outer surface of a host cell membrane. Host cell surface proteins can serve as receptors for viruses, bacteria, hormones, and other molecules, allowing them to bind to the cell membrane and initiate specific cellular responses such as infection. These proteins are also key targets for therapeutic applications. In particular, said host cell surface protein is a tumor marker.
  • a subject matter of the invention thus concerns the use of a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein as described above, said host cell surface protein being a tumor marker, for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus” (IRPOV) specifically directed against said host cell surface protein from an “Infectious and Replicative Oncolytic Virus” (IROV), the native envelope glycoprotein of said IROV being inactivated (resulting in the loss of its natural tropism) in said IRPOV and said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV:
  • IRPOV Infectious, Replicative and Pseudotyped Oncolytic Virus
  • ⁇ is capable of allowing the production of an IRPOV
  • ⁇ has retained its native targeting capacity for said host cell surface protein
  • ⁇ has retained its native infectious activity permitting to said IRPOV to entry into a host tumor cell expressing said tumor marker.
  • Tumor marker also known as a biomarker or cancer marker, means a substance or molecule produced by a tumor or by the body in response to the presence of cancer.
  • said tumor marker is present on the cell membrane of cancer cells and expressed (whereas absent in normal equivalent cells or expressed in a mutated form with respect to the expression in normal equivalent cells) or overexpressed in tumor cells as compared to normal equivalent cells (e.g. median fold change T/N > 2).
  • tumor marker expression levels can be determined by RT-qPCR analysis of biological samples, or determined by Western Blot analysis of biological samples. These cell membrane-associated tumor markers can thus be used to specifically target host tumor cells for killing them (therapeutic approach). Examples of tumor markers are:
  • MMPZL1 Myelin protein zero-like 1
  • a subject matter of the invention thus concerns the use of a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein as described above, said host cell surface protein being a tumor marker and said tumor marker being chosen among: the “Alanine, Serine, Cysteine Transporter 2” (ASCT2), the “Myelin protein zero-like 1” (MPZL1) and the “ubiquitous vertebrate glucose transporter 1” (GLUT1).
  • ASCT2 Alanine, Serine, Cysteine Transporter 2
  • MPZL1 Myelin protein zero-like 1
  • GLUT1 ubiquitous vertebrate glucose transporter 1
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said IRPOV is devoid of a nucleic acid encoding a retroviral GAG protein.
  • a subject matter of the invention relates to the use of a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2” (ASCT2) or the “Myelin protein zero-like 1” (MPZL1), for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus” (IRPOV) specifically directed against said host cell surface protein from an “Infectious and Replicative Oncolytic Virus” (IROV), the native envelope glycoprotein of said IROV being inactivated in said IRPOV and said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV:
  • ⁇ is capable of allowing the production of an IRPOV
  • ⁇ has retained its native targeting capacity for said host cell surface protein
  • ⁇ has retained its native infectious activity permitting to said IRPOV to entry into a host cell expressing said host cell surface protein.
  • a subject matter of the invention thus concerns the use of a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein as described above, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2” (ASCT2; also named SLC1a5), for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus” (IRPOV) specifically directed against said host cell surface protein from an “Infectious and Replicative Oncolytic Virus” (IROV), the native envelope glycoprotein of said IROV being inactivated (resulting in the loss of its natural tropism) in said IRPOV and said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV:
  • ⁇ is capable of allowing the production of an IRPOV
  • Pseudotyped Oncolytic Virus means virus particle(s) whose native surface glycoproteins have been replaced by those derived from heterologous enveloped viruses to permit virus entry and alter its tropism (or to interrogate the ability of the glycoproteins to permit entry).
  • a Pseudotyped Oncolytic Virus is produced by reverse genetics from a genetically modified genome in which the native envelope is replaced by a heterologous one. Its new host cell surface protein specificity and tropism can be assessed in infection experiments using non permissive cells in culture and host cell surface protein overexpression (see hereafter).
  • “Infectious Oncolytic Virus” means an oncolytic virus which is able to infect (i.e. to entry into) host (tumor) cells for which said oncolytic virus has a natural or modified tropism.
  • Replicative Oncolytic Virus means an oncolytic virus which is able to replicate, or to reproduce, within host (tumor) cells, leading to the destruction of those host (tumor) cells.
  • IROV Infectious and Replicative Oncolytic Virus
  • IROV infected host (tumor) cell.
  • IRPOV Infectious, Replicative and Pseudotyped Oncolytic Virus
  • IRPOV infectious virus
  • IRPOV specifically directed against said host cell surface protein means that the pseudotyping of an IROV to obtain an IRPOV according to invention leads to a viral particle (i.e. IRPOV) having a tropism specifically directed against a host cell surface protein of interest.
  • the native envelope glycoprotein of said IROV being inactivated means that the pseudotyping of an IROV to obtain an IRPOV according to invention involves inactivating the native envelope glycoprotein of said IROV so that it is not expressed or at least not functional.
  • retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV is capable of allowing the production of an IRPOV” means that the retroviral envelope glycoprotein or a fragment thereof used according to the invention allows the production of a functional viral particles.
  • retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV has retained its native targeting capacity for said host cell surface protein means that the retroviral envelope glycoprotein or a fragment thereof used according to the invention keeps its native targeting capacity for said host cell surface protein (e.g. tumor marker) and thus gives the IRPOV according to the invention a new tropism directed against said host cell surface protein (e.g. tumor marker).
  • This capacity can be evaluated according to classical methods known in the art, wherein the capacity of the retroviral envelope glycoprotein or a fragment thereof at the virion surface to bind to a cell surface protein (its receptor) and promote virus infection is measured (see Examples).
  • such assays can be performed using VSVAG particules pseudotyped with the retroviral envelope glycoprotein (Ferlin A et al. 2014. Characterization of pH-sensitive molecular switches that trigger the structural transition of vesicular stomatitis virus glycoprotein from the postfusion state toward the prefusion state. J Virol 88:13396-13409) to transduce target cells expressing or not the cell surface protein (its receptor) as illustrated in the examples. Briefly, 2.10 6 BSR cells seeded in a 6 cm dish are transfected with 2pg of the expression vector for the retroviral envelope glycoprotein using jetPrime® (PolyPlus, France).
  • transfected cells are infected with VSVAG-GVGFP particles, which contains a gene coding for GFP and have the glycoprotein G on its surface -but not at the genome level- at a MOI (Multiplicity Of Infection) of 3 for 4h. Cells are then washed four times to remove residual virus. Fourty four hours later, cell supernatant containing the particles pseudotyped with retroviral envelope glycoproteins are harvested and added to target cells. Titers are determined by flow cytometry analysis based on the percent of GFP+ cells. The targeting capacity of retroviral envelope glycoproteins was estimated by comparing the titers obtained with target cells overexpressing the receptor with that obtained with target cells expressing low levels of the receptor or laking it (KO cells).
  • “Said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV has retained its native infectious activity permitting to said IRPOV to entry into a host (tumor) cell” means that the retroviral envelope glycoprotein or a fragment thereof used according to the invention keeps its native infectious activity and thus gives the IRPOV according to the invention the capacity to infect host (tumor) cell.
  • This infectious activity can be evaluated through infectious assays wherein the capacity of the retroviral envelope glycoprotein or a fragment thereof at the virion surface to bind to a cell surface protein (its receptor) and promote virus infection is measured (see Examples). For instance, such infectious assays can be performed using VSVAG particules pseudotyped with the retroviral envelope glycoprotein (Ferlin A et al. 2014.
  • transfected cells are infected with VSVAG-G*/GFP particles, which contains a gene coding for GFP and have the glycoprotein G on its surface -but not at the genome level- at a MOI (Multiplicity Of Infection) of 3 for 4h. Cells are then washed four times to remove residual virus. Fourty four hours later, cell supernatant containing the particles pseudotyped with retroviral envelope glycoproteins are harvested and added to target cells expressing the receptor. Titers are determined by flow cytometry analysis based on the percent of GFP+ cells.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof when present in the genome of said IRPOV has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium.
  • Fusogenic properties of a given retroviral envelope glycoprotein may be assessed in fusion assays based on alpha- complementation of the beta galactosidase, as described in Bacquin et al. (Cell Fusion- Based Screening Method Identifies Glycosylphosphatidylinositol-Anchored Protein Ly6e as the Receptor for Mouse Endogenous Retroviral Envelope Syncytin-A. J Virol. 2017 Aug 24;91 (18):e00832-17.).
  • a first group of cells are transfected, for example by using Lipofectamine (Invitrogen), with vectors encoding the beta-galactosidase fragment Gal-alpha, and a second group of cells are cotransfected with vectors encoding the beta-galactosidase fragment Gal-omega and a retroviral envelope glycoprotein at a ratio of 1 :1.
  • the two groups of cells were cocultured at a ratio of 1 :1.
  • cells were fixed and stained with X-Gal to identify, by microscopy analysis, blue syncytia formed following fusion of cells from both groups. The number and mean area of syncytia are analyzed in randomly chosen fields by Imaged.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
  • said modification which encompasses partial or total truncation of the intracytoplasmic tail of said retroviral envelope glycoprotein or a fragment thereof, advantageously allows to enhance the cell-cell fusion activity and/or to improve the production of the IRPOV.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof.
  • Endogenous retroviral envelope glycoprotein means that the retroviral envelope glycoprotein or a fragment thereof used according to the invention comes from an endogenous retrovirus.
  • Endogenous retroviruses are a class of retroviruses that have integrated their genetic material into the genome of an organism. Unlike exogenous retroviruses, which are external and can infect host cells from outside the body, endogenous retroviruses are inherited from generation to generation as part of the host DNA.
  • an endogenous retroviral envelope glycoprotein having a native targeting capacity for the “Alanine, Serine, Cysteine Transporter?” can be chosen among: Syncytin-1 , Syncytin-Ory1 , Dasy Env1.1 , RD114, BaEV, ptERV-W, ggERV-W, paERV-W(3), paERV- W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1, MMERV-B6, mfuERV, GeERV and SERV-2.
  • exogenous retroviruses exist, which are a type of retrovirus that are found outside of the host organism genome. Unlike endogenous retroviruses (ERVs), which have integrated their genetic material into the host DNA over evolutionary time, exogenous retroviruses are separated and independent entities that can infect host cells from outside the host body.
  • a subject matter of the invention concerns thus the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an exogenous retroviral envelope glycoprotein or a fragment thereof.
  • an exogenous retroviral envelope glycoprotein having a native targeting capacity for the “Alanine, Serine, Cysteine Transporter 2" can be chosen among: MPMV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, SRV-6 and SRV-7.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S-[D/N]-[G/R]- X1GX2X3X4X5X6X7” (SEQ ID NOs: 131 to 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X 2 V or P
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 131) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDRX1GX2X3X4X5X6X7” (SEQ ID NO: 132) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNGXIGX2X 3 X 4 X 5 X6X7” (SEQ ID NO: 133) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNRX1GX2X3X4X5X6X7” (SEQ ID NO: 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S-[D/N]-[G/R]- XIGX2X3X4XSX6X7” (SEQ ID NOs: 135 to 138) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises:
  • ⁇ Xi G or R or no amino acid
  • ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein
  • ⁇ Xi G or R or no amino acid
  • ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein
  • ⁇ Xi G or R or no amino acid, or
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNRX1GX2X3X4X5X6X7” (SEQ ID NO: 138) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S-[D/N]-[G/R]- XIGX2X3X4XSX6X7” (SEQ ID NOs: 139 to 142) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises:
  • ⁇ Xi G or R or no amino acid
  • ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein
  • ⁇ Xi G or R or no amino acid
  • ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein
  • ⁇ Xi G or R or no amino acid, or
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 139) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDRX1GX2X3X4X5X6X7” (SEQ ID NO: 140) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNGXIGX2X 3 X 4 X 5 X6X7” (SEQ ID NO: 141) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNRX1GX2X3X4X5X6X7” (SEQ ID NO: 142) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGPX1DX2X3R” (SEQ ID NO: 4) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi L or Q or T
  • X2 Q or T or K or A or M or no amino acid
  • X3 T or A or V or I.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGX1X2DX3X4X5” (SEQ ID NO: 5) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ X3 Q or T or K
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGPX1DX2X3R” (SEQ ID NO: 7) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 11, 13, 21 to 35 and 145.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 12, 14 to 20 and 36.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 13 and 15.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 11 and 13.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 12 and 15.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppERV- W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1, MMERV-B6, mfuERV, GeERV, SRV-6, SRV-7, SERV-2 and their orthologs.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-1 , SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, REV/CSV/SNV/DIAV bis , ptERV-W, ggERV-W, paERV-W(3), paERV- W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1 , TvERV, DrERV, CfERV-Fc1 , SERV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Das
  • % of identity means that the percentage determined by direct comparison of two oligonucleotide sequences (nucleic acid sequence), by determining the number of identical nucleotides between the two sequences, then dividing it by the number of nucleotides of the longer of the two sequences, and multiplying the result by 100.
  • has at least 90% of identity is therefore meant that the aforementioned percentage of identity is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100%.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, BaEV, ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1, MMERV-B6, mfuERV, GeERV, SERV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, BaEV, ptERV-W, ggERV-W, paERV-W(3), paERV-W
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: MPMV, SRV-1, SRV-2, SRV- 4, SRV-5, SMRV, REV/CSV/SNV/DIAV, REV/CSV/SNV/DIAV bis and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV and REV/CSV/SNV/DIAV bis , the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 45, 49, 51, 53, 55, 57, 59 and 61, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 46, 50, 52, 54, 56,
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1 , RD114, MPMV, BaEV, SRV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1 , RD114, MPMV, BaEV and SRV-2, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47 and 51 , or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48 and 52; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 ,
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, BaEV and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114 and BaEV, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43 and 47, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44 and 48; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114 and BaEV, the nucleic acid of
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: MPMV, SRV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV and SRV-2, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 45 and 51, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 46 and 52; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV and SRV-2, the nucleic acid of which is respectively the sequences SEQ ID NOs: 45 and 51 , or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 46 and 52.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 85, 87, 89, 91 , 93, 95 and 97, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 86, 88, 90, 92, 94, 96 and 98.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which is respectively the sequences SEQ ID NOs: 85, 87, 89, 91, 93, 95 and 97, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 86, 88, 90, 92, 94, 96 and 98.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, and BaEVA18, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 85, 87, 89, 91 and 95, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 86, 88, 90, 92 and 96.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18 and BaEVA18, the nucleic acid of which is respectively the sequences SEQ ID NOs: 85, 87, 89, 91 and 95, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 86, 88, 90, 92 and 96.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: MPMVA23 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 93 and 97, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 94 and 98.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: MPMVA23 and SRV-2A23, the nucleic acid of which is respectively the sequences SEQ ID NOs: 93 and 97, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 94 and 98.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein as described above, said host cell surface protein being a tumor marker and said tumor marker being the “Myelin protein zero-like 1” (MPZL1), for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus” (IRPOV) specifically directed against said host cell surface protein from an “Infectious and Replicative Oncolytic Virus” (IROV), the native envelope glycoprotein of said IROV being inactivated (resulting in the loss of its natural tropism) in said IRPOV and said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV:
  • ⁇ is capable of allowing the production of an IRPOV
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof when present in the genome of said IRPOV has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof.
  • an endogenous retroviral envelope glycoprotein having a native targeting capacity for the “Myelin protein zero-like 1" (MPZL1) can be Syn-Mab1.
  • the subject matter of the invention can thus concern the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn-Mab1 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof is Syn-Mab1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 146, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 147; and in particular said retroviral envelope glycoprotein or a fragment thereof is Syn-Mab1, the nucleic acid of which is the sequence SEQ ID NO: 146, or the amino acid sequence of which is the sequence SEQ ID NO: 147.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an exogenous retroviral envelope glycoprotein or a fragment thereof.
  • a subject matter of the invention thus concerns the use of a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein as described above, said host cell surface protein being a tumor marker and said tumor marker being the “ubiquitous vertebrate glucose transporter 1” (GLUT1), for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus” (IRPOV) specifically directed against said host cell surface protein from an “Infectious and Replicative Oncolytic Virus” (IROV), the native envelope glycoprotein of said IROV being inactivated (resulting in the loss of its natural tropism) in said IRPOV and said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV:
  • IRPOV Infectious, Replicative and Pseudotyped Oncolytic Virus
  • ⁇ is capable of allowing the production of an IRPOV
  • ⁇ has retained its native targeting capacity for GLUT 1 ; and has retained its native infectious activity permitting to said IRPOV to entry into a host tumor cell expressing GLUT 1.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said IRPOV specifically directed against GLUT1 is devoid of a nucleic acid encoding a retroviral GAG protein.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof when present in the genome of said IRPOV has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof.
  • an endogenous retroviral envelope glycoprotein having a native targeting capacity for the ““ubiquitous vertebrate glucose transporter 1" (GLUT1) can be HTLV-1 Env.
  • the subject matter of the invention can thus concern the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: HTLV-1 Env and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof is HTLV-1 Env, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 148, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 149; and in particular said retroviral envelope glycoprotein or a fragment thereof is HTLV-1 Env, the nucleic acid of which is the sequence SEQ ID NO: 148, or the amino acid sequence of which is the sequence SEQ ID NO: 149.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an exogenous retroviral envelope glycoprotein or a fragment thereof.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said native envelope glycoprotein of said IROV is inactivated by addition, deletion or substitution of at least one nucleotide of the nucleic acid sequence encoding it, in particular said native envelope glycoprotein of said IROV being inactivated by substitution of said nucleic acid sequence encoding it by the nucleic acid sequence encoding said retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said native envelope glycoprotein of said IROV is inactivated by substitution of said nucleic acid sequence encoding it by the nucleic acid sequence encoding said retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said IROV is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • ⁇ a paramyxovirus, in particular said IROV being a VSV.
  • VSV-N polypeptide a VSV-N polypeptide
  • VSV-P polypeptide a VSV-M polypeptide
  • the retroviral envelope glycoprotein of the invention or a fragment thereof optionally a marker polypeptide (lacZ, or a gene coding the sodium iodide transporter, or a gene coding for fluorescent protein GFP, RFP...) and a VSV-L polypeptide.
  • the native VSV-G polypeptide is inactivated and the retroviral envelope glycoprotein of the invention or a fragment thereof is introduced.
  • the VSV-M polypeptide can comprise mutations which reduce the cytopathogenicity of the VSV. Examples of mutations are amino acid substitutions M51A or M51R, V221 F, and S226R or deletions Mdelta51.
  • MV Middle Virus
  • N nucleocapsid
  • P phosphoprotein
  • M matrix
  • F fusion
  • H hemagglutinin attachment
  • L viral polymerase
  • MV nucleic acid molecule of which encodes a nucleocapsid (N) polypeptide, a phosphoprotein (P) polypeptide, a matrix (M) polypeptide and the retroviral envelope glycoprotein of the invention or a fragment thereof.
  • a marker polypeptide (lacZ, or a gene coding the sodium iodide transporter, or a gene coding for fluorescent protein GFP, RFP...) can be also inserted between the N-P, the P-M or the Env-L genes.
  • the native MV-F and/or MV-H polypeptides are inactivated and the retroviral envelope glycoprotein of the invention or a fragment thereof is introduced.
  • SeV Sendai Virus
  • mPIV-1 murine parainfluenza virus type 1
  • the SeV genome is a single molecule of negative - sense RNA that encodes several polypeptides: a nucleoprotein (NP), a phosphoprotein (P), a matrix protein (M), a fusion protein (F), a hemagglutininneuraminidase protein (HN), and a large polymerase protein (L).
  • NP nucleoprotein
  • P phosphoprotein
  • M matrix protein
  • F fusion protein
  • HN hemagglutininneuraminidase protein
  • L large polymerase protein
  • a pseudotyped SeV the nucleic acid molecule of which encodes a SeV-NP polypeptide, a SeV-P polypeptide, a SeV-M polypeptide, the retroviral envelope glycoprotein of the invention or a fragment thereof and a SeV-L polypeptide.
  • a marker polypeptide (lacZ, or a gene coding the sodium iodide transporter, or a gene coding for fluorescent protein GFP, RFP...) can be also inserted between the N-P, the P-M or the Env-L genes
  • the native SeV-F and/or SeV-HN polypeptides are inactivated and the retroviral envelope glycoprotein of the invention or a fragment thereof is introduced.
  • NDV Newcastlele Disease Virus
  • APMV-1 avian paramyxovirus serotype 1
  • NP nucleoprotein
  • P phosphoprotein
  • M matrix protein
  • F fusion protein
  • HN hemagglutininneuraminidase protein
  • L large polymerase protein
  • NDV nucleic acid molecule of which encodes a NDV-NP polypeptide, a NDV-P polypeptide, a NDV-M polypeptide, the retroviral envelope glycoprotein of the invention or a fragment thereof and a NDV-L polypeptide.
  • a marker polypeptide (lacZ, or a gene coding the sodium iodide transporter, or a gene coding for fluorescent protein GFP, RFP). can be also inserted between the N-P, the P-M or the Env-L genes
  • the native NDV-F and/or NDV-HN polypeptides are inactivated and the retroviral envelope glycoprotein of the invention or a fragment thereof is introduced.
  • CDV Canine distemper virus
  • N nucleocapsid
  • P phosphoprotein
  • M matrix
  • F fusion
  • H hemagglutinin attachment
  • L viral polymerase
  • a pseudotyped CDV the nucleic acid molecule of which encodes a CDV-N polypeptide, a CDV-P polypeptide, a CDV-M polypeptide, the retroviral envelope glycoprotein of the invention or a fragment thereof and a CDV-L polypeptide.
  • a marker polypeptide (lacZ, or a gene coding the sodium iodide transporter, or a gene coding for fluorescent protein GFP, RFP...) can be also inserted between the N-P, the P-M or the Env-L genes.
  • the native CDV-F and/or CDV-H polypeptides are inactivated and the retroviral envelope glycoprotein of the invention or a fragment thereof is introduced.
  • Paramyxovirus means a member of the paramyxovirus family.
  • the paramyxovirus genome is composed of the nucleocapsid (N) polypeptide, a phosphoprotein (P) polypeptide, a matrix (M) polypeptide, a fusion (F) polypeptide, a hemagglutinin attachment (H) polypeptide and a viral polymerase (L) polypeptide.
  • N nucleocapsid
  • P phosphoprotein
  • M matrix
  • F fusion
  • H hemagglutinin attachment
  • L viral polymerase
  • a pseudotyped paramyxovirus the nucleic acid molecule of which encodes a N polypeptide, a P polypeptide, a M polypeptide, the retroviral envelope glycoprotein of the invention or a fragment thereof and a L polypeptide.
  • a marker polypeptide (lacZ, or a gene coding the sodium iodide transporter, or a gene coding for fluorescent protein GFP, RFP).
  • the native F and/or H polypeptides are inactivated and the retroviral envelope glycoprotein of the invention or a fragment thereof is introduced.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a Syncytin-1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syncytin-1 , the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 37, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 38.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a Syncytin-Ory1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syncytin-Ory1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 39, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 40.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a Dasy Env1.1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Dasy Env1.1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 41 , or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 42.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a RD114 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a RD114, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 43, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 44.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a MPMV or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a MPMV, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 45, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 46.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a BaEV or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a BaEV, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 47, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 48.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • a paramyxovirus and - said retroviral envelope glycoprotein or a fragment thereof is a SRV-2 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a SRV-2, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 51, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 52.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a Syn1A53 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syn1A53, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 85, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 86.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a SynOry1A25 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a SynOry1A25, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 87, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 88.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a DasyEnvA28 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a DasyEnvA28, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 89, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 90.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a RD114A18 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a RD114A18, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 91 , or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 92.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a MPMVA23 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a MPMVA23, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 93, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 94.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a BaEVA18 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a BaEVA18, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 95, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 96.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • paramyxovirus a Newcastle Disease Virus
  • said retroviral envelope glycoprotein or a fragment thereof is a SRV-2A23 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a SRV-2A23, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 97, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 98.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a Syn-Mab1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syn-Mab1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 146, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 147.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a HTLV-1 Env or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a HTLV-1 Env, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 148, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 149.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • VSV Vesicular Stomatitis Virus
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95
  • said IROV is a Measles Virus (MV).
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • said IROV is a Sendai Virus (SeV).
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • said IROV is a Newcastle Disease Virus (NDV).
  • NDV Newcastle Disease Virus
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • said IROV is a Canine distemper virus (CDV).
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • said IROV is a paramyxovirus
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • said IROV is a Vesicular Stomatitis Virus (VSV).
  • VSV Vesicular Stomatitis Virus
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • said IROV is a Measles Virus (MV).
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • said IROV is a Sendai Virus (SeV).
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • said IROV is a Newcastle Disease Virus (NDV); and - said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
  • NDV Newcastle Disease Virus
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • said IROV is a Canine distemper virus (CDV).
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
  • a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
  • said IROV is a paramyxovirus
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
  • a subject matter of the invention concerns an IRPOV genome encoding an IRPOV specifically directed against a host cell surface protein, said IRPOV genome comprising an IROV genome comprising:
  • ⁇ a nucleic acid sequence encoding a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein, said retroviral envelope glycoprotein or a fragment thereof:
  • IRPOV genome means that the nucleic acid molecule encodes and allows the production of the viral particle according to the invention (i.e. the IRPOV according to the invention).
  • Said IRPOV genome is obtained by means of classic cloning techniques, which use restriction enzymes, plasmids, PCR amplification, etc.
  • said nucleic acid sequence encoding said retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein is amplified by PCR and cloned into the plasmid comprising the IROV genome at the place of the native native envelope glycoprotein of the IROV via, e.g. Mlul/Avrll, restriction sites.
  • IROV genome means that the nucleic acid molecule that encodes the virus from which the IRPOV genome according to the invention is obtained by pseudotyping.
  • a subject matter of the invention concerns the IRPOV genome encoding an IRPOV specifically directed against a host cell surface protein as described above, said host cell surface protein being a tumor marker, said IRPOV genome comprising an IROV genome comprising:
  • ⁇ a nucleic acid sequence encoding a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein, said retroviral envelope glycoprotein or a fragment thereof:
  • a subject matter of the invention thus concerns the IRPOV genome encoding an IRPOV specifically directed against a host cell surface protein as described above, said host cell surface protein being a tumor marker and said tumor marker being chosen among: the “Alanine, Serine, Cysteine Transporter 2” (ASCT2), the “Myelin protein zero-like 1” (MPZL1) and the “ubiquitous vertebrate glucose transporter 1” (GLLIT1).
  • ASCT2 Alanine, Serine, Cysteine Transporter 2
  • MPZL1 Myelin protein zero-like 1
  • GLLIT1 ubiquitous vertebrate glucose transporter 1
  • a subject matter of the invention relates to an IRPOV genome encoding an IRPOV specifically directed against a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2” (ASCT2) or the “Myelin protein zero-like 1” (MPZL1), said IRPOV genome comprising an IROV genome comprising:
  • ⁇ a nucleic acid sequence encoding a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein, said retroviral envelope glycoprotein or a fragment thereof:
  • a subject matter of the invention concerns the IRPOV genome encoding an IRPOV specifically directed against a host cell surface protein as described above, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2” (ASCT2), said IRPOV genome comprising an IROV genome comprising:
  • ⁇ a nucleic acid sequence encoding a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein, said retroviral envelope glycoprotein or a fragment thereof:
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an exogenous retroviral envelope glycoprotein or a fragment thereof.
  • a subject matter of the invention concerns the IRPOV genome as described above wherein said retroviral envelope glycoprotein or a fragment thereof has a native targeting capacity for a tumor marker being chosen among: the “Alanine, Serine, Cysteine Transporter 2" (ASCT2), the “Myelin protein zero-like 1” (MPZL1) and the “ubiquitous vertebrate glucose transporter 1" (GLLIT1), in particular said tumor marker being the “Alanine, Serine, Cysteine Transporter?” (ASCT2) and said retroviral envelope glycoprotein or a fragment thereof comprising the conserved “S- [D/N]-[G/R]-XIGX 2 X3X4X 5 X6X7” (SEQ ID NOS: 131 to 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S-[D/N]-[G/R]-XIGX 2 X3X 4 X 5 X6X7” (SEQ ID NOs: 131 to 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises: - the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 131) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 131) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDRX1GX2X3X4X5X6X7” (SEQ ID NO: 132) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNGX1GX2X3X4X5X6X7” (SEQ ID NO: 133) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNRX1GX2X3X4X5X6X7” (SEQ ID NO: 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S-[D/N]-[G/R]-XIGX 2 X3X4X 5 X6X7” (SEQ ID NOs: 135 to 138) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises:
  • ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein
  • ⁇ Xi G or R or no amino acid
  • ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein
  • ⁇ Xi G or R or no amino acid, or
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S 00X10X2X3X4X5X6X7” (SEQ ID NO: 135) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNGX1GX2X3X4X5X6X7” (SEQ ID NO: 137) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNRXIGX 2 X3X 4 X5X6X7” (SEQ ID NO: 138) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S-[D/N]-[G/R]-XIGX 2 X 3 X 4 X 5 X6X7” (SEQ ID NOs: 139 to 142) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 Q or L or T or I
  • ⁇ X 4 D or N
  • ⁇ Xs Q or T or K or E
  • ⁇ Xs A or L or K or V or I or R or E, and
  • ⁇ X? R or T or S or G or K;
  • ⁇ Xi G or R or no amino acid
  • ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein
  • ⁇ Xi G or R or no amino acid, or
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 139) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDRX1GX2X3X4X5X6X7” (SEQ ID NO: 140) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 Q or L or T or I
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNGX1GX2X3X4X5X6X7” (SEQ ID NO: 141) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNRX1GX2X3X4X5X6X7” (SEQ ID NO: 142) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGPX1DX2X3R” (SEQ ID NO: 4) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ X 2 Q or T or K or A or M or no amino acid
  • ⁇ X3 T or A or V or I.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGX1X2DX3X4X5” (SEQ ID NO: 5) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ X3 Q or T or K
  • ⁇ X 4 A or L or K or T
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGX1X2DX3X4X5” (SEQ ID NO: 6) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ X3 Q or T or K
  • ⁇ X 4 A or L or K
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGPX1DX2X3R” (SEQ ID NO: 7) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 36 and 145.
  • SEQ ID NOs: 8 to 36 and 145 it means that the sequence chosen sequence can be the sequence SEQ ID NO: 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36 or 145.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 11, 13, 21 to 35 and 145.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 12, 14 to 20 and 36.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 13 and 15.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 11 and 13.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 12 and 15.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-1 , SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1, MMERV-B6, mfuERV, GeERV, SRV-6, SRV-7, SERV-2 and their orthologs.
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-1 , SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, REV/CSV/SNV/DIAV bis , ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1, SERV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114, MPMV,
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114, BaEV, ptERV-W, ggERV- W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1, MMERV-B6, mfuERV, GeERV, SERV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, BaEV, ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W,
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: MPMV, SRV-1 , SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, REV/CSV/SNV/DIAV bis and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV and REV/CSV/SNV/DIAV bis , the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 45, 49, 51, 53, 55, 57, 59 and 61, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 46, 50, 52, 54, 56, 58, 60 and 62; and in
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1 , RD114, MPMV, BaEV and SRV-2, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41 , 43, 45, 47 and 51 , or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48 and 52; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1,
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114, BaEV and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114 and BaEV, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43 and 47, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44 and 48; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114 and BaEV, the nucleic acid of which is respectively the sequences SEQ ID NO
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: MPMV, SRV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV and SRV-2, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 45 and 51, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 46 and 52; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV and SRV-2, the nucleic acid of which is respectively the sequences SEQ ID NOs: 45 and 51 , or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 46 and 52.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 85, 87, 89, 91 , 93, 95 and 97, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 86, 88, 90, 92, 94, 96 and 98.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which is respectively the sequences SEQ ID NOs: 85, 87, 89, 91, 93, 95 and 97, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 86, 88, 90, 92, 94, 96 and 98.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, and BaEVA18, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 85, 87, 89, 91 and 95, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 86, 88, 90, 92 and 96.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18 and BaEVA18, the nucleic acid of which is respectively the sequences SEQ ID NOs: 85, 87, 89, 91 and 95, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 86, 88, 90, 92 and 96.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: MPMVA23 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 93 and 97, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 94 and 98.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: MPMVA23 and SRV-2A23, the nucleic acid of which is respectively the sequences SEQ ID NOs: 93 and 97, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 94 and 98.
  • a subject matter of the invention concerns the IRPOV genome encoding an IRPOV specifically directed against a host cell surface protein as described above, said host cell surface protein being a tumor marker and said tumor marker being the “Myelin protein zero-like 1” (MPZL1), for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus” (IRPOV) specifically directed against said host cell surface protein from an “Infectious and Replicative Oncolytic Virus” (IROV), the native envelope glycoprotein of said IROV being inactivated (resulting in the loss of its natural tropism) in said IRPOV and said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV:
  • ⁇ is capable of allowing the production of an IRPOV
  • ⁇ has retained its native targeting capacity for MPZL1 ; and has retained its native infectious activity permitting to said IRPOV to entry into a host tumor cell expressing MPZL1.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof.
  • an endogenous retroviral envelope glycoprotein having a native targeting capacity for the “Myelin protein zero-like 1" (MPZL1) can be Syn-Mab1.
  • the subject matter of the invention can thus concern the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn-Mab1 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof is Syn-Mab1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 146, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 147; and in particular said retroviral envelope glycoprotein or a fragment thereof is Syn-Mab1, the nucleic acid of which is the sequence SEQ ID NO: 146, or the amino acid sequence of which is the sequence SEQ ID NO: 147.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a native targeting capacity for MPZL1, in particular said retroviral envelope glycoprotein or a fragment thereof is Syn-Mab1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 146, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 147.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an exogenous retroviral envelope glycoprotein or a fragment thereof.
  • a subject matter of the invention thus concerns the IRPOV genome encoding an IRPOV specifically directed against a host cell surface protein as described above, said host cell surface protein being a tumor marker and said tumor marker being the “ubiquitous vertebrate glucose transporter 1” (GLUT1), for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus” (IRPOV) specifically directed against said host cell surface protein from an “Infectious and Replicative Oncolytic Virus” (IROV), the native envelope glycoprotein of said IROV being inactivated (resulting in the loss of its natural tropism) in said IRPOV and said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV:
  • ⁇ is capable of allowing the production of an IRPOV
  • ⁇ has retained its native infectious activity permitting to said IRPOV to entry into a host tumor cell expressing GLUT 1.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof when present in the genome of said IRPOV has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof.
  • an endogenous retroviral envelope glycoprotein having a native targeting capacity for the ““ubiquitous vertebrate glucose transporter 1" (GLUT1) can be HTLV-1 Env.
  • the subject matter of the invention can thus concern the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: HTLV-1 Env and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof is HTLV-1 Env, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 148, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 149; and in particular said retroviral envelope glycoprotein or a fragment thereof is HTLV-1 Env, the nucleic acid of which is the sequence SEQ ID NO: 148, or the amino acid sequence of which is the sequence SEQ ID NO: 149.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an exogenous retroviral envelope glycoprotein or a fragment thereof.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome results from addition, deletion or substitution of at least one of its nucleotides, in particular said inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome results from its replacement by said nucleic acid sequence encoding said retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome results from its replacement by said nucleic acid sequence encoding said retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein said IROV genome is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • a paramyxovirus genome in particular said IROV genome being a VSV genome.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • I ROV genome is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a Syncytin-1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syncytin-1 , the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 37, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 38.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • I ROV genome is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a Syncytin-Ory1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syncytin-Ory1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 39, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 40.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • I ROV genome is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a Dasy Env1.1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Dasy Env1.1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 41 , or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 42.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • I ROV genome is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a RD114 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a RD114, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 43, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 44.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • I ROV genome is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a MPMV or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a MPMV, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 45, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 46.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • I ROV genome is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a BaEV or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a BaEV, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 47, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 48.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • I ROV genome is chosen among:
  • VSV Vesicular Stomatitis Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a SRV-2 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a SRV-2, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 51, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 52.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • I ROV genome is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a Syn1A53 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syn1A53, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 85, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 86.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • I ROV genome is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a SynOry1A25 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a SynOry1A25, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 87, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 88.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • I ROV genome is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a DasyEnvA28 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a DasyEnvA28, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 89, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 90.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • I ROV genome is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a RD114A18 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a RD114A18, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 91 , or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 92.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • I ROV genome is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a MPMVA23 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a MPMVA23, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 93, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 94.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • I ROV genome is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a BaEVA18 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a BaEVA18, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 95, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 96.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • I ROV genome is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • retroviral envelope glycoprotein or a fragment thereof is a SRV-2A23 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a SRV-2A23, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 97, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 98.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • I ROV genome is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a Syn-Mab1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syn-Mab1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 146, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 147.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • I ROV genome is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a HTLV-1 Env or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a HTLV-1 Env, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 148, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 149.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • said I ROV genome is a Vesicular Stomatitis Virus (VSV) genome
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein: - said IROV genome is a Measles Virus (MV) genome; and
  • MV Measles Virus
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-0ry1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • said IROV genome is a Sendai Virus (SeV) genome
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • said IROV genome is a Newcastle Disease Virus (NDV) genome
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • said IROV genome is a Canine distemper virus (CDV) genome
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • said IROV genome is a paramyxovirus genome
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • said IROV genome is a Vesicular Stomatitis Virus (VSV) genome
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • said IROV genome is a Measles Virus (MV) genome
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • said IROV genome is a Sendai Virus (SeV) genome
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • said IROV genome is a Newcastle Disease Virus (NDV) genome
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • said IROV genome is a Canine distemper virus (CDV) genome
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
  • a subject matter of the invention concerns the IRPOV genome as described above, wherein:
  • said IROV genome is a paramyxovirus genome
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
  • the IRPOV genome according to the invention can further comprise genes to be delivered to host tumor cells encoding, for instance:
  • ⁇ immune checkpoint inhibitors such as antibodies
  • Immuno stimulating factors means proteins that can stimulate production, maturation or activation of cells of the immune system for anti-tumorigenic effects.
  • Immuno checkpoint inhibitors means antibodies that block checkpoint inhibitory proteins that stop the immune system from attacking the cancer cells.
  • factors enhancing fusogenic activity of the IRPOV means proteins that has a capacity to trigger the fusion of tumor cells infected by the IRPOV with adjacent host (tumor) cells for a better anti-tumoral effect.
  • a subject matter of the invention concerns a plasmid comprising the IRPOV genome as described above and means for expressing it.
  • a subject matter of the invention concerns a use of the IRPOV genome as described above or the plasmid as described above for producing a viral particle, said viral particle being an IRPOV and comprising the IRPOV genome as described above.
  • a subject matter of the invention concerns a method of production of a viral particle, said viral particle being an IRPOV and comprising the IRPOV genome as described above, said method comprising at least the steps of: a. co-transfection of an animal eukaryotic cell, in particular said animal eukaryotic cell constitutively expressing the T7 RNA polymerase ⁇ e.g. BSR- T7), with: i. a plasmid as described above; and ii.
  • helper plasmids encoding for structural proteins and enzymes of IRPOV and the means for expressing it, in particular said structural proteins and enzymes of IRPOV being under T7 promoter control and contain an IRES element allowing cap-independent translation of IRPOV proteins, to obtain a transfected animal eukaryotic cell; b. co-culturing said transfected eukaryotic animal cell with Vero-NK cells expressing a host cell surface protein, to enable the production of a viral particle, said host cell surface protein being a tumor marker and said tumor marker being the ASCT2; and c. harvesting and purifying said viral particle.
  • the plasmid as described above is employed in transfection experiments along with helper plasmids (encoding structural proteins and enzymes of VSV being under T7 promoter control and contain an IRES element).
  • the T7 RNA polymerase is expressed constitutively from BSR-T7 cells, allowing vaccinia virus-free recovery of VSV.
  • the helper vectors containing an IRES element allows cap-independent translation of VSV proteins in T7 RNA polymerase- expressing cells.
  • BSR-T7 cells at approximately 80% confluency are transfected with 10pg of the plasmid according to the invention, 1-5pg of helper plasmids using Fugene6 (Promega, Germany).
  • the cells are trypsinized, pellet for 10 minutes at 3000 rpm, and overlaid on permissive Vero-NK cells expressing endogenous ASCT2. From 48h of co-culture, green fluorescent and/or syncytia formation iss noticed. Virus-containing supernatant is then harvested, clarified, and stored at -80°C. To produce large amounts of IRPOV, IRPOV according to the invention are incubated on Vero-NK cells at a MOI of 0.00001. Supernatant is harvested at 3 days after infection, filtered using 0.22 pM filter and stored at -80°C. To concentrate the virus, supernatants are ultracentrifuged at 25 000 rpm for 2h at 4°C (Beckman Coulter, USA).
  • a subject matter of the invention concerns the method of production of a viral particle as described above, wherein the animal eukaryotic cell of step a. constitutively expressing the T7 RNA polymerase.
  • a subject matter of the invention concerns the method of production of a viral particle as described above, wherein the animal eukaryotic cell of step a. is a BSR-T7 cell.
  • a subject matter of the invention concerns a viral particle, said viral particle being an IRPOV, the IRPOV genome of which comprises the IRPOV genome as described above.
  • viral particle also named IRPOV, means the complete infectious form of a virus outside of a host cell. It has to be pointed out that said viral particle belongs to a first set of viral particles according to the invention.
  • a subject matter of the invention concerns the viral particle as described above, wherein the retroviral envelope glycoprotein or a fragment thereof encoded by said IRPOV genome and expressed to the surface of said viral particle has a native targeting capacity for a host cell surface protein.
  • a subject matter of the invention concerns the viral particle as described above, wherein the retroviral envelope glycoprotein or a fragment thereof encoded by said IRPOV genome and expressed to the surface of said viral particle has a native targeting capacity for a host cell surface protein, said host cell surface protein being a tumor marker.
  • a subject matter of the invention concerns the viral particle as described above, wherein the retroviral envelope glycoprotein or a fragment thereof encoded by said IRPOV genome and expressed to the surface of said viral particle has a native targeting capacity for a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being chosen among: the “Alanine, Serine, Cysteine Transporter 2” (ASCT2), the “Myelin protein zero-like 1” (MPZL1) and the “ubiquitous vertebrate glucose transporter 1” (GLLIT1).
  • ASCT2 Alanine, Serine, Cysteine Transporter 2
  • MPZL1 Myelin protein zero-like 1
  • GLLIT1 ubiquitous vertebrate glucose transporter 1
  • a subject matter of the invention concerns the viral particle as described above, wherein the retroviral envelope glycoprotein or a fragment thereof encoded by said IRPOV genome and expressed to the surface of said viral particle has a native targeting capacity for a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2” (ASCT2).
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an exogenous retroviral envelope glycoprotein or a fragment thereof.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S-[D/N]-[G/R]-XIGX 2 X3X4X 5 X6X7” (SEQ ID NOs: 131 to 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X 4 D or N
  • ⁇ Xs any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “800X10X2X3X4X5X6X7” (SEQ ID NO: 131) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X 2 V or P
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDRX1GX2X3X4X5X6X7” (SEQ ID NO: 132) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNGXiGX ⁇ X ⁇ sXeX/ (SEQ ID NO: 133) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNRXIGX 2 X3X 4 X5X6X7” (SEQ ID NO: 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ⁇ X3 any amino acid
  • ⁇ X5 any amino acid or no amino acid
  • ⁇ X7 any amino acid.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S-[D/N]-[G/R]-XIGX 2 X 3 X 4 X 5 X6X7” (SEQ ID NOs: 135 to 138) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises:
  • ⁇ Xi G or R or no amino acid
  • ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein
  • ⁇ Xi G or R or no amino acid
  • ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein
  • ⁇ Xi G or R or no amino acid, or
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 135) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • X? R or T or S or G or K.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDRX1GX2X3X4X5X6X7” (SEQ ID NO: 136) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNGX1GX2X3X4X5X6X7” (SEQ ID NO: 137) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNRX1GX2X3X4X5X6X7” (SEQ ID NO: 138) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S-[D/N]-[G/R]-XIGX 2 X3X4X 5 X6X7” (SEQ ID NOs: 139 to 142) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises: - the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 139) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein
  • ⁇ Xi G or R or no amino acid
  • ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein
  • ⁇ Xi G or R or no amino acid, or
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 139) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDRX1GX2X3X4X5X6X7” (SEQ ID NO: 140) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNGX1GX2X3X4X5X6X7” (SEQ ID NO: 141) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNRX1GX2X3X4X5X6X7” (SEQ ID NO: 142) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ Xi G or R or no amino acid
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGPX1DX2X3R” (SEQ ID NO: 4) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ X 2 Q or T or K or A or M or no amino acid
  • ⁇ X3 T or A or V or I.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGX1X2DX3X4X5” (SEQ ID NO: 5) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ X3 Q or T or K
  • ⁇ X 4 A or L or K or T
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGX1X2DX3X4X5” (SEQ ID NO: 6) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • ⁇ X3 Q or T or K
  • ⁇ X4 A or L or K
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGPX1DX2X3R” (SEQ ID NO: 7) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 36 and 145.
  • SEQ ID NOs: 8 to 36 and 145 it means that the sequence chosen sequence can be the sequence SEQ ID NO: 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36 or 145.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 11, 13, 21 to 35 and 145.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 12, 14 to 20 and 36.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 13 and 15.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 11 and 13.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 12 and 15.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-1 , SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1, MMERV-B6, mfuERV, GeERV, SRV-6, SRV-7, SERV-2 and their orthologs.
  • said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1,
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-1 , SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, REV/CSV/SNV/DIAV bis , ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1, SERV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SR
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114, BaEV, ptERV-W, ggERV- W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1, MMERV-B6, mfuERV, GeERV, SERV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, BaEV, ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppER
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: MPMV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, REV/CSV/SNV/DIAV bis and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV and REV/CSV/SNV/DIAV bis , the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 45, 49, 51, 53, 55, 57, 59 and 61, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 46, 50, 52, 54, 56, 58, 60 and 62; and in particular said retroviral envelope
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1 , RD114, MPMV, BaEV and SRV-2, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41 , 43, 45, 47 and 51 , or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48 and 52; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114, BaEV and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114 and BaEV, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43 and 47, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44 and 48; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114 and BaEV, the nucleic acid of which is respectively the sequences SEQ ID NOs:
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: MPMV, SRV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV and SRV-2, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 45 and 51, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 46 and 52; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV and SRV-2, the nucleic acid of which is respectively the sequences SEQ ID NOs: 45 and 51 , or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 46 and 52.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 85, 87, 89, 91 , 93, 95 and 97, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 86, 88, 90, 92, 94, 96 and 98.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which is respectively the sequences SEQ ID NOs: 85, 87, 89, 91, 93, 95 and 97, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 86, 88, 90, 92, 94, 96 and 98.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, and BaEVA18, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 85, 87, 89, 91 and 95, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 86, 88, 90, 92 and 96.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18 and BaEVA18, the nucleic acid of which is respectively the sequences SEQ ID NOs: 85, 87, 89, 91 and 95, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 86, 88, 90, 92 and 96.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: MPMVA23 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 93 and 97, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 94 and 98.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: MPMVA23 and SRV-2A23, the nucleic acid of which is respectively the sequences SEQ ID NOs: 93 and 97, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 94 and 98.
  • a subject matter of the invention concerns the viral particle as described above, wherein the retroviral envelope glycoprotein or a fragment thereof encoded by said IRPOV genome and expressed to the surface of said viral particle has a native targeting capacity for a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “Myelin protein zero-like 1” (MPZL1).
  • MPZL1 Myelin protein zero-like 1
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof when present in the genome of said IRPOV has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof.
  • an endogenous retroviral envelope glycoprotein having a native targeting capacity for the “Myelin protein zero-like 1" (MPZL1) can be Syn-Mab1.
  • the subject matter of the invention can thus concern the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn-Mab1 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof is Syn-Mab1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 146, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 147; and in particular said retroviral envelope glycoprotein or a fragment thereof is Syn-Mab1, the nucleic acid of which is the sequence SEQ ID NO: 146, or the amino acid sequence of which is the sequence SEQ ID NO: 147.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an exogenous retroviral envelope glycoprotein or a fragment thereof.
  • a subject matter of the invention concerns the viral particle as described above, wherein the retroviral envelope glycoprotein or a fragment thereof encoded by said IRPOV genome and expressed to the surface of said viral particle has a native targeting capacity for a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “ubiquitous vertebrate glucose transporter 1” (GLUT1).
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof when present in the genome of said IRPOV has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof.
  • an endogenous retroviral envelope glycoprotein having a native targeting capacity for the ““ubiquitous vertebrate glucose transporter 1” (GLUT1) can be HTLV-1 Env.
  • the subject matter of the invention can thus concern the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: HTLV-1 Env and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof is HTLV-1 Env, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 148, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 149; and in particular said retroviral envelope glycoprotein or a fragment thereof is HTLV-1 Env, the nucleic acid of which is the sequence SEQ ID NO: 148, or the amino acid sequence of which is the sequence SEQ ID NO: 149.
  • a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an exogenous retroviral envelope glycoprotein or a fragment thereof.
  • a subject matter of the invention concerns the viral particle as described above, wherein said viral particle is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • ⁇ a pseudotyped paramyxovirus, in particular said viral particle being a pseudotyped VSV.
  • a subject matter of the invention concerns the viral particle as described above, wherein:
  • - said viral particle is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus
  • NDV Newcastle Disease Virus
  • CDV Canine distemper virus
  • said retroviral envelope glycoprotein or a fragment thereof is a Syncytin-1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syncytin-1 , the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 37, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 38.
  • a subject matter of the invention concerns the viral particle as described above, wherein:
  • - said viral particle is chosen among:
  • VSV Vesicular Stomatitis Virus
  • MV Measles Virus

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Abstract

The invention concerns new oncolytic viruses and their uses In particular, the invention is a viral particle for its use in the prevention and/or the treatment of a tumor, said viral particle comprising: an inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome; and a nucleic acid sequence encoding a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein.

Description

TITLE: ONCOLYTIC VIRUSES AND THEIR USES
CONTEXT OF THE INVENTION
The invention concerns new oncolytic viruses and their uses.
PRIOR ART
Cancer is a leading cause of death worldwile. Over the past several years, in addition to conventional treatment, new generations of cancer therapy have been developed to deal with the limitations of current treatments.
Oncolytic virotherapy has recently emerged as a promising alternative to conventional cancer treatments. The idea is to employ infectious viruses to selectively infect and efficiently kill tumor cells. Oncolytic viruses (OVs) are able to induce cell death via their natural lysis activity on tumor cells as well as via indirect effects, which mediate immune responses directed against the tumor. T-VEC, which is an HSV1-derived OV, demonstrated anticancer potency and has been approved by FDA for the treatment of melanoma (Andtbacka RHI, et al. 2015. Talimogene Laherparepvec Improves Durable Response Rate in Patients With Advanced Melanoma. J Clin Oncol Off J Am Soc Clin Oncol 33:2780-2788).
In parallel, many approaches have been investigated to develop optimized viruses whose tumor specificity and anti-tumor efficiency are promoted. For example, restriction of virus replication in tumor cells can be achieved through genetic engineering to modify receptor recognition.
Despite these investigations, the development of a safe and effective therapeutic tool using optimized oncolytic viruses has not been achieved.
BRIEF SUMMARY OF THE INVENTION
In this context, the inventors have developed an easily adaptable, safe, effective and improved “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV) having a specific tropism for tumor cells and being able to induce cell death via cell-cell fusion in addition of its natural lysis activity, by taking advantage of the capacity of a retroviral envelope glycoprotein or a fragment thereof to target a tumor marker. Consequently, a first subject matter of the invention concerns the use of said retroviral envelope glycoprotein or a fragment thereof which target a tumor marker for producing an IRPOV specifically directed against said tumor marker. Of this subject matter, it follows that the invention also concerns an IRPOV genome encoding said IRPOV. Another subject matter of the invention relates to a plasmid comprising said IRPOV genome and its use for producing a viral particle, said viral particle being said IRPOV. Another subject matter of the invention thus concerns said IRPOV as such or a pharmaceutical composition comprising it and its use as a drug, in particular for its use in the prevention and/or the treatment of a tumor.
DETAILLED DESCRIPTION
In a first aspect, a subject matter of the invention concerns a use of a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein, for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV) specifically directed against said host cell surface protein from an “Infectious and Replicative Oncolytic Virus" (IROV), the native envelope glycoprotein of said IROV being inactivated (resulting in the loss of its natural tropism) in said IRPOV and said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV: ■ is capable of allowing the production of an IRPOV;
■ has retained its native targeting capacity for said host cell surface protein; and
■ has retained its native infectious activity permitting to said IRPOV to entry into a host cell expressing said host cell surface protein.
“Retroviral envelope glycoprotein” means a glycoprotein located on the surface of retroviruses, a type of RNA virus. This protein (Env) is essential for the virus ability to enter and infect host cells. It plays a crucial role in the viral life cycle by mediating the fusion of the viral membrane with the host cell membrane, allowing the virus to enter the host cell. The retroviral Env protein is typically composed of two subunits: Sil (surface glycoprotein) and TM (transmembrane glycoprotein). Sil is responsible for binding to specific receptors on the host cell surface, while TM triggers the fusion process between the viral and host cell membranes together. The interaction between the retroviral envelope protein and host cell receptors is a critical step in retroviral infection and determines the virus tropism, i.e. ability to infect specific cell types.
“Virus tropism” means the capability of a virus to infect and replicate within specific types of cells, tissues, or organisms. It depends on various factors, including the virus surface proteins or receptors, which determine the host cells or organisms it can target.
“Fragment thereof” means a portion or segment of said retroviral envelope glycoprotein. It consists of a sequence of amino acids (or nucleic acids encoding said fragment) and represents only a part of the complete protein structure. These fragments can vary in size and keep the same function as the retroviral envelope glycoprotein from which it comes from.
“Host cell surface protein” means a protein molecule located on the outer surface of a host cell membrane. Host cell surface proteins can serve as receptors for viruses, bacteria, hormones, and other molecules, allowing them to bind to the cell membrane and initiate specific cellular responses such as infection. These proteins are also key targets for therapeutic applications. In particular, said host cell surface protein is a tumor marker.
In another embodiment, a subject matter of the invention thus concerns the use of a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein as described above, said host cell surface protein being a tumor marker, for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV) specifically directed against said host cell surface protein from an “Infectious and Replicative Oncolytic Virus" (IROV), the native envelope glycoprotein of said IROV being inactivated (resulting in the loss of its natural tropism) in said IRPOV and said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV:
■ is capable of allowing the production of an IRPOV;
■ has retained its native targeting capacity for said host cell surface protein; and
■ has retained its native infectious activity permitting to said IRPOV to entry into a host tumor cell expressing said tumor marker.
“Tumor marker”, also known as a biomarker or cancer marker, means a substance or molecule produced by a tumor or by the body in response to the presence of cancer. In the invention, said tumor marker is present on the cell membrane of cancer cells and expressed (whereas absent in normal equivalent cells or expressed in a mutated form with respect to the expression in normal equivalent cells) or overexpressed in tumor cells as compared to normal equivalent cells (e.g. median fold change T/N > 2). For example, tumor marker expression levels can be determined by RT-qPCR analysis of biological samples, or determined by Western Blot analysis of biological samples. These cell membrane-associated tumor markers can thus be used to specifically target host tumor cells for killing them (therapeutic approach). Examples of tumor markers are:
■ the “Alanine, Serine, Cysteine Transporter 2” (ASCT2), also known as Slc1a5, which is a principal transporter of glutamine in cancer cells;
■ “Myelin protein zero-like 1" (MPZL1), a transmembrane glycoprotein that is implicated in extracellular matrix-induced signal transduction and enhances proliferation, migration and invasion of cancer cells; and
■ the “ubiquitous vertebrate glucose transporter 1" (GLUT 1).
In another embodiment, a subject matter of the invention thus concerns the use of a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein as described above, said host cell surface protein being a tumor marker and said tumor marker being chosen among: the “Alanine, Serine, Cysteine Transporter 2” (ASCT2), the “Myelin protein zero-like 1” (MPZL1) and the “ubiquitous vertebrate glucose transporter 1” (GLUT1).
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said IRPOV is devoid of a nucleic acid encoding a retroviral GAG protein.
In another embodiment, a subject matter of the invention relates to the use of a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2” (ASCT2) or the “Myelin protein zero-like 1" (MPZL1), for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV) specifically directed against said host cell surface protein from an “Infectious and Replicative Oncolytic Virus" (IROV), the native envelope glycoprotein of said IROV being inactivated in said IRPOV and said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV:
■ is capable of allowing the production of an IRPOV;
■ has retained its native targeting capacity for said host cell surface protein; and
■ has retained its native infectious activity permitting to said IRPOV to entry into a host cell expressing said host cell surface protein.
In another embodiment, a subject matter of the invention thus concerns the use of a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein as described above, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2” (ASCT2; also named SLC1a5), for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV) specifically directed against said host cell surface protein from an “Infectious and Replicative Oncolytic Virus" (IROV), the native envelope glycoprotein of said IROV being inactivated (resulting in the loss of its natural tropism) in said IRPOV and said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV:
■ is capable of allowing the production of an IRPOV;
■ has retained its native targeting capacity for ASCT2; and
■ has retained its native infectious activity permitting to said IRPOV to entry into a host tumor cell expressing ASCT2.
“Having a native targeting capacity for a host cell surface protein” means that the retroviral envelope glycoprotein of the invention has a natural or inherent ability to specifically interact with and bind to host cell surface protein. This interaction is highly specific. “Oncolytic Virus” means a type of virus that has been genetically modified or naturally evolved to selectively infect and destroy cancer cells while sparing normal, healthy cells.
“Pseudotyped Oncolytic Virus” means virus particle(s) whose native surface glycoproteins have been replaced by those derived from heterologous enveloped viruses to permit virus entry and alter its tropism (or to interrogate the ability of the glycoproteins to permit entry). A Pseudotyped Oncolytic Virus is produced by reverse genetics from a genetically modified genome in which the native envelope is replaced by a heterologous one. Its new host cell surface protein specificity and tropism can be assessed in infection experiments using non permissive cells in culture and host cell surface protein overexpression (see hereafter).
“Infectious Oncolytic Virus” means an oncolytic virus which is able to infect (i.e. to entry into) host (tumor) cells for which said oncolytic virus has a natural or modified tropism.
“Replicative Oncolytic Virus” means an oncolytic virus which is able to replicate, or to reproduce, within host (tumor) cells, leading to the destruction of those host (tumor) cells.
“Infectious and Replicative Oncolytic Virus (IROV)” thus means virus particle(s):
■ the infectious capacity of which is functional; and
■ the replicative capacity of which is functional.
Consequently, said IROV can entry into a host (tumor) cell and allows formation of new (infectious and replicative) virions by the infected host (tumor) cell.
“Infectious, Replicative and Pseudotyped Oncolytic Virus (IRPOV)” thus means virus particle(s):
■ the infectious capacity of which is conserved despite the introduction of a heterologous retroviral envelope glycoprotein or a fragment thereof; and
■ the replicative capacity of which is conserved despite the introduction of a heterologous retroviral envelope glycoprotein or a fragment thereof.
Consequently, said IRPOV can entry into a host (tumor) cell and allows formation of new (infectious, replicative and pseudotyped) virions by the infected host (tumor) cell.
“IRPOV specifically directed against said host cell surface protein” means that the pseudotyping of an IROV to obtain an IRPOV according to invention leads to a viral particle (i.e. IRPOV) having a tropism specifically directed against a host cell surface protein of interest.
“The native envelope glycoprotein of said IROV being inactivated” means that the pseudotyping of an IROV to obtain an IRPOV according to invention involves inactivating the native envelope glycoprotein of said IROV so that it is not expressed or at least not functional.
“Said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV is capable of allowing the production of an IRPOV” means that the retroviral envelope glycoprotein or a fragment thereof used according to the invention allows the production of a functional viral particles.
“Said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV has retained its native targeting capacity for said host cell surface protein” means that the retroviral envelope glycoprotein or a fragment thereof used according to the invention keeps its native targeting capacity for said host cell surface protein (e.g. tumor marker) and thus gives the IRPOV according to the invention a new tropism directed against said host cell surface protein (e.g. tumor marker). This capacity can be evaluated according to classical methods known in the art, wherein the capacity of the retroviral envelope glycoprotein or a fragment thereof at the virion surface to bind to a cell surface protein (its receptor) and promote virus infection is measured (see Examples). For instance, such assays can be performed using VSVAG particules pseudotyped with the retroviral envelope glycoprotein (Ferlin A et al. 2014. Characterization of pH-sensitive molecular switches that trigger the structural transition of vesicular stomatitis virus glycoprotein from the postfusion state toward the prefusion state. J Virol 88:13396-13409) to transduce target cells expressing or not the cell surface protein (its receptor) as illustrated in the examples. Briefly, 2.106 BSR cells seeded in a 6 cm dish are transfected with 2pg of the expression vector for the retroviral envelope glycoprotein using jetPrime® (PolyPlus, France). The following day, transfected cells are infected with VSVAG-GVGFP particles, which contains a gene coding for GFP and have the glycoprotein G on its surface -but not at the genome level- at a MOI (Multiplicity Of Infection) of 3 for 4h. Cells are then washed four times to remove residual virus. Fourty four hours later, cell supernatant containing the particles pseudotyped with retroviral envelope glycoproteins are harvested and added to target cells. Titers are determined by flow cytometry analysis based on the percent of GFP+ cells. The targeting capacity of retroviral envelope glycoproteins was estimated by comparing the titers obtained with target cells overexpressing the receptor with that obtained with target cells expressing low levels of the receptor or laking it (KO cells).
“Said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV has retained its native infectious activity permitting to said IRPOV to entry into a host (tumor) cell” means that the retroviral envelope glycoprotein or a fragment thereof used according to the invention keeps its native infectious activity and thus gives the IRPOV according to the invention the capacity to infect host (tumor) cell. This infectious activity can be evaluated through infectious assays wherein the capacity of the retroviral envelope glycoprotein or a fragment thereof at the virion surface to bind to a cell surface protein (its receptor) and promote virus infection is measured (see Examples). For instance, such infectious assays can be performed using VSVAG particules pseudotyped with the retroviral envelope glycoprotein (Ferlin A et al. 2014. Characterization of pH-sensitive molecular switches that trigger the structural transition of vesicular stomatitis virus glycoprotein from the postfusion state toward the prefusion state. J Virol 88:13396-13409) to transduce target cells expressing the cell surface protein (its receptor) as illustrated in the examples. Briefly, 2.106 BSR cells seeded in a 6 cm dish are transfected with 2pg of the expression vector for the retroviral envelope glycoprotein using jetPrime® (PolyPlus, France). The following day, transfected cells are infected with VSVAG-G*/GFP particles, which contains a gene coding for GFP and have the glycoprotein G on its surface -but not at the genome level- at a MOI (Multiplicity Of Infection) of 3 for 4h. Cells are then washed four times to remove residual virus. Fourty four hours later, cell supernatant containing the particles pseudotyped with retroviral envelope glycoproteins are harvested and added to target cells expressing the receptor. Titers are determined by flow cytometry analysis based on the percent of GFP+ cells.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof when present in the genome of said IRPOV has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium.
“Said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium” means that the retroviral envelope glycoprotein or a fragment thereof used according to the invention keeps its native fusiogenic activity. Said fusogenic activity refers to the capacity of said retroviral envelope glycoprotein or a fragment thereof to fuse the plasma membranes of two or more adjacent cells leading to the formation of a large multinucleated cell, called syncytium. Fusogenic properties of a given retroviral envelope glycoprotein may be assessed in fusion assays based on alpha- complementation of the beta galactosidase, as described in Bacquin et al. (Cell Fusion- Based Screening Method Identifies Glycosylphosphatidylinositol-Anchored Protein Ly6e as the Receptor for Mouse Endogenous Retroviral Envelope Syncytin-A. J Virol. 2017 Aug 24;91 (18):e00832-17.). Briefly, a first group of cells are transfected, for example by using Lipofectamine (Invitrogen), with vectors encoding the beta-galactosidase fragment Gal-alpha, and a second group of cells are cotransfected with vectors encoding the beta-galactosidase fragment Gal-omega and a retroviral envelope glycoprotein at a ratio of 1 :1. At 4 to 24 h after plasmid transfection, the two groups of cells were cocultured at a ratio of 1 :1. At 16 to 48 h after cell mixing, cells were fixed and stained with X-Gal to identify, by microscopy analysis, blue syncytia formed following fusion of cells from both groups. The number and mean area of syncytia are analyzed in randomly chosen fields by Imaged.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail. It has to be pointed out that said modification, which encompasses partial or total truncation of the intracytoplasmic tail of said retroviral envelope glycoprotein or a fragment thereof, advantageously allows to enhance the cell-cell fusion activity and/or to improve the production of the IRPOV.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof.
“Endogenous retroviral envelope glycoprotein” means that the retroviral envelope glycoprotein or a fragment thereof used according to the invention comes from an endogenous retrovirus. Endogenous retroviruses (ERVs) are a class of retroviruses that have integrated their genetic material into the genome of an organism. Unlike exogenous retroviruses, which are external and can infect host cells from outside the body, endogenous retroviruses are inherited from generation to generation as part of the host DNA. For instance, an endogenous retroviral envelope glycoprotein having a native targeting capacity for the “Alanine, Serine, Cysteine Transporter?" (ASCT2) can be chosen among: Syncytin-1 , Syncytin-Ory1 , Dasy Env1.1 , RD114, BaEV, ptERV-W, ggERV-W, paERV-W(3), paERV- W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1, MMERV-B6, mfuERV, GeERV and SERV-2.
As mentioned, exogenous retroviruses exist, which are a type of retrovirus that are found outside of the host organism genome. Unlike endogenous retroviruses (ERVs), which have integrated their genetic material into the host DNA over evolutionary time, exogenous retroviruses are separated and independent entities that can infect host cells from outside the host body. In another embodiment, a subject matter of the invention concerns thus the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an exogenous retroviral envelope glycoprotein or a fragment thereof. For instance, an exogenous retroviral envelope glycoprotein having a native targeting capacity for the “Alanine, Serine, Cysteine Transporter 2" (ASCT2) can be chosen among: MPMV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, SRV-6 and SRV-7.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S-[D/N]-[G/R]- X1GX2X3X4X5X6X7” (SEQ ID NOs: 131 to 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid, ■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises:
- the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 131) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid;
- the conserved “SDRXIGX2X3X4X5X6X7” (SEQ ID NO: 132) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid;
- the conserved “SNGXIGX2X3X4X5X6X7” (SEQ ID NO: 133) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid; or
- the conserved “SNRXIGX2X3X4X5X6X7” (SEQ ID NO: 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 131) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDRX1GX2X3X4X5X6X7” (SEQ ID NO: 132) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNGXIGX2X3X4X5X6X7” (SEQ ID NO: 133) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNRX1GX2X3X4X5X6X7” (SEQ ID NO: 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S-[D/N]-[G/R]- XIGX2X3X4XSX6X7” (SEQ ID NOs: 135 to 138) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000010_0001
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises:
- the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 135) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
- th
Figure imgf000010_0002
O: 136) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
- th
Figure imgf000010_0003
O: 137) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000010_0004
or
- the conserved “SNRXIGX2X3X4X5X6X7” (SEQ ID NO: 138) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000010_0005
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 135) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000011_0001
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDRX1GX2X3X4X5X6X7” (SEQ ID NO: 136) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000011_0002
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNGXIGX2X3X4X5X6X7” (SEQ ID NO: 137) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000011_0003
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNRX1GX2X3X4X5X6X7” (SEQ ID NO: 138) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000011_0004
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S-[D/N]-[G/R]- XIGX2X3X4XSX6X7” (SEQ ID NOs: 139 to 142) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000012_0001
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises:
- the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 139) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
- th
Figure imgf000012_0004
140) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
- th
Figure imgf000012_0005
141) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000012_0002
or
- the conserved “SNRXIGX2X3X4X5X6X7” (SEQ ID NO: 142) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000012_0003
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 139) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000013_0001
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDRX1GX2X3X4X5X6X7” (SEQ ID NO: 140) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000013_0002
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNGXIGX2X3X4X5X6X7” (SEQ ID NO: 141) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000013_0003
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNRX1GX2X3X4X5X6X7” (SEQ ID NO: 142) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000013_0004
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGPX1DX2X3R” (SEQ ID NO: 4) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = L or Q or T, X2 = Q or T or K or A or M or no amino acid, and X3 = T or A or V or I.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGX1X2DX3X4X5” (SEQ ID NO: 5) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = V or P,
■ X2 = Q or L,
■ X3 = Q or T or K,
■ X4 = A or L or K or T, and
■ X5 = R or T.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGX1X2DX3X4X5” (SEQ ID NO: 6) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = V or P,
■ X2 = Q or L,
■ X3 = Q or T or K,
■ X4 = A or L or K, and
■ X5 = R or T.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGPX1DX2X3R” (SEQ ID NO: 7) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = Q or L,
■ X2 = T or K, and
■ X3 = T or A.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 36 and 145. By “SEQ ID NOs: 8 to 36 and 145”, it means that the sequence chosen sequence can be the sequence SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36 or 145.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 11, 13, 21 to 35 and 145.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 12, 14 to 20 and 36. In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 13 and 15.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 11 and 13.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 12 and 15.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppERV- W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1, MMERV-B6, mfuERV, GeERV, SRV-6, SRV-7, SERV-2 and their orthologs.
By “their orthologs”, it means that the retroviral envelope glycoproteins or a fragment thereof found in different species that originated from a common ancestral gene through speciation which lead to the above listed retroviral envelope glycoproteins. These retroviral envelope glycoproteins or a fragment thereof typically share a similar function and have evolved to perform equivalent roles in their respective organisms.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-1 , SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, REV/CSV/SNV/DIAVbis, ptERV-W, ggERV-W, paERV-W(3), paERV- W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1 , TvERV, DrERV, CfERV-Fc1 , SERV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, REV/CSV/SNV/DIAVbis, ptERV-W, ggERV-W, paERV- W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1 and SERV-2, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41 , 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83 and 143, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 and 144; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, REV/CSV/SNV/DIAVbis, ptERV-W, ggERV-W, paERV- W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1 and SERV-2, the nucleic acid of which is respectively the sequences SEQ ID NOs: 37, 39, 41 , 43, 45, 47,
49, 51 , 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81 , 83 and 143, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48,
50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 and 144.
By “% of identity”, it means that the percentage determined by direct comparison of two oligonucleotide sequences (nucleic acid sequence), by determining the number of identical nucleotides between the two sequences, then dividing it by the number of nucleotides of the longer of the two sequences, and multiplying the result by 100. By "has at least 90% of identity" is therefore meant that the aforementioned percentage of identity is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or is 100%. In this respect, it should be noted that this definition applies to all embodiments of the invention, including when a direct comparison of two polypeptide sequences (amino acids sequence) is involved. In addition, it is understood that sequences having at least 90% of identity with a reference sequence conserved the same activity and the same function has said reference sequence, in particular said activity and said function can be improved.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, BaEV, ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1, MMERV-B6, mfuERV, GeERV, SERV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, BaEV, ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1 and SERV-2, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41 , 43, 47, 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81, 83 and 143, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 48, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 and 144; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, BaEV, ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1 and SERV-2, the nucleic acid of which is respectively the sequences SEQ ID NOs: 37, 39, 41 , 43, 47, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 and 143, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 38, 40, 42, 44, 48, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 and 144.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: MPMV, SRV-1, SRV-2, SRV- 4, SRV-5, SMRV, REV/CSV/SNV/DIAV, REV/CSV/SNV/DIAVbis and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV and REV/CSV/SNV/DIAVbis, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 45, 49, 51, 53, 55, 57, 59 and 61, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 46, 50, 52, 54, 56, 58, 60 and 62; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV and REV/CSV/SNV/DIAVbis, the nucleic acid of which is respectively the sequences SEQ ID NOs: 45, 49, 51 , 53, 55, 57, 59 and 61 , or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 46, 50, 52, 54, 56, 58, 60 and 62.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1 , RD114, MPMV, BaEV, SRV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1 , RD114, MPMV, BaEV and SRV-2, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47 and 51 , or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48 and 52; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1 , RD114, MPMV, BaEV and SRV-2, the nucleic acid of which is respectively the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47 and 51 , or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48 and 52.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, BaEV and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114 and BaEV, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43 and 47, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44 and 48; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114 and BaEV, the nucleic acid of which is respectively the sequences SEQ ID NOs: 37, 39, 41 , 43 and 47, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 38, 40, 42, 44 and 48.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: MPMV, SRV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV and SRV-2, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 45 and 51, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 46 and 52; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV and SRV-2, the nucleic acid of which is respectively the sequences SEQ ID NOs: 45 and 51 , or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 46 and 52.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 85, 87, 89, 91 , 93, 95 and 97, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 86, 88, 90, 92, 94, 96 and 98.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which is respectively the sequences SEQ ID NOs: 85, 87, 89, 91, 93, 95 and 97, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 86, 88, 90, 92, 94, 96 and 98.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, and BaEVA18, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 85, 87, 89, 91 and 95, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 86, 88, 90, 92 and 96.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18 and BaEVA18, the nucleic acid of which is respectively the sequences SEQ ID NOs: 85, 87, 89, 91 and 95, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 86, 88, 90, 92 and 96.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: MPMVA23 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 93 and 97, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 94 and 98.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: MPMVA23 and SRV-2A23, the nucleic acid of which is respectively the sequences SEQ ID NOs: 93 and 97, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 94 and 98.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein as described above, said host cell surface protein being a tumor marker and said tumor marker being the “Myelin protein zero-like 1” (MPZL1), for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV) specifically directed against said host cell surface protein from an “Infectious and Replicative Oncolytic Virus" (IROV), the native envelope glycoprotein of said IROV being inactivated (resulting in the loss of its natural tropism) in said IRPOV and said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV:
■ is capable of allowing the production of an IRPOV;
■ has retained its native targeting capacity for MPZL1 ; and
■ has retained its native infectious activity permitting to said IRPOV to entry into a host tumor cell expressing MPZL1.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof when present in the genome of said IRPOV has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof. For instance, an endogenous retroviral envelope glycoprotein having a native targeting capacity for the “Myelin protein zero-like 1" (MPZL1) can be Syn-Mab1. The subject matter of the invention can thus concern the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn-Mab1 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof is Syn-Mab1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 146, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 147; and in particular said retroviral envelope glycoprotein or a fragment thereof is Syn-Mab1, the nucleic acid of which is the sequence SEQ ID NO: 146, or the amino acid sequence of which is the sequence SEQ ID NO: 147.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an exogenous retroviral envelope glycoprotein or a fragment thereof.
In another embodiment, a subject matter of the invention thus concerns the use of a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein as described above, said host cell surface protein being a tumor marker and said tumor marker being the “ubiquitous vertebrate glucose transporter 1” (GLUT1), for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV) specifically directed against said host cell surface protein from an “Infectious and Replicative Oncolytic Virus" (IROV), the native envelope glycoprotein of said IROV being inactivated (resulting in the loss of its natural tropism) in said IRPOV and said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV:
■ is capable of allowing the production of an IRPOV;
■ has retained its native targeting capacity for GLUT 1 ; and has retained its native infectious activity permitting to said IRPOV to entry into a host tumor cell expressing GLUT 1.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said IRPOV specifically directed against GLUT1 is devoid of a nucleic acid encoding a retroviral GAG protein.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof when present in the genome of said IRPOV has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof. For instance, an endogenous retroviral envelope glycoprotein having a native targeting capacity for the ““ubiquitous vertebrate glucose transporter 1" (GLUT1) can be HTLV-1 Env. The subject matter of the invention can thus concern the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: HTLV-1 Env and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof is HTLV-1 Env, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 148, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 149; and in particular said retroviral envelope glycoprotein or a fragment thereof is HTLV-1 Env, the nucleic acid of which is the sequence SEQ ID NO: 148, or the amino acid sequence of which is the sequence SEQ ID NO: 149.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an exogenous retroviral envelope glycoprotein or a fragment thereof.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said native envelope glycoprotein of said IROV is inactivated by addition, deletion or substitution of at least one nucleotide of the nucleic acid sequence encoding it, in particular said native envelope glycoprotein of said IROV being inactivated by substitution of said nucleic acid sequence encoding it by the nucleic acid sequence encoding said retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein.
By “inactivated by addition, deletion or substitution of at least one nucleotide of the nucleic acid sequence encoding it”, it means that the nucleic acid sequence encoding the native envelope glycoprotein of said IROV is mutated in order to cancel its expression or at least lead to a protein nonfunctional. This can be achieved by addition, deletion or substitution of at least one nucleotide (at least 2, at least 3, at least 4 or at least 5 nucleotides) in the nucleic acid sequence encoding it, which can result, for instance, in creating an early stop codon. Another solution can be to completely substitute or replace said nucleic acid sequence by another one (e.g. the one coding for the retroviral envelope glycoprotein of the invention or a fragment thereof). It has to be pointed out that said inactivation results in the loss of the natural tropism of said IROV, which in the IRPOV becomes consequently the tropism of the retroviral envelope glycoprotein or a fragment thereof introduced by the pseudotyping.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said native envelope glycoprotein of said IROV is inactivated by substitution of said nucleic acid sequence encoding it by the nucleic acid sequence encoding said retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said IROV is chosen among:
■ a Vesicular Stomatitis Virus (VSV);
■ a Measles Virus (MV);
■ a Sendai Virus (SeV);
■ a Newcastle Disease Virus (NDV);
■ a Canine distemper virus (CDV); and
■ a paramyxovirus, in particular said IROV being a VSV.
“Vesicular Stomatitis Virus (VSV)” is a member of the Rhabdoviridae family. The VSV genome is a single molecule of negative - sense RNA that encodes five major polypeptides: a nucleocapsid (N) polypeptide, a phosphoprotein (P) polypeptide, a matrix (M) polypeptide, a glycoprotein (G) polypeptide, and a viral polymerase (L) polypeptide. In particular, it has to be pointed out that the subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said IROV is a VSV. This leads to the production of a pseudotyped VSV, the nucleic acid molecule of which encodes a VSV-N polypeptide, a VSV-P polypeptide, a VSV-M polypeptide, the retroviral envelope glycoprotein of the invention or a fragment thereof, optionally a marker polypeptide (lacZ, or a gene coding the sodium iodide transporter, or a gene coding for fluorescent protein GFP, RFP...) and a VSV-L polypeptide. Here, the native VSV-G polypeptide is inactivated and the retroviral envelope glycoprotein of the invention or a fragment thereof is introduced. Interestingly, the VSV-M polypeptide can comprise mutations which reduce the cytopathogenicity of the VSV. Examples of mutations are amino acid substitutions M51A or M51R, V221 F, and S226R or deletions Mdelta51.
“Measles Virus (MV)” is a member of the paramyxovirus family. The MV genome is composed of the nucleocapsid (N) polypeptide, a phosphoprotein (P) polypeptide, a matrix (M) polypeptide, a fusion (F) polypeptide, a hemagglutinin attachment (H) polypeptide and a viral polymerase (L) polypeptide. In particular, it has to be pointed out that the subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said IROV is a MV. This leads to the production of a pseudotyped MV, the nucleic acid molecule of which encodes a nucleocapsid (N) polypeptide, a phosphoprotein (P) polypeptide, a matrix (M) polypeptide and the retroviral envelope glycoprotein of the invention or a fragment thereof. A marker polypeptide (lacZ, or a gene coding the sodium iodide transporter, or a gene coding for fluorescent protein GFP, RFP...) can be also inserted between the N-P, the P-M or the Env-L genes. Here, the native MV-F and/or MV-H polypeptides are inactivated and the retroviral envelope glycoprotein of the invention or a fragment thereof is introduced. “Sendai Virus (SeV)”, also known as murine parainfluenza virus type 1 (mPIV-1), is a paramyxovirus belonging to the Paramyxoviridae family. The SeV genome is a single molecule of negative - sense RNA that encodes several polypeptides: a nucleoprotein (NP), a phosphoprotein (P), a matrix protein (M), a fusion protein (F), a hemagglutininneuraminidase protein (HN), and a large polymerase protein (L). In particular, it has to be pointed out that the subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said IROV is a SeV. This leads to the production of a pseudotyped SeV, the nucleic acid molecule of which encodes a SeV-NP polypeptide, a SeV-P polypeptide, a SeV-M polypeptide, the retroviral envelope glycoprotein of the invention or a fragment thereof and a SeV-L polypeptide. A marker polypeptide (lacZ, or a gene coding the sodium iodide transporter, or a gene coding for fluorescent protein GFP, RFP...) can be also inserted between the N-P, the P-M or the Env-L genes Here, the native SeV-F and/or SeV-HN polypeptides are inactivated and the retroviral envelope glycoprotein of the invention or a fragment thereof is introduced.
“Newcastle Disease Virus (NDV)”, also known as avian paramyxovirus serotype 1 (APMV-1), is a paramyxovirus belonging to the Paramyxoviridae family. The NDV genome is a single molecule of negative - sense RNA that encodes several polypeptides: a nucleoprotein (NP), a phosphoprotein (P), a matrix protein (M), a fusion protein (F), a hemagglutininneuraminidase protein (HN), and a large polymerase protein (L). In particular, it has to be pointed out that the subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said IROV is a NDV. This leads to the production of a pseudotyped NDV, the nucleic acid molecule of which encodes a NDV-NP polypeptide, a NDV-P polypeptide, a NDV-M polypeptide, the retroviral envelope glycoprotein of the invention or a fragment thereof and a NDV-L polypeptide. A marker polypeptide (lacZ, or a gene coding the sodium iodide transporter, or a gene coding for fluorescent protein GFP, RFP...) can be also inserted between the N-P, the P-M or the Env-L genes Here, the native NDV-F and/or NDV-HN polypeptides are inactivated and the retroviral envelope glycoprotein of the invention or a fragment thereof is introduced.
“Canine distemper virus (CDV)” is a member of the paramyxovirus family. The CDV genome is composed of the nucleocapsid (N) polypeptide, a phosphoprotein (P) polypeptide, a matrix (M) polypeptide, a fusion (F) polypeptide, a hemagglutinin attachment (H) polypeptide and a viral polymerase (L) polypeptide. In particular, it has to be pointed out that the subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said IROV is a CDV. This leads to the production of a pseudotyped CDV, the nucleic acid molecule of which encodes a CDV-N polypeptide, a CDV-P polypeptide, a CDV-M polypeptide, the retroviral envelope glycoprotein of the invention or a fragment thereof and a CDV-L polypeptide. A marker polypeptide (lacZ, or a gene coding the sodium iodide transporter, or a gene coding for fluorescent protein GFP, RFP...) can be also inserted between the N-P, the P-M or the Env-L genes. Here, the native CDV-F and/or CDV-H polypeptides are inactivated and the retroviral envelope glycoprotein of the invention or a fragment thereof is introduced.
“Paramyxovirus” means a member of the paramyxovirus family. The paramyxovirus genome is composed of the nucleocapsid (N) polypeptide, a phosphoprotein (P) polypeptide, a matrix (M) polypeptide, a fusion (F) polypeptide, a hemagglutinin attachment (H) polypeptide and a viral polymerase (L) polypeptide. In particular, it has to be pointed out that the subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein said IROV is a CDV. This leads to the production of a pseudotyped paramyxovirus, the nucleic acid molecule of which encodes a N polypeptide, a P polypeptide, a M polypeptide, the retroviral envelope glycoprotein of the invention or a fragment thereof and a L polypeptide. A marker polypeptide (lacZ, or a gene coding the sodium iodide transporter, or a gene coding for fluorescent protein GFP, RFP...) can be also inserted between the N-P, the P-M or the Env-L genes. Here, the native F and/or H polypeptides are inactivated and the retroviral envelope glycoprotein of the invention or a fragment thereof is introduced.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said I ROV is chosen among:
■ a Vesicular Stomatitis Virus (VSV);
■ a Measles Virus (MV);
■ a Sendai Virus (SeV);
■ a Newcastle Disease Virus (NDV);
■ a Canine distemper virus (CDV); and
■ a paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a Syncytin-1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syncytin-1 , the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 37, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 38.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said I ROV is chosen among:
■ a Vesicular Stomatitis Virus (VSV);
■ a Measles Virus (MV);
■ a Sendai Virus (SeV);
■ a Newcastle Disease Virus (NDV);
■ a Canine distemper virus (CDV); and
■ a paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a Syncytin-Ory1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syncytin-Ory1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 39, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 40.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said I ROV is chosen among:
■ a Vesicular Stomatitis Virus (VSV);
■ a Measles Virus (MV);
■ a Sendai Virus (SeV);
■ a Newcastle Disease Virus (NDV);
■ a Canine distemper virus (CDV); and
■ a paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a Dasy Env1.1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Dasy Env1.1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 41 , or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 42.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said I ROV is chosen among:
■ a Vesicular Stomatitis Virus (VSV); ■ a Measles Virus (MV);
■ a Sendai Virus (SeV);
■ a Newcastle Disease Virus (NDV);
■ a Canine distemper virus (CDV); and
■ a paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a RD114 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a RD114, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 43, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 44.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said I ROV is chosen among:
■ a Vesicular Stomatitis Virus (VSV);
■ a Measles Virus (MV);
■ a Sendai Virus (SeV);
■ a Newcastle Disease Virus (NDV);
■ a Canine distemper virus (CDV); and
■ a paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a MPMV or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a MPMV, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 45, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 46.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said I ROV is chosen among:
■ a Vesicular Stomatitis Virus (VSV);
■ a Measles Virus (MV);
■ a Sendai Virus (SeV);
■ a Newcastle Disease Virus (NDV);
■ a Canine distemper virus (CDV); and
■ a paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a BaEV or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a BaEV, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 47, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 48.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said I ROV is chosen among:
■ a Vesicular Stomatitis Virus (VSV);
■ a Measles Virus (MV);
■ a Sendai Virus (SeV);
■ a Newcastle Disease Virus (NDV);
■ a Canine distemper virus (CDV); and
■ a paramyxovirus, and - said retroviral envelope glycoprotein or a fragment thereof is a SRV-2 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a SRV-2, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 51, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 52.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said I ROV is chosen among:
■ a Vesicular Stomatitis Virus (VSV);
■ a Measles Virus (MV);
■ a Sendai Virus (SeV);
■ a Newcastle Disease Virus (NDV);
■ a Canine distemper virus (CDV); and
■ a paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a Syn1A53 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syn1A53, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 85, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 86.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said I ROV is chosen among:
■ a Vesicular Stomatitis Virus (VSV);
■ a Measles Virus (MV);
■ a Sendai Virus (SeV);
■ a Newcastle Disease Virus (NDV);
■ a Canine distemper virus (CDV); and
■ a paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a SynOry1A25 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a SynOry1A25, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 87, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 88.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said I ROV is chosen among:
■ a Vesicular Stomatitis Virus (VSV);
■ a Measles Virus (MV);
■ a Sendai Virus (SeV);
■ a Newcastle Disease Virus (NDV);
■ a Canine distemper virus (CDV); and
■ a paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a DasyEnvA28 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a DasyEnvA28, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 89, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 90. In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said I ROV is chosen among:
■ a Vesicular Stomatitis Virus (VSV);
■ a Measles Virus (MV);
■ a Sendai Virus (SeV);
■ a Newcastle Disease Virus (NDV);
■ a Canine distemper virus (CDV); and
■ a paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a RD114A18 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a RD114A18, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 91 , or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 92.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said I ROV is chosen among:
■ a Vesicular Stomatitis Virus (VSV);
■ a Measles Virus (MV);
■ a Sendai Virus (SeV);
■ a Newcastle Disease Virus (NDV);
■ a Canine distemper virus (CDV); and
■ a paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a MPMVA23 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a MPMVA23, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 93, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 94.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said I ROV is chosen among:
■ a Vesicular Stomatitis Virus (VSV);
■ a Measles Virus (MV);
■ a Sendai Virus (SeV);
■ a Newcastle Disease Virus (NDV);
■ a Canine distemper virus (CDV); and
■ a paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a BaEVA18 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a BaEVA18, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 95, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 96.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said I ROV is chosen among:
■ a Vesicular Stomatitis Virus (VSV);
■ a Measles Virus (MV);
■ a Sendai Virus (SeV);
■ a Newcastle Disease Virus (NDV); a Canine distemper virus (CDV); and a paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a SRV-2A23 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a SRV-2A23, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 97, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 98.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said I ROV is chosen among:
■ a Vesicular Stomatitis Virus (VSV);
■ a Measles Virus (MV);
■ a Sendai Virus (SeV);
■ a Newcastle Disease Virus (NDV);
■ a Canine distemper virus (CDV); and
■ a paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a Syn-Mab1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syn-Mab1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 146, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 147.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said I ROV is chosen among:
■ a Vesicular Stomatitis Virus (VSV);
■ a Measles Virus (MV);
■ a Sendai Virus (SeV);
■ a Newcastle Disease Virus (NDV);
■ a Canine distemper virus (CDV); and
■ a paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a HTLV-1 Env or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a HTLV-1 Env, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 148, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 149.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said I ROV is a Vesicular Stomatitis Virus (VSV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95 and 97 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 52, 85, 87, 89, 91, 93, 95 and 97. In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said IROV is a Measles Virus (MV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95 and 97 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 52, 85, 87, 89, 91, 93, 95 and 97.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said IROV is a Sendai Virus (SeV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95 and 97 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 52, 85, 87, 89, 91, 93, 95 and 97.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said IROV is a Newcastle Disease Virus (NDV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95 and 97 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 52, 85, 87, 89, 91, 93, 95 and 97.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said IROV is a Canine distemper virus (CDV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95 and 97 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 52, 85, 87, 89, 91, 93, 95 and 97.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said IROV is a paramyxovirus; and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95 and 97 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 52, 85, 87, 89, 91, 93, 95 and 97.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said IROV is a Vesicular Stomatitis Virus (VSV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said IROV is a Measles Virus (MV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said IROV is a Sendai Virus (SeV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said IROV is a Newcastle Disease Virus (NDV); and - said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said IROV is a Canine distemper virus (CDV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
In another embodiment, a subject matter of the invention concerns the use of a retroviral envelope glycoprotein or a fragment thereof as described above, wherein:
- said IROV is a paramyxovirus; and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
In a second aspect, a subject matter of the invention concerns an IRPOV genome encoding an IRPOV specifically directed against a host cell surface protein, said IRPOV genome comprising an IROV genome comprising:
■ an inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome (resulting in the loss of its natural tropism); and
■ a nucleic acid sequence encoding a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein, said retroviral envelope glycoprotein or a fragment thereof:
- being capable of allowing the production of an IRPOV;
- having retained its native targeting capacity for said host cell surface protein; and
- having retained its native infectious activity permitting to said IRPOV to entry into a host cell expressing said host cell surface protein.
“IRPOV genome” means that the nucleic acid molecule encodes and allows the production of the viral particle according to the invention (i.e. the IRPOV according to the invention). Said IRPOV genome is obtained by means of classic cloning techniques, which use restriction enzymes, plasmids, PCR amplification, etc. For instance, for cloning into the IROV genome, said nucleic acid sequence encoding said retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein is amplified by PCR and cloned into the plasmid comprising the IROV genome at the place of the native native envelope glycoprotein of the IROV via, e.g. Mlul/Avrll, restriction sites.
“IROV genome” means that the nucleic acid molecule that encodes the virus from which the IRPOV genome according to the invention is obtained by pseudotyping. In another embodiment, a subject matter of the invention concerns the IRPOV genome encoding an IRPOV specifically directed against a host cell surface protein as described above, said host cell surface protein being a tumor marker, said IRPOV genome comprising an IROV genome comprising:
■ an inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome (resulting in the loss of its natural tropism); and
■ a nucleic acid sequence encoding a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein, said retroviral envelope glycoprotein or a fragment thereof:
- being capable of allowing the production of an IRPOV;
- having retained its native targeting capacity for said host cell surface protein; and
- having retained its native infectious activity permitting to said IRPOV to entry into a host tumor cell expressing said tumor marker.
In another embodiment, a subject matter of the invention thus concerns the IRPOV genome encoding an IRPOV specifically directed against a host cell surface protein as described above, said host cell surface protein being a tumor marker and said tumor marker being chosen among: the “Alanine, Serine, Cysteine Transporter 2” (ASCT2), the “Myelin protein zero-like 1” (MPZL1) and the “ubiquitous vertebrate glucose transporter 1” (GLLIT1).
In another embodiment, a subject matter of the invention relates to an IRPOV genome encoding an IRPOV specifically directed against a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2” (ASCT2) or the “Myelin protein zero-like 1" (MPZL1), said IRPOV genome comprising an IROV genome comprising:
■ an inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome; and
■ a nucleic acid sequence encoding a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein, said retroviral envelope glycoprotein or a fragment thereof:
- being capable of allowing the production of an IRPOV;
- having retained its native targeting capacity for said host cell surface protein; and
- having retained its native infectious activity permitting to said IRPOV to entry into a host cell expressing said host cell surface protein.
In another embodiment, a subject matter of the invention concerns the IRPOV genome encoding an IRPOV specifically directed against a host cell surface protein as described above, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2” (ASCT2), said IRPOV genome comprising an IROV genome comprising:
■ an inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome (resulting in the loss of its natural tropism); and
■ a nucleic acid sequence encoding a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein, said retroviral envelope glycoprotein or a fragment thereof:
- being capable of allowing the production of an IRPOV;
- having retained its native targeting capacity for ASCT2; and having retained its native infectious activity permitting to said IRPOV to entry into a host tumor cell expressing ASCT2.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an exogenous retroviral envelope glycoprotein or a fragment thereof.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above wherein said retroviral envelope glycoprotein or a fragment thereof has a native targeting capacity for a tumor marker being chosen among: the “Alanine, Serine, Cysteine Transporter 2" (ASCT2), the “Myelin protein zero-like 1" (MPZL1) and the “ubiquitous vertebrate glucose transporter 1" (GLLIT1), in particular said tumor marker being the “Alanine, Serine, Cysteine Transporter?" (ASCT2) and said retroviral envelope glycoprotein or a fragment thereof comprising the conserved “S- [D/N]-[G/R]-XIGX2X3X4X5X6X7” (SEQ ID NOS: 131 to 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S-[D/N]-[G/R]-XIGX2X3X4X5X6X7” (SEQ ID NOs: 131 to 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises: - the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 131) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid;
- the conserved “SDRX1GX2X3X4X5X6X7” (SEQ ID NO: 132) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid;
- the conserved “SNGX1GX2X3X4X5X6X7” (SEQ ID NO: 133) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid; or
- the conserved “SNRX1GX2X3X4X5X6X7” (SEQ ID NO: 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 131) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid. In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDRX1GX2X3X4X5X6X7” (SEQ ID NO: 132) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNGX1GX2X3X4X5X6X7” (SEQ ID NO: 133) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNRX1GX2X3X4X5X6X7” (SEQ ID NO: 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S-[D/N]-[G/R]-XIGX2X3X4X5X6X7” (SEQ ID NOs: 135 to 138) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000034_0001
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises:
- the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 135) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein: ■ Xi = G or R or no amino acid,
- th
Figure imgf000035_0001
O: 136) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
- th
Figure imgf000035_0002
O: 137) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000035_0003
or
- the conserved “SNRXIGX2X3X4X5X6X7” (SEQ ID NO: 138) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000035_0004
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S 00X10X2X3X4X5X6X7” (SEQ ID NO: 135) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000035_0005
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDRXIGX2X3X4X5X6X7” (SEQ ID NO: 136) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein: ■ Xi = G or R or no amino acid,
Figure imgf000036_0002
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNGX1GX2X3X4X5X6X7” (SEQ ID NO: 137) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000036_0003
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNRXIGX2X3X4X5X6X7” (SEQ ID NO: 138) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000036_0004
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S-[D/N]-[G/R]-XIGX2X3X4X5X6X7” (SEQ ID NOs: 139 to 142) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000036_0001
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises:
- the conserved “SDGXIGX2X3X4X5X6X7” (SEQ ID NO: 139) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = Q or L or T or I,
■ X4 = D or N, ■ Xs = Q or T or K or E,
■ Xs = A or L or K or V or I or R or E, and
■ X? = R or T or S or G or K;
- the conserved “SDRX1GX2X3X4X5X6X7” (SEQ ID NO: 140) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
- th
Figure imgf000037_0004
141) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000037_0001
or
- the conserved “SNRX1GX2X3X4X5X6X7” (SEQ ID NO: 142) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000037_0002
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 139) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000037_0003
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDRX1GX2X3X4X5X6X7” (SEQ ID NO: 140) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = Q or L or T or I,
■ X4 = D or N,
Figure imgf000038_0001
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNGX1GX2X3X4X5X6X7” (SEQ ID NO: 141) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000038_0002
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNRX1GX2X3X4X5X6X7” (SEQ ID NO: 142) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000038_0003
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGPX1DX2X3R” (SEQ ID NO: 4) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = L or Q or T,
■ X2 = Q or T or K or A or M or no amino acid, and
■ X3 = T or A or V or I.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGX1X2DX3X4X5” (SEQ ID NO: 5) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = V or P,
■ X2 = Q or L,
■ X3 = Q or T or K,
■ X4 = A or L or K or T, and
■ X5 = R or T.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGX1X2DX3X4X5” (SEQ ID NO: 6) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = V or P,
■ X2 = Q or L,
■ X3 = Q or T or K,
■ X4 = A or L or K, and
■ X5 = R or T. In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGPX1DX2X3R” (SEQ ID NO: 7) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = Q or L,
■ X2 = T or K, and
■ X3 = T or A.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 36 and 145. By “SEQ ID NOs: 8 to 36 and 145”, it means that the sequence chosen sequence can be the sequence SEQ ID NO: 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36 or 145.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 11, 13, 21 to 35 and 145.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 12, 14 to 20 and 36.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 13 and 15.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 11 and 13.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 12 and 15.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-1 , SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1, MMERV-B6, mfuERV, GeERV, SRV-6, SRV-7, SERV-2 and their orthologs.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-1 , SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, REV/CSV/SNV/DIAVbis, ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1, SERV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, REV/CSV/SNV/DIAVbis, ptERV-W, ggERV-W, paERV- W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1 and SERV-2, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83 and 143, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 and 144; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, REV/CSV/SNV/DIAVbis, ptERV-W, ggERV-W, paERV- W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1 and SERV-2, the nucleic acid of which is respectively the sequences SEQ ID NOs: 37, 39, 41 , 43, 45, 47,
49, 51 , 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81 , 83 and 143, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48,
50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 and 144.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114, BaEV, ptERV-W, ggERV- W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1, MMERV-B6, mfuERV, GeERV, SERV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, BaEV, ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1 and SERV-2, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41 , 43, 47, 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81, 83 and 143, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 48, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 and 144; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, BaEV, ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1 and SERV-2, the nucleic acid of which is respectively the sequences SEQ ID NOs: 37, 39, 41 , 43, 47, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 and 143, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 38, 40, 42, 44, 48, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 and 144.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: MPMV, SRV-1 , SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, REV/CSV/SNV/DIAVbis and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV and REV/CSV/SNV/DIAVbis, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 45, 49, 51, 53, 55, 57, 59 and 61, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 46, 50, 52, 54, 56, 58, 60 and 62; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV and REV/CSV/SNV/DIAVbis, the nucleic acid of which is respectively the sequences SEQ ID NOs: 45, 49, 51 , 53, 55, 57, 59 and 61 , or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 46, 50, 52, 54, 56, 58, 60 and 62.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1 , RD114, MPMV, BaEV and SRV-2, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41 , 43, 45, 47 and 51 , or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48 and 52; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1 , RD114, MPMV, BaEV and SRV-2, the nucleic acid of which is respectively the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47 and 51 , or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48 and 52.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114, BaEV and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114 and BaEV, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43 and 47, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44 and 48; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114 and BaEV, the nucleic acid of which is respectively the sequences SEQ ID NOs: 37, 39, 41 , 43 and 47, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 38, 40, 42, 44 and 48.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: MPMV, SRV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV and SRV-2, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 45 and 51, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 46 and 52; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV and SRV-2, the nucleic acid of which is respectively the sequences SEQ ID NOs: 45 and 51 , or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 46 and 52.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 85, 87, 89, 91 , 93, 95 and 97, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 86, 88, 90, 92, 94, 96 and 98.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which is respectively the sequences SEQ ID NOs: 85, 87, 89, 91, 93, 95 and 97, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 86, 88, 90, 92, 94, 96 and 98.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, and BaEVA18, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 85, 87, 89, 91 and 95, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 86, 88, 90, 92 and 96.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18 and BaEVA18, the nucleic acid of which is respectively the sequences SEQ ID NOs: 85, 87, 89, 91 and 95, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 86, 88, 90, 92 and 96.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: MPMVA23 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 93 and 97, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 94 and 98.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: MPMVA23 and SRV-2A23, the nucleic acid of which is respectively the sequences SEQ ID NOs: 93 and 97, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 94 and 98.
In another embodiment, a subject matter of the invention concerns the IRPOV genome encoding an IRPOV specifically directed against a host cell surface protein as described above, said host cell surface protein being a tumor marker and said tumor marker being the “Myelin protein zero-like 1” (MPZL1), for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV) specifically directed against said host cell surface protein from an “Infectious and Replicative Oncolytic Virus" (IROV), the native envelope glycoprotein of said IROV being inactivated (resulting in the loss of its natural tropism) in said IRPOV and said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV:
■ is capable of allowing the production of an IRPOV;
■ has retained its native targeting capacity for MPZL1 ; and has retained its native infectious activity permitting to said IRPOV to entry into a host tumor cell expressing MPZL1.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof. For instance, an endogenous retroviral envelope glycoprotein having a native targeting capacity for the “Myelin protein zero-like 1" (MPZL1) can be Syn-Mab1. The subject matter of the invention can thus concern the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn-Mab1 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof is Syn-Mab1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 146, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 147; and in particular said retroviral envelope glycoprotein or a fragment thereof is Syn-Mab1, the nucleic acid of which is the sequence SEQ ID NO: 146, or the amino acid sequence of which is the sequence SEQ ID NO: 147.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a native targeting capacity for MPZL1, in particular said retroviral envelope glycoprotein or a fragment thereof is Syn-Mab1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 146, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 147.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an exogenous retroviral envelope glycoprotein or a fragment thereof.
In another embodiment, a subject matter of the invention thus concerns the IRPOV genome encoding an IRPOV specifically directed against a host cell surface protein as described above, said host cell surface protein being a tumor marker and said tumor marker being the “ubiquitous vertebrate glucose transporter 1” (GLUT1), for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV) specifically directed against said host cell surface protein from an “Infectious and Replicative Oncolytic Virus" (IROV), the native envelope glycoprotein of said IROV being inactivated (resulting in the loss of its natural tropism) in said IRPOV and said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV:
■ is capable of allowing the production of an IRPOV;
■ has retained its native targeting capacity for GLUT 1 ; and
■ has retained its native infectious activity permitting to said IRPOV to entry into a host tumor cell expressing GLUT 1. In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof when present in the genome of said IRPOV has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof. For instance, an endogenous retroviral envelope glycoprotein having a native targeting capacity for the ““ubiquitous vertebrate glucose transporter 1" (GLUT1) can be HTLV-1 Env. The subject matter of the invention can thus concern the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: HTLV-1 Env and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof is HTLV-1 Env, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 148, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 149; and in particular said retroviral envelope glycoprotein or a fragment thereof is HTLV-1 Env, the nucleic acid of which is the sequence SEQ ID NO: 148, or the amino acid sequence of which is the sequence SEQ ID NO: 149.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an exogenous retroviral envelope glycoprotein or a fragment thereof.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome results from addition, deletion or substitution of at least one of its nucleotides, in particular said inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome results from its replacement by said nucleic acid sequence encoding said retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome results from its replacement by said nucleic acid sequence encoding said retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein said IROV genome is chosen among:
■ a Vesicular Stomatitis Virus (VSV) genome;
■ a Measles Virus (MV) genome;
■ a Sendai Virus (SeV) genome;
■ a Newcastle Disease Virus (NDV) genome;
■ a Canine distemper virus (CDV) genome; and
■ a paramyxovirus genome, in particular said IROV genome being a VSV genome. In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said I ROV genome is chosen among:
■ a Vesicular Stomatitis Virus (VSV) genome;
■ a Measles Virus (MV) genome;
■ a Sendai Virus (SeV) genome;
■ a Newcastle Disease Virus (NDV) genome;
■ a Canine distemper virus (CDV) genome; and
■ a paramyxovirus genome, and
- said retroviral envelope glycoprotein or a fragment thereof is a Syncytin-1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syncytin-1 , the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 37, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 38.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said I ROV genome is chosen among:
■ a Vesicular Stomatitis Virus (VSV) genome;
■ a Measles Virus (MV) genome;
■ a Sendai Virus (SeV) genome;
■ a Newcastle Disease Virus (NDV) genome;
■ a Canine distemper virus (CDV) genome; and
■ a paramyxovirus genome, and
- said retroviral envelope glycoprotein or a fragment thereof is a Syncytin-Ory1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syncytin-Ory1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 39, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 40.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said I ROV genome is chosen among:
■ a Vesicular Stomatitis Virus (VSV) genome;
■ a Measles Virus (MV) genome;
■ a Sendai Virus (SeV) genome;
■ a Newcastle Disease Virus (NDV) genome;
■ a Canine distemper virus (CDV) genome; and
■ a paramyxovirus genome, and
- said retroviral envelope glycoprotein or a fragment thereof is a Dasy Env1.1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Dasy Env1.1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 41 , or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 42.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said I ROV genome is chosen among:
■ a Vesicular Stomatitis Virus (VSV) genome;
■ a Measles Virus (MV) genome;
■ a Sendai Virus (SeV) genome;
■ a Newcastle Disease Virus (NDV) genome; a Canine distemper virus (CDV) genome; and a paramyxovirus genome, and
- said retroviral envelope glycoprotein or a fragment thereof is a RD114 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a RD114, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 43, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 44.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said I ROV genome is chosen among:
■ a Vesicular Stomatitis Virus (VSV) genome;
■ a Measles Virus (MV) genome;
■ a Sendai Virus (SeV) genome;
■ a Newcastle Disease Virus (NDV) genome;
■ a Canine distemper virus (CDV) genome; and
■ a paramyxovirus genome, and
- said retroviral envelope glycoprotein or a fragment thereof is a MPMV or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a MPMV, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 45, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 46.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said I ROV genome is chosen among:
■ a Vesicular Stomatitis Virus (VSV) genome;
■ a Measles Virus (MV) genome;
■ a Sendai Virus (SeV) genome;
■ a Newcastle Disease Virus (NDV) genome;
■ a Canine distemper virus (CDV) genome; and
■ a paramyxovirus genome, and
- said retroviral envelope glycoprotein or a fragment thereof is a BaEV or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a BaEV, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 47, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 48.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said I ROV genome is chosen among:
■ a Vesicular Stomatitis Virus (VSV) genome;
■ a Measles Virus (MV) genome;
■ a Sendai Virus (SeV) genome;
■ a Newcastle Disease Virus (NDV) genome;
■ a Canine distemper virus (CDV) genome; and
■ a paramyxovirus genome, and
- said retroviral envelope glycoprotein or a fragment thereof is a SRV-2 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a SRV-2, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 51, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 52.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said I ROV genome is chosen among:
■ a Vesicular Stomatitis Virus (VSV) genome;
■ a Measles Virus (MV) genome;
■ a Sendai Virus (SeV) genome;
■ a Newcastle Disease Virus (NDV) genome;
■ a Canine distemper virus (CDV) genome; and
■ a paramyxovirus genome, and
- said retroviral envelope glycoprotein or a fragment thereof is a Syn1A53 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syn1A53, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 85, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 86.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said I ROV genome is chosen among:
■ a Vesicular Stomatitis Virus (VSV) genome;
■ a Measles Virus (MV) genome;
■ a Sendai Virus (SeV) genome;
■ a Newcastle Disease Virus (NDV) genome;
■ a Canine distemper virus (CDV) genome; and
■ a paramyxovirus genome, and
- said retroviral envelope glycoprotein or a fragment thereof is a SynOry1A25 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a SynOry1A25, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 87, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 88.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said I ROV genome is chosen among:
■ a Vesicular Stomatitis Virus (VSV) genome;
■ a Measles Virus (MV) genome;
■ a Sendai Virus (SeV) genome;
■ a Newcastle Disease Virus (NDV) genome;
■ a Canine distemper virus (CDV) genome; and
■ a paramyxovirus genome, and
- said retroviral envelope glycoprotein or a fragment thereof is a DasyEnvA28 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a DasyEnvA28, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 89, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 90.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said I ROV genome is chosen among:
■ a Vesicular Stomatitis Virus (VSV) genome; ■ a Measles Virus (MV) genome;
■ a Sendai Virus (SeV) genome;
■ a Newcastle Disease Virus (NDV) genome;
■ a Canine distemper virus (CDV) genome; and
■ a paramyxovirus genome, and
- said retroviral envelope glycoprotein or a fragment thereof is a RD114A18 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a RD114A18, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 91 , or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 92.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said I ROV genome is chosen among:
■ a Vesicular Stomatitis Virus (VSV) genome;
■ a Measles Virus (MV) genome;
■ a Sendai Virus (SeV) genome;
■ a Newcastle Disease Virus (NDV) genome;
■ a Canine distemper virus (CDV) genome; and
■ a paramyxovirus genome, and
- said retroviral envelope glycoprotein or a fragment thereof is a MPMVA23 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a MPMVA23, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 93, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 94.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said I ROV genome is chosen among:
■ a Vesicular Stomatitis Virus (VSV) genome;
■ a Measles Virus (MV) genome;
■ a Sendai Virus (SeV) genome;
■ a Newcastle Disease Virus (NDV) genome;
■ a Canine distemper virus (CDV) genome; and
■ a paramyxovirus genome, and
- said retroviral envelope glycoprotein or a fragment thereof is a BaEVA18 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a BaEVA18, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 95, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 96.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said I ROV genome is chosen among:
■ a Vesicular Stomatitis Virus (VSV) genome;
■ a Measles Virus (MV) genome;
■ a Sendai Virus (SeV) genome;
■ a Newcastle Disease Virus (NDV) genome;
■ a Canine distemper virus (CDV) genome; and
■ a paramyxovirus genome, and - said retroviral envelope glycoprotein or a fragment thereof is a SRV-2A23 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a SRV-2A23, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 97, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 98.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said I ROV genome is chosen among:
■ a Vesicular Stomatitis Virus (VSV) genome;
■ a Measles Virus (MV) genome;
■ a Sendai Virus (SeV) genome;
■ a Newcastle Disease Virus (NDV) genome;
■ a Canine distemper virus (CDV) genome; and
■ a paramyxovirus genome, and
- said retroviral envelope glycoprotein or a fragment thereof is a Syn-Mab1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syn-Mab1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 146, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 147.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said I ROV genome is chosen among:
■ a Vesicular Stomatitis Virus (VSV) genome;
■ a Measles Virus (MV) genome;
■ a Sendai Virus (SeV) genome;
■ a Newcastle Disease Virus (NDV) genome;
■ a Canine distemper virus (CDV) genome; and
■ a paramyxovirus genome, and
- said retroviral envelope glycoprotein or a fragment thereof is a HTLV-1 Env or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a HTLV-1 Env, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 148, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 149.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said I ROV genome is a Vesicular Stomatitis Virus (VSV) genome; and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95 and 97 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 52, 85, 87, 89, 91, 93, 95 and 97.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein: - said IROV genome is a Measles Virus (MV) genome; and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-0ry1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95 and 97 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 52, 85, 87, 89, 91, 93, 95 and 97.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said IROV genome is a Sendai Virus (SeV) genome; and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95 and 97 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 52, 85, 87, 89, 91, 93, 95 and 97.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said IROV genome is a Newcastle Disease Virus (NDV) genome; and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95 and 97 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 52, 85, 87, 89, 91, 93, 95 and 97.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said IROV genome is a Canine distemper virus (CDV) genome; and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95 and 97 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 52, 85, 87, 89, 91, 93, 95 and 97.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said IROV genome is a paramyxovirus genome; and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95 and 97 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 52, 85, 87, 89, 91, 93, 95 and 97.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said IROV genome is a Vesicular Stomatitis Virus (VSV) genome; and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said IROV genome is a Measles Virus (MV) genome; and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said IROV genome is a Sendai Virus (SeV) genome; and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said IROV genome is a Newcastle Disease Virus (NDV) genome; and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said IROV genome is a Canine distemper virus (CDV) genome; and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
In another embodiment, a subject matter of the invention concerns the IRPOV genome as described above, wherein:
- said IROV genome is a paramyxovirus genome; and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
Interestingly, the IRPOV genome according to the invention can further comprise genes to be delivered to host tumor cells encoding, for instance:
■ immune stimulating factors; or
■ immune checkpoint inhibitors, such as antibodies;
■ factors enhancing fusogenic activity of the IRPOV.
“Immune stimulating factors” means proteins that can stimulate production, maturation or activation of cells of the immune system for anti-tumorigenic effects.
“Immune checkpoint inhibitors” means antibodies that block checkpoint inhibitory proteins that stop the immune system from attacking the cancer cells.
"factors enhancing fusogenic activity of the IRPOV" means proteins that has a capacity to trigger the fusion of tumor cells infected by the IRPOV with adjacent host (tumor) cells for a better anti-tumoral effect.
In a third aspect, a subject matter of the invention concerns a plasmid comprising the IRPOV genome as described above and means for expressing it.
In a fourth aspect, a subject matter of the invention concerns a use of the IRPOV genome as described above or the plasmid as described above for producing a viral particle, said viral particle being an IRPOV and comprising the IRPOV genome as described above. In another aspect, a subject matter of the invention concerns a method of production of a viral particle, said viral particle being an IRPOV and comprising the IRPOV genome as described above, said method comprising at least the steps of: a. co-transfection of an animal eukaryotic cell, in particular said animal eukaryotic cell constitutively expressing the T7 RNA polymerase {e.g. BSR- T7), with: i. a plasmid as described above; and ii. helper plasmids encoding for structural proteins and enzymes of IRPOV and the means for expressing it, in particular said structural proteins and enzymes of IRPOV being under T7 promoter control and contain an IRES element allowing cap-independent translation of IRPOV proteins, to obtain a transfected animal eukaryotic cell; b. co-culturing said transfected eukaryotic animal cell with Vero-NK cells expressing a host cell surface protein, to enable the production of a viral particle, said host cell surface protein being a tumor marker and said tumor marker being the ASCT2; and c. harvesting and purifying said viral particle.
Such a method is illustrated in the examples below and is accessible to the person skilled in the art in view of the literature at his disposal (e.g. Harty RN et al. Vaccinia virus-free recovery of vesicular stomatitis virus. Journal of Molecular Microbiology and Biotechnology. 2001 Oct;3(4):513-517. PMID: 11545270). For instance, such method can be performed as follows:
The plasmid as described above is employed in transfection experiments along with helper plasmids (encoding structural proteins and enzymes of VSV being under T7 promoter control and contain an IRES element). The T7 RNA polymerase is expressed constitutively from BSR-T7 cells, allowing vaccinia virus-free recovery of VSV. The helper vectors containing an IRES element allows cap-independent translation of VSV proteins in T7 RNA polymerase- expressing cells. Briefly, BSR-T7 cells at approximately 80% confluency are transfected with 10pg of the plasmid according to the invention, 1-5pg of helper plasmids using Fugene6 (Promega, Germany). At 24 hours postransfection, the cells are trypsinized, pellet for 10 minutes at 3000 rpm, and overlaid on permissive Vero-NK cells expressing endogenous ASCT2. From 48h of co-culture, green fluorescent and/or syncytia formation iss noticed. Virus-containing supernatant is then harvested, clarified, and stored at -80°C. To produce large amounts of IRPOV, IRPOV according to the invention are incubated on Vero-NK cells at a MOI of 0.00001. Supernatant is harvested at 3 days after infection, filtered using 0.22 pM filter and stored at -80°C. To concentrate the virus, supernatants are ultracentrifuged at 25 000 rpm for 2h at 4°C (Beckman Coulter, USA).
In another embodiment, a subject matter of the invention concerns the method of production of a viral particle as described above, wherein the animal eukaryotic cell of step a. constitutively expressing the T7 RNA polymerase. In particular, a subject matter of the invention concerns the method of production of a viral particle as described above, wherein the animal eukaryotic cell of step a. is a BSR-T7 cell.
In another aspect, a subject matter of the invention concerns a viral particle, said viral particle being an IRPOV, the IRPOV genome of which comprises the IRPOV genome as described above.
Alternatively, this aspect also encompasses a viral particle liable to be produced with the method as described above. “Viral particle”, also named IRPOV, means the complete infectious form of a virus outside of a host cell. It has to be pointed out that said viral particle belongs to a first set of viral particles according to the invention.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein the retroviral envelope glycoprotein or a fragment thereof encoded by said IRPOV genome and expressed to the surface of said viral particle has a native targeting capacity for a host cell surface protein.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein the retroviral envelope glycoprotein or a fragment thereof encoded by said IRPOV genome and expressed to the surface of said viral particle has a native targeting capacity for a host cell surface protein, said host cell surface protein being a tumor marker.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein the retroviral envelope glycoprotein or a fragment thereof encoded by said IRPOV genome and expressed to the surface of said viral particle has a native targeting capacity for a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being chosen among: the “Alanine, Serine, Cysteine Transporter 2” (ASCT2), the “Myelin protein zero-like 1” (MPZL1) and the “ubiquitous vertebrate glucose transporter 1” (GLLIT1).
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein the retroviral envelope glycoprotein or a fragment thereof encoded by said IRPOV genome and expressed to the surface of said viral particle has a native targeting capacity for a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2” (ASCT2).
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an exogenous retroviral envelope glycoprotein or a fragment thereof.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S-[D/N]-[G/R]-XIGX2X3X4X5X6X7” (SEQ ID NOs: 131 to 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid, ■ X4 = D or N,
■ Xs = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises:
- the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 131) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid;
- the conserved “SDRXIGX2X3X4X5X6X7” (SEQ ID NO: 132) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid;
- the conserved “SNGXIGX2X3X4X5X6X7” (SEQ ID NO: 133) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid; or
- the conserved “SNRXIGX2X3X4X5X6X7” (SEQ ID NO: 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “800X10X2X3X4X5X6X7” (SEQ ID NO: 131) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid, ■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDRX1GX2X3X4X5X6X7” (SEQ ID NO: 132) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNGXiGX^X^sXeX/ (SEQ ID NO: 133) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNRXIGX2X3X4X5X6X7” (SEQ ID NO: 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S-[D/N]-[G/R]-XIGX2X3X4X5X6X7” (SEQ ID NOs: 135 to 138) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000056_0001
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises:
- the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 135) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
- th
Figure imgf000057_0001
O: 136) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
- th
Figure imgf000057_0002
O: 137) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000057_0003
or
- the conserved “SNRX1GX2X3X4X5X6X7” (SEQ ID NO: 138) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000057_0004
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 135) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid, ,
Figure imgf000057_0005
X? = R or T or S or G or K.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDRX1GX2X3X4X5X6X7” (SEQ ID NO: 136) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000058_0002
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNGX1GX2X3X4X5X6X7” (SEQ ID NO: 137) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000058_0003
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNRX1GX2X3X4X5X6X7” (SEQ ID NO: 138) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000058_0004
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “S-[D/N]-[G/R]-XIGX2X3X4X5X6X7” (SEQ ID NOs: 139 to 142) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000058_0001
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises: - the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 139) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
- th
Figure imgf000059_0004
140) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
- th
Figure imgf000059_0005
141) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000059_0001
or
- the conserved “SNRX1GX2X3X4X5X6X7” (SEQ ID NO: 142) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000059_0002
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGX1GX2X3X4X5X6X7” (SEQ ID NO: 139) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000059_0003
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDRX1GX2X3X4X5X6X7” (SEQ ID NO: 140) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000060_0001
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNGX1GX2X3X4X5X6X7” (SEQ ID NO: 141) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000060_0002
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SNRX1GX2X3X4X5X6X7” (SEQ ID NO: 142) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
Figure imgf000060_0003
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGPX1DX2X3R” (SEQ ID NO: 4) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = L or Q or T,
■ X2 = Q or T or K or A or M or no amino acid, and
■ X3 = T or A or V or I.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGX1X2DX3X4X5” (SEQ ID NO: 5) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = V or P,
■ X2 = Q or L,
■ X3 = Q or T or K,
■ X4 = A or L or K or T, and
■ X5 = R or T. In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGX1X2DX3X4X5” (SEQ ID NO: 6) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = V or P,
■ X2 = Q or L,
■ X3 = Q or T or K,
■ X4 = A or L or K, and
■ X5 = R or T.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises the conserved “SDGGGPX1DX2X3R” (SEQ ID NO: 7) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = Q or L,
■ X2 = T or K, and
■ X3 = T or A.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 36 and 145. By “SEQ ID NOs: 8 to 36 and 145”, it means that the sequence chosen sequence can be the sequence SEQ ID NO: 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36 or 145.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 11, 13, 21 to 35 and 145.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 12, 14 to 20 and 36.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 13 and 15.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 8 to 11 and 13.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof comprises an ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein chosen among the sequences SEQ ID NOs: 12 and 15.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-1 , SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1, MMERV-B6, mfuERV, GeERV, SRV-6, SRV-7, SERV-2 and their orthologs.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-1 , SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, REV/CSV/SNV/DIAVbis, ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1, SERV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, REV/CSV/SNV/DIAVbis, ptERV-W, ggERV-W, paERV- W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1 and SERV-2, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83 and 143, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 and 144; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, REV/CSV/SNV/DIAVbis, ptERV-W, ggERV-W, paERV- W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1 and SERV-2, the nucleic acid of which is respectively the sequences SEQ ID NOs: 37, 39, 41 , 43, 45, 47,
49, 51 , 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81 , 83 and 143, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48,
50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 and 144.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114, BaEV, ptERV-W, ggERV- W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1, MMERV-B6, mfuERV, GeERV, SERV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, BaEV, ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1 and SERV-2, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41 , 43, 47, 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81, 83 and 143, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 48, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 and 144; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, BaEV, ptERV-W, ggERV-W, paERV-W(3), paERV-W(5), nIERV-W, ppERV-W, hmERV-W, ame-ERV-Fc1, TvERV, DrERV, CfERV-Fc1 and SERV-2, the nucleic acid of which is respectively the sequences SEQ ID NOs: 37, 39, 41 , 43, 47, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83 and 143, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 38, 40, 42, 44, 48, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84 and 144.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: MPMV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV, REV/CSV/SNV/DIAVbis and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV and REV/CSV/SNV/DIAVbis, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 45, 49, 51, 53, 55, 57, 59 and 61, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 46, 50, 52, 54, 56, 58, 60 and 62; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV, SRV-1, SRV-2, SRV-4, SRV-5, SMRV, REV/CSV/SNV/DIAV and REV/CSV/SNV/DIAVbis, the nucleic acid of which is respectively the sequences SEQ ID NOs: 45, 49, 51 , 53, 55, 57, 59 and 61 , or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 46, 50, 52, 54, 56, 58, 60 and 62.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1 , RD114, MPMV, BaEV and SRV-2, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41 , 43, 45, 47 and 51 , or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48 and 52; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1 , RD114, MPMV, BaEV and SRV-2, the nucleic acid of which is respectively the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47 and 51 , or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48 and 52.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114, BaEV and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114 and BaEV, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43 and 47, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44 and 48; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1 , Syncytin-Ory1, Dasy Env1.1, RD114 and BaEV, the nucleic acid of which is respectively the sequences SEQ ID NOs: 37, 39, 41 , 43 and 47, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 38, 40, 42, 44 and 48.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: MPMV, SRV-2 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV and SRV-2, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 45 and 51, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 46 and 52; and in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: MPMV and SRV-2, the nucleic acid of which is respectively the sequences SEQ ID NOs: 45 and 51 , or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 46 and 52.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 85, 87, 89, 91 , 93, 95 and 97, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 86, 88, 90, 92, 94, 96 and 98.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which is respectively the sequences SEQ ID NOs: 85, 87, 89, 91, 93, 95 and 97, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 86, 88, 90, 92, 94, 96 and 98.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, and BaEVA18, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 85, 87, 89, 91 and 95, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 86, 88, 90, 92 and 96.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: Syn1A53, SynOry1A25, DasyEnvA28, RD114A18 and BaEVA18, the nucleic acid of which is respectively the sequences SEQ ID NOs: 85, 87, 89, 91 and 95, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 86, 88, 90, 92 and 96.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: MPMVA23 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 93 and 97, or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 94 and 98.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail and is chosen among: MPMVA23 and SRV-2A23, the nucleic acid of which is respectively the sequences SEQ ID NOs: 93 and 97, or the amino acid sequence of which is respectively the sequences SEQ ID NOs: 94 and 98.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein the retroviral envelope glycoprotein or a fragment thereof encoded by said IRPOV genome and expressed to the surface of said viral particle has a native targeting capacity for a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “Myelin protein zero-like 1” (MPZL1).
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof when present in the genome of said IRPOV has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof. For instance, an endogenous retroviral envelope glycoprotein having a native targeting capacity for the “Myelin protein zero-like 1" (MPZL1) can be Syn-Mab1. The subject matter of the invention can thus concern the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn-Mab1 and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof is Syn-Mab1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 146, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 147; and in particular said retroviral envelope glycoprotein or a fragment thereof is Syn-Mab1, the nucleic acid of which is the sequence SEQ ID NO: 146, or the amino acid sequence of which is the sequence SEQ ID NO: 147.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an exogenous retroviral envelope glycoprotein or a fragment thereof.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein the retroviral envelope glycoprotein or a fragment thereof encoded by said IRPOV genome and expressed to the surface of said viral particle has a native targeting capacity for a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “ubiquitous vertebrate glucose transporter 1” (GLUT1).
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof when present in the genome of said IRPOV has retained its native capacity to fuse the plasma membranes of two or more adjacent host (tumor) cells leading to the formation of a syncytium.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof. For instance, an endogenous retroviral envelope glycoprotein having a native targeting capacity for the ““ubiquitous vertebrate glucose transporter 1” (GLUT1) can be HTLV-1 Env. The subject matter of the invention can thus concern the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is chosen among: HTLV-1 Env and their orthologs; in particular said retroviral envelope glycoprotein or a fragment thereof is HTLV-1 Env, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 148, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 149; and in particular said retroviral envelope glycoprotein or a fragment thereof is HTLV-1 Env, the nucleic acid of which is the sequence SEQ ID NO: 148, or the amino acid sequence of which is the sequence SEQ ID NO: 149.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an exogenous retroviral envelope glycoprotein or a fragment thereof.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein said viral particle is chosen among:
■ a pseudotyped Vesicular Stomatitis Virus (VSV);
■ a pseudotyped Measles Virus (MV);
■ a pseudotyped Sendai Virus (SeV);
■ a pseudotyped Newcastle Disease Virus (NDV);
■ a pseudotyped Canine distemper virus (CDV); and
■ a pseudotyped paramyxovirus, in particular said viral particle being a pseudotyped VSV.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is chosen among:
■ a pseudotyped Vesicular Stomatitis Virus (VSV);
■ a pseudotyped Measles Virus (MV);
■ a pseudotyped Sendai Virus (SeV);
■ a pseudotyped Newcastle Disease Virus (NDV);
■ a pseudotyped Canine distemper virus (CDV); and
■ a pseudotyped paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a Syncytin-1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syncytin-1 , the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 37, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 38.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is chosen among:
■ a pseudotyped Vesicular Stomatitis Virus (VSV);
■ a pseudotyped Measles Virus (MV);
■ a pseudotyped Sendai Virus (SeV);
■ a pseudotyped Newcastle Disease Virus (NDV);
■ a pseudotyped Canine distemper virus (CDV); and
■ a pseudotyped paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a Syncytin-Ory1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syncytin-Ory1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 39, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 40.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is chosen among:
■ a pseudotyped Vesicular Stomatitis Virus (VSV);
■ a pseudotyped Measles Virus (MV);
■ a pseudotyped Sendai Virus (SeV);
■ a pseudotyped Newcastle Disease Virus (NDV);
■ a pseudotyped Canine distemper virus (CDV); and
■ a pseudotyped paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a Dasy Env1.1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Dasy Env1.1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 41 , or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 42.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is chosen among:
■ a pseudotyped Vesicular Stomatitis Virus (VSV);
■ a pseudotyped Measles Virus (MV);
■ a pseudotyped Sendai Virus (SeV);
■ a pseudotyped Newcastle Disease Virus (NDV);
■ a pseudotyped Canine distemper virus (CDV); and
■ a pseudotyped paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a RD114 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a RD114, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 43, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 44.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is chosen among:
■ a pseudotyped Vesicular Stomatitis Virus (VSV);
■ a pseudotyped Measles Virus (MV);
■ a pseudotyped Sendai Virus (SeV);
■ a pseudotyped Newcastle Disease Virus (NDV);
■ a pseudotyped Canine distemper virus (CDV); and
■ a pseudotyped paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a MPMV or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a MPMV, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 45, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 46.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is chosen among: ■ a pseudotyped Vesicular Stomatitis Virus (VSV);
■ a pseudotyped Measles Virus (MV);
■ a pseudotyped Sendai Virus (SeV);
■ a pseudotyped Newcastle Disease Virus (NDV);
■ a pseudotyped Canine distemper virus (CDV); and
■ a pseudotyped paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a BaEV or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a BaEV, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 47, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 48.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is chosen among:
■ a pseudotyped Vesicular Stomatitis Virus (VSV);
■ a pseudotyped Measles Virus (MV);
■ a pseudotyped Sendai Virus (SeV);
■ a pseudotyped Newcastle Disease Virus (NDV);
■ a pseudotyped Canine distemper virus (CDV); and
■ a pseudotyped paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a SRV-2 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a SRV-2, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 51, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 52.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is chosen among:
■ a pseudotyped Vesicular Stomatitis Virus (VSV);
■ a pseudotyped Measles Virus (MV);
■ a pseudotyped Sendai Virus (SeV);
■ a pseudotyped Newcastle Disease Virus (NDV);
■ a pseudotyped Canine distemper virus (CDV); and
■ a pseudotyped paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a Syn1A53 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syn1A53, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 85, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 86.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is chosen among:
■ a pseudotyped Vesicular Stomatitis Virus (VSV); a pseudotyped Measles Virus (MV); a pseudotyped Sendai Virus (SeV); a pseudotyped Newcastle Disease Virus (NDV); a pseudotyped Canine distemper virus (CDV); and a pseudotyped paramyxovirus, and - said retroviral envelope glycoprotein or a fragment thereof is a SynOry1A25 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a SynOry1A25, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 87, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 88.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is chosen among:
■ a pseudotyped Vesicular Stomatitis Virus (VSV);
■ a pseudotyped Measles Virus (MV);
■ a pseudotyped Sendai Virus (SeV);
■ a pseudotyped Newcastle Disease Virus (NDV);
■ a pseudotyped Canine distemper virus (CDV); and
■ a pseudotyped paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a DasyEnvA28 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a DasyEnvA28, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 89, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 90.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is chosen among:
■ a pseudotyped Vesicular Stomatitis Virus (VSV);
■ a pseudotyped Measles Virus (MV);
■ a pseudotyped Sendai Virus (SeV);
■ a pseudotyped Newcastle Disease Virus (NDV);
■ a pseudotyped Canine distemper virus (CDV); and
■ a pseudotyped paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a RD114A18 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a RD114A18, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 91 , or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 92.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is chosen among:
■ a pseudotyped Vesicular Stomatitis Virus (VSV);
■ a pseudotyped Measles Virus (MV);
■ a pseudotyped Sendai Virus (SeV);
■ a pseudotyped Newcastle Disease Virus (NDV);
■ a pseudotyped Canine distemper virus (CDV); and
■ a pseudotyped paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a MPMVA23 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a MPMVA23, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 93, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 94. In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is chosen among:
■ a pseudotyped Vesicular Stomatitis Virus (VSV);
■ a pseudotyped Measles Virus (MV);
■ a pseudotyped Sendai Virus (SeV);
■ a pseudotyped Newcastle Disease Virus (NDV);
■ a pseudotyped Canine distemper virus (CDV); and
■ a pseudotyped paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a BaEVA18 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a BaEVA18, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 95, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 96.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is chosen among:
■ a pseudotyped Vesicular Stomatitis Virus (VSV);
■ a pseudotyped Measles Virus (MV);
■ a pseudotyped Sendai Virus (SeV);
■ a pseudotyped Newcastle Disease Virus (NDV);
■ a pseudotyped Canine distemper virus (CDV); and
■ a pseudotyped paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a SRV-2A23 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a SRV-2A23, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 97, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 98.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is chosen among:
■ a pseudotyped Vesicular Stomatitis Virus (VSV);
■ a pseudotyped Measles Virus (MV);
■ a pseudotyped Sendai Virus (SeV);
■ a pseudotyped Newcastle Disease Virus (NDV);
■ a pseudotyped Canine distemper virus (CDV); and
■ a pseudotyped paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a Syn-Mab1 or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a Syn-Mab1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 146, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 147.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is chosen among:
■ a pseudotyped Vesicular Stomatitis Virus (VSV);
■ a pseudotyped Measles Virus (MV);
■ a pseudotyped Sendai Virus (SeV);
■ a pseudotyped Newcastle Disease Virus (NDV); a pseudotyped Canine distemper virus (CDV); and a pseudotyped paramyxovirus, and
- said retroviral envelope glycoprotein or a fragment thereof is a HTLV-1 Env or an ortholog thereof, in particular said retroviral envelope glycoprotein or a fragment thereof being a HTLV-1 Env, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 148, or the amino acid sequence of which has respectively at least 90% of identity with the sequence SEQ ID NO: 149.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is a pseudotyped Vesicular Stomatitis Virus (VSV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95 and 97 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 52, 85, 87, 89, 91, 93, 95 and 97.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is a pseudotyped Measles Virus (MV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95 and 97 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 52, 85, 87, 89, 91, 93, 95 and 97.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is a pseudotyped Sendai Virus (SeV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95 and 97 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 52, 85, 87, 89, 91, 93, 95 and 97. In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is a pseudotyped Newcastle Disease Virus (NDV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95 and 97 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 52, 85, 87, 89, 91, 93, 95 and 97.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is a pseudotyped Canine distemper virus (CDV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95 and 97 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 52, 85, 87, 89, 91, 93, 95 and 97.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is a pseudotyped paramyxovirus; and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syncytin-1, Syncytin-Ory1, Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18, SRV-2A23 and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syncytin-1, Syncytin-Ory1 , Dasy Env1.1, RD114, MPMV, BaEV, SRV-2, Syn1A53, SynOry1A25, DasyEnvA28, RD114A18, MPMVA23, BaEVA18 and SRV-2A23, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 37, 39, 41, 43, 45, 47, 51, 85, 87, 89, 91 , 93, 95 and 97 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 38, 40, 42, 44, 46, 48, 52, 85, 87, 89, 91, 93, 95 and 97.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is a pseudotyped Vesicular Stomatitis Virus (VSV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149. In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is a pseudotyped Measles Virus (MV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is a pseudotyped Sendai Virus (SeV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is a pseudotyped Newcastle Disease Virus (NDV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is a pseudotyped Canine distemper virus (CDV); and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, wherein:
- said viral particle is a pseudotyped paramyxovirus; and
- said retroviral envelope glycoprotein or a fragment thereof is chosen among: Syn- Mab1, HTLV-1 Env and their orthologs, in particular said retroviral envelope glycoprotein or a fragment thereof being chosen among: Syn-Mab1 and HTLV-1 Env, the nucleic acid of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 146 and 148 or the amino acid sequence of which has respectively at least 90% of identity with the sequences SEQ ID NOs: 147 and 149.
In another aspect, a subject matter of the invention concerns a viral particle as described above for its use as a drug. In particular, a subject matter of the invention relates to the viral particle of the invention for its use as a drug, said viral particle being an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV), the IRPOV genome of which encodes an IRPOV specifically directed against a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2” (ASCT2), the “Myelin protein zero-like 1” (MPZL1), or the “ubiquitous vertebrate glucose transporter 1” (GLLIT1), said IRPOV genome comprising an “Infectious and Replicative Oncolytic Virus" (IROV) genome comprising:
■ an inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome; and
■ a nucleic acid sequence encoding a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein, said retroviral envelope glycoprotein or a fragment thereof:
- being capable of allowing the production of an IRPOV;
- having retained its native targeting capacity for said host cell surface protein; and
- having retained its native infectious activity permitting to said IRPOV to entry into a host cell expressing said host cell surface protein, and at least a pharmaceutically acceptable vehicle.
In another embodiment, a subject matter of the invention concerns the viral particle of the invention for its use as a drug, wherein the viral particle comprising an IRPOV genome encoding an IRPOV is devoid of a nucleic acid encoding a retroviral GAG protein. In particular, a subject matter of the invention concerns the viral particle of the invention for its use as a drug, wherein the viral particle comprising an IRPOV genome encoding an IRPOV specifically directed against GLLIT1 is devoid of a nucleic acid encoding a retroviral GAG protein.
In the context of the invention, it is important to point out that the viral particle of the invention for its use as a drug concerns both human and veterinary medicines (e.g. dog, horse or cat). Consequently, a subject matter of the invention concerns the viral particle of the invention for its use as a human drug or as a veterinary drug. Indeed, as illustrated in the examples below, viral particle of the invention is able to infect both human and animal cells by targeting the tumor marker of the invention. In particular, viral particle of the invention designed to target human ASCT2 also target dog ASCT2.
In another aspect, a subject matter of the invention concerns a pharmaceutical composition comprising the viral particle as described above (as an active substance) and at least a pharmaceutically acceptable vehicle.
As previously mentioned, the invention relates to human and veterinary medicines (e.g. dog, horse or cat). A subject matter of the invention thus concerns a human or veterinary pharmaceutical composition comprising the viral particle as described above (as an active substance) and at least a pharmaceutically acceptable vehicle.
“Pharmaceutically acceptable vehicle” means a substance or formulation that is safe and suitable for use as a carrier or base in the preparation of pharmaceutical or medicinal products. These vehicles are often inert or non-reactive substances that serve as a medium for delivering the active pharmaceutical ingredient (API) to the patient in a stable, consistent, and effective manner. The choice of a pharmaceutically acceptable vehicle depends on factors like the intended route of administration, the physical and chemical properties of the drug, and patient safety considerations. Common pharmaceutically acceptable vehicles include: ■ Oral Formulations: Vehicles for oral medications often include water, oils, emulsifiers, and sweeteners to create liquids, suspensions, tablets, or capsules;
■ Injectable Formulations: Injectable vehicles may involve sterile water, saline, or specialized solvents to create solutions or suspensions for intravenous, intramuscular, or subcutaneous administration; and
■ Inhalation: Aerosolized drug vehicles often contain propellants and solvents suitable for inhalation.
Others means to deliver the viral particle as described above can be through liposomal or exosomal formulations, wherein a liposome vesicle or an exosome vesicle are used as a pharmaceutical acceptable carrier.
In another embodiment, a subject matter of the invention concerns the pharmaceutical composition as described above, said pharmaceutical composition comprising a (unit) dose from 5 x 106 TCID50 to 5 x 1O10 TCID50 of the viral particle according to the invention.
“TCID50” means the quantity of viral particle required to destroy or induce any other type of cytopathic effect in 50% of infected cells or cultures. By “from 5 x 106 TCID50 to 5 x 1O10 TCID50”, it is also encompassed from 5 x 106 TCID50 to 5 x 107 TCID50, from 5 x 106 TCID50 to 5 x 108 TCID50, from 5 x 106 TCID50 to 5 x 109 TCID50, from 5 x 107 TCID50 to 5 x 101° TCID50, from 5 x 108 TCID50 to 5 x 101° TCID50 and from 5 x 109 TCID50 to 5 x 101° TCID50.
In another aspect, a subject matter of the invention concerns a kit-of-parts comprising at least two viral particles as described above for the separate or sequential administration of said at least two viral particles, each viral particle comprising a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein.
In another aspect, a subject matter of the invention concerns a kit-of-parts comprising at least two pharmaceutical compositions as described above for the separate or sequential administration of said at least two pharmaceutical compositions, each pharmaceutical composition comprising a viral particle according to the invention comprising a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein.
“Kit-of-parts" means that the invention may implement different IRPOVs according to the invention (at least two) with the provision that the nature of the retroviral envelope glycoprotein is different between said at least two IRPOVs. In this way, supplying the kit-of- parts of the invention makes it possible to adapt the therapeutic strategy as soon as immunization of the host to the first IRPOV administered occurs, by administering a second IRPOV that is different from the first.
By “the retroviral envelope glycoprotein is different between said at least two IRPOVs”, it means either:
■ that the kit-of-parts of the invention implements two viral particles each carrying a retroviral envelope glycoprotein targeting distinct host cell surface proteins, or
■ that the kit-of-parts of the invention implements two viral particles each carrying a retroviral envelope glycoprotein targeting the same host cell surface protein.
In other words, a subject matter of the invention concerns:
(A) a kit-of-parts comprising at least two viral particles according to the invention for the separate or sequential administration of said at least two viral particles, each viral particle comprising a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein distinct from the other, said viral particle being an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV), the IRPOV genome of which encodes an IRPOV specifically directed against a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2" (ASCT2), the “Myelin protein zero-like 1" (MPZL1), or the “ubiquitous vertebrate glucose transporter 1” (GLLIT1), said IRPOV genome comprising an “Infectious and Replicative Oncolytic Virus" (IROV) genome comprising:
■ an inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome; and
■ a nucleic acid sequence encoding a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein, said retroviral envelope glycoprotein or a fragment thereof:
- being capable of allowing the production of an IRPOV;
- having retained its native targeting capacity for said host cell surface protein; and
- having retained its native infectious activity permitting to said IRPOV to entry into a host cell expressing said host cell surface protein; and
(B) a kit-of-parts comprising at least two viral particles according to the invention for the separate or sequential administration of said at least two viral particles, each viral particle comprising a retroviral envelope glycoprotein distinct from the other or a fragment thereof distinct from the other having a native targeting capacity for the same host cell surface protein, said viral particle being an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV), the IRPOV genome of which encodes an IRPOV specifically directed against a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2” (ASCT2), the “Myelin protein zero-like 1” (MPZL1), or the “ubiquitous vertebrate glucose transporter 1" (GLLIT1), said IRPOV genome comprising an “Infectious and Replicative Oncolytic Virus" (IROV) genome comprising:
■ an inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome; and
■ a nucleic acid sequence encoding a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein, said retroviral envelope glycoprotein or a fragment thereof:
- being capable of allowing the production of an IRPOV;
- having retained its native targeting capacity for said host cell surface protein; and having retained its native infectious activity permitting to said IRPOV to entry into a host cell expressing said host cell surface protein.
Interestingly, it is understood that with the “kit-of-parts" provided by the invention it is possible to have at least two viral particles as described above or at least two pharmaceutical compositions as described above, wherein said at least two viral particles have a pseudotyped targeting capacity for at least two distinct host cell surface proteins. Such strategy may optimize the prevention and/or the treatment of a tumor. For example, the kit- of-parts as described above may implement:
■ a first viral particle having a pseudotyped targeting capacity for ASCT2 and a second viral particle having a pseudotyped targeting capacity for MPZL1;
■ a first viral particle having a pseudotyped targeting capacity for ASCT2 and a second viral particle having a pseudotyped targeting capacity for GLLIT1; or
■ a first viral particle having a pseudotyped targeting capacity for GLLIT1 and a second viral particle having a pseudotyped targeting capacity for MPZL1. It is also understood that with the “kit-of-parts" provided by the invention it is possible to have at least two viral particles as described above or at least two pharmaceutical compositions as described above, wherein said at least two viral particles have a pseudotyped targeting capacity for the same host cell surface protein, in particular said host cell surface protein being ASCT2. Such strategy may also optimize the prevention and/or the treatment of a tumor. For instance, it can be firstly administered to a patient in need a pseudotyped VSV according to the invention expressing Syncytin-1 (targeting ASCT2) and if immunization of the host to the said pseudotyped VSV occurs, another pseudotyped VSV according to the invention expressing a different retroviral envelope glycoprotein but still targeting ASCT2 (e.g. Syncytin-Ory1) according to the invention, is secondly administered.
In this context to target host cell surface proteins, an alternative subject matter of the invention concerns a kit-of-parts comprising for the separate or sequential administration at least:
■ a viral particle as described above comprising a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein, said host cell surface protein being in particular a tumor marker; and
■ a viral particle comprising either a viral envelope glycoprotein or a fragment thereof having a native targeting capacity for ASTC2, or a mutated and/or modified viral envelope glycoprotein or a fragment thereof having a targeting capacity for ASTC2.
In another embodiment, said alternative subject matter of the invention concerns the kit-of- parts as described above comprising for the separate or sequential administration at least:
■ a viral particle as described above comprising a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for ASTC2; and
■ a viral particle comprising either a viral envelope glycoprotein or a fragment thereof having a native targeting capacity for ASTC2, or a mutated and/or modified viral envelope glycoprotein or a fragment thereof having a targeting capacity for ASTC2.
Consequently, it is understood that the invention also encompasses a use of either a viral envelope glycoprotein or a fragment thereof having a native targeting capacity for ASTC2, or a mutated and/or modified viral envelope glycoprotein or a fragment thereof having a targeting capacity for ASTC2, for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV) specifically directed against said ASCT2 from an “Infectious and Replicative Oncolytic Virus" (IROV), the native envelope glycoprotein of said IROV being inactivated (resulting in the loss of its natural tropism) in said IRPOV and said viral envelope glycoprotein or a fragment thereof having a native targeting capacity for ASTC2, or said mutated and/or modified viral envelope glycoprotein or a fragment thereof having a targeting capacity for ASTC2 when present in said IRPOV:
■ is capable of allowing the production of an IRPOV;
■ has retained its (native) targeting capacity for ASTC2; and
■ has retained its (native) infectious activity permitting to said IRPOV to entry into a host tumor cell expressing ASCT2.
Here, the produced IRPOV being also a viral particle, it has to be pointed out that said viral particle according to this alternative belongs to a second (different) set of viral particles.
As the aforementioned use allows the production of an alternative IRPOV, another subject matter of the invention thus concerns an IRPOV genome encoding an IRPOV specifically directed against ASCT2, said IRPOV genome comprising an IROV genome comprising: ■ an inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome (resulting in the loss of its natural tropism); and
■ a nucleic acid sequence encoding a viral envelope glycoprotein or a fragment thereof having a native targeting capacity for ASTC2, or a mutated and/or modified viral envelope glycoprotein or a fragment thereof having a targeting capacity for ASTC2, said (mutated and/or modified) viral envelope glycoprotein or a fragment thereof:
- being capable of allowing the production of an IRPOV;
- having retained its (native) targeting capacity for ASCT2; and
- having retained its (native) infectious activity permitting to said IRPOV to entry into a host tumor cell expressing ASCT2.
“Viral envelope glycoprotein or a fragment thereof having a native targeting capacity for ASTC2” corresponds to viral envelope glycoprotein or a fragment thereof having a natural or inherent ability to specifically interact with and bind to ASCT2. This interaction is highly specific.
“Mutated and/or modified viral envelope glycoprotein or a fragment thereof having a targeting capacity for ASTC2” corresponds to viral envelope glycoprotein or a fragment thereof, the natural tropism of which is inactivated and replaced by a tropism for ASCT2. To achieve this, it can be taken a VSV virus as IROV to produce an IRPOV wherein the native VSV-G envelope is replaced by:
■ a mutated VSV-G envelope or a mutated MV-H (in combination with MV-F) fused with a fragment having the capacity to bind to ASCT2 (i.e. Receptor Binding Domain or RBD), and comprising the conserved “S-[D/N]-[G/R]-XIGX2X3X4X5X6X7” (SEQ ID NOs: 131 to 134) ASCT2 binding motif; or
■ a mutated VSV-G envelope or mutated MV-H (in combination with MV-F) fused with a molecule or protein having a native targeting capacity for ASTC2, such as an antibody or a fragment thereof, or a suppressyn protein.
For instance, the fragment having the capacity to bind to ASCT2 (i.e. Receptor Binding Domain or RBD) and comprising the conserved “S-[D/N]-[G/R]-XIGX2X3X4XSX6X7” (SEQ ID NOs: 131 to 134) ASCT2 binding motif can be the RBD of Syncytinl (SEQ ID NOs: 150 and 151). This 124aa fragment can be fused to MV-H mutated for its native binding capacity (Hmut-Syn1-RBD; SEQ ID NOs: 154 and 155). The Hmut-Syn1-RBD protein, along with the MV-F protein, can be expressed at the cell surface of the infected (tumor) cells and can trigger the fusion of these cells with cells expressing the ASCT2 receptor. This is illustrated by the following examples section.
For instance, the fragment having a native targeting capacity to ASCT2 can be the Suppressyn protein (SEQ ID NOs: 152 and 153). This 160aa fragment can be fused to MV-H mutated for its native binding capacity (Hmut-Suppressyn; SEQ ID NOs: 156 and 157). The Hmut-Suppressyn protein, along with the MV-F protein, can be expressed at the cell surface of the infected (tumor) cells and can trigger the fusion of these cells with cells expressing the ASCT2 receptor. This is illustrated by the following examples section.
In another embodiment, a subject matter of the invention thus concerns the use of either a Hmut-Syn1-RBD protein (SEQ ID NO: 155) or Hmut-Suppressyn protein (SEQ ID NO: 157) having a targeting capacity for ASTC2, for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV) specifically directed against said ASCT2 from an “Infectious and Replicative Oncolytic Virus" (IROV), the native envelope glycoprotein of said IROV being inactivated (resulting in the loss of its natural tropism) in said IRPOV and said Hmut-Syn1-RBD protein (SEQ ID NO: 155) or Hmut- Suppressyn protein (SEQ ID NO: 157) when present in said IRPOV:
■ is capable of allowing the production of an IRPOV;
■ has retained its (native) targeting capacity for ASCT2; and
■ has retained its (native) infectious activity permitting to said IRPOV to entry into a host tumor cell expressing ASCT2.
In another embodiment, a subject matter of the invention concerns the IRPOV genome encoding an IRPOV specifically directed against ASCT2, said IRPOV genome comprising an IROV genome comprising:
■ an inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome (resulting in the loss of its natural tropism); and
■ a nucleic acid sequence having the sequence SEQ ID NO: 154 or 156 respectively encoding a Hmut-Syn1-RBD protein (SEQ ID NO: 155) or Hmut- Suppressyn protein (SEQ ID NO: 157) having a targeting capacity for ASTC2, said Hmut-Syn1-RBD protein (SEQ ID NO: 155) or Hmut-Suppressyn protein (SEQ ID NO: 157):
- being capable of allowing the production of an IRPOV;
- having retained its (native) targeting capacity for ASTC2; and
- having retained its (native) infectious activity permitting to said IRPOV to entry into a host tumor cell expressing ASCT2.
Plasmid comprising the alternative IRPOV genome as described above and means for expressing it, as its use for producing a viral particle and the method for producing said viral particle, are also encompassed by the invention.
The produced viral particle as such, comprising either a viral envelope glycoprotein or a fragment thereof having a native targeting capacity for ASTC2, or a mutated and/or modified viral envelope glycoprotein or a fragment thereof having a targeting capacity for ASTC2, is one of the subject matters of the invention, its use as a drug or as an active substance in a pharmaceutical composition too.
In another aspect, a subject matter of the invention concerns a viral particle according to the invention for its use in the prevention and/or the treatment of a tumor, said viral particle being an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV), the IRPOV genome of which encodes an IRPOV specifically directed against a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2" (ASCT2), the “Myelin protein zero-like 1" (MPZL1), or the “ubiquitous vertebrate glucose transporter 1” (GLLIT1), said IRPOV genome comprising an “Infectious and Replicative Oncolytic Virus" (IROV) genome comprising:
■ an inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome; and
■ a nucleic acid sequence encoding a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein, said retroviral envelope glycoprotein or a fragment thereof:
- being capable of allowing the production of an IRPOV;
- having retained its native targeting capacity for said host cell surface protein; and
- having retained its native infectious activity permitting to said IRPOV to entry into a host cell expressing said host cell surface protein. In another embodiment, a subject matter of the invention concerns the viral particle of the invention for its use as described above, wherein the viral particle comprising an IRPOV genome encoding an IRPOV is devoid of a nucleic acid encoding a retroviral GAG protein. In particular, the subject matter of the invention concerns the viral particle of the invention for its use as described above, wherein the viral particle comprising an IRPOV genome encoding an IRPOV specifically directed against GLLIT1 is devoid of a nucleic acid encoding a retroviral GAG protein.
In another embodiment, a subject matter of the invention concerns the viral particle of the invention for its use as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has retained its native capacity to fuse the plasma membranes of two or more adjacent host cells leading to the formation of a syncytium.
In another embodiment, a subject matter of the invention concerns the viral particle of the invention for its use as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
In another embodiment, a subject matter of the invention concerns the viral particle of the invention for its use as described above, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof.
In another embodiment, a subject matter of the invention concerns the viral particle of the invention for its use as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a native targeting capacity for ASCT2, in particular said retroviral envelope glycoprotein or a fragment thereof comprising the conserved “S-[D/N]-[G/R]- X1GX2X3X4X5X6X7” (SEQ ID NOs: 131 to 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
In another embodiment, a subject matter of the invention concerns the viral particle of the invention for its use as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a native targeting capacity for MPZL1 , in particular said retroviral envelope glycoprotein or a fragment thereof is Syn-Mab1 , the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 146, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 147.
In another embodiment, a subject matter of the invention concerns the viral particle of the invention for its use as described above, wherein said retroviral envelope glycoprotein or a fragment thereof has a native targeting capacity for GLLIT1 , in particular said retroviral envelope glycoprotein or a fragment thereof is HTLV-1 Env, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 148, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 149.
In another embodiment, a subject matter of the invention concerns the viral particle of the invention for its use as described above, said tumor being one of the following group: digestive tumors, prostate tumors, brain tumors, head and neck tumors, lung tumors and women's cancers. In the same aspect, a subject matter of the invention also concerns a viral particle as described above, or pharmaceutical composition as described above, or kit-of-parts as described above for its use in the prevention and/or the treatment of a tumor, in particular said tumor being one of the following group: digestive tumors (esophageal, gastric, colorectal, hepatic, bile duct, pancreatic, renal, rectum), prostate tumors, brain tumors, head and neck tumors, lung tumors and women's cancers (breast, ovary, uterus, cervix).
As previously mentioned, the invention relates to human and veterinary medicines {e.g. dog, horse or cat). A subject matter of the invention thus concerns a viral particle as described above, or pharmaceutical composition as described above, or kit-of-parts as described above for its use in the prevention and/or the treatment of a human tumor or a veterinary tumor (in particular a dog, horse or cat tumor).
In another embodiment, a subject matter of the invention concerns the viral particle as described above, or pharmaceutical composition as described above, or kit-of-parts as described above for its use as described above, wherein the administration of said viral particle and/or pharmaceutical composition is systemic, intravenous, intra-arterial, via injection into tumor, and/or via intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, epidural, intracardiac, intraarticular, intracavernous, intracerebral, intracerebroventricular and intravitreal injection(s).
In another embodiment, a subject matter of the invention concerns the viral particle as described above, or pharmaceutical composition as described above, or kit-of-parts as described above for its use as described above, said tumor being a digestive tumor, in particular an esophageal, gastric, colorectal, hepatic, bile duct, pancreatic, renal or rectum tumor.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, or pharmaceutical composition as described above, or kit-of-parts as described above for its use as described above, said tumor being a prostate tumor.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, or pharmaceutical composition as described above, or kit-of-parts as described above for its use as described above, said tumor being a brain tumor.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, or pharmaceutical composition as described above, or kit-of-parts as described above for its use as described above, said tumor being a head and neck tumor.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, or pharmaceutical composition as described above, or kit-of-parts as described above for its use as described above, said tumor being a lung tumor.
In another embodiment, a subject matter of the invention concerns the viral particle as described above, or pharmaceutical composition as described above, or kit-of-parts as described above for its use as described above, said tumor being a women's cancers, in particular a breast, ovary, uterus or cervix tumor.
In another embodiment, a subject matter of the invention concerns the viral particle as described above for its use as described above.
In another embodiment, a subject matter of the invention concerns the pharmaceutical composition as described above for its use as described above. In another embodiment, a subject matter of the invention concerns the kit-of-parts as described above for its use as described above.
Alternatively, this aspect also encompasses a method of prevention and/or treatment of a tumor comprising the administration to a patient in need of an effective amount of the viral particle as described above or the pharmaceutical composition as described above.
Interestingly, the viral particle as described above, the pharmaceutical composition as described above or the kit-of-parts as described above for its use according to the invention can be combined with other tools in the fight against cancer, such as: CAR-T cells, immunotherapy (anti-PD1, antibody drug conjugate [ADC], etc.) and/or radiotherapy.
In any event, it should be noted that the various aspects of the invention, like the various embodiments thereof, are interdependent. They can therefore be combined with each other as many times as necessary to obtain aspects and/or preferred embodiments of the invention not explicitly described. This also applies to all the definitions provided in this description, which apply to all aspects of the invention and its embodiments.
In addition, the present invention is illustrated by, but not limited to, the following figures and examples.
LIST OF FIGURES
Figure 1 : ASCT2 is overexpressed in primary solid tumors.
(A-B) Boxplots of normalized (TPM) and Iog2-transformed expression of ASCT2 in tumor (T; filled grey boxplots) and control (C; empty black boxplots) adjacent tissue samples retrieved from TCGA (The Cancer Genome Atlas)-Recount2. The names of the cohorts are given in Table 1. Empty grey boxes correspond to basal expression in normal tissues (N) from GTEx (Genotype-Tissue Expression)-Recount2. The number of samples analyzed in each group (N, C, T) is shown on the x axis. The (N, T) and (C, T) P-values are shown as asterisks above the boxplots: *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001 Mann-Whitney U- test. Cohorts are grouped according to body systems; HEMATO., hematological tumors. Data are shown as mean with 25-75th percentile range (box) and 10-90th percentile range (whiskers). Mild outliers are depicted as black dots.
Figure 2: Detection of ASCT2 transcripts and proteins in normal and tumor samples.
(A) Expression levels of ASCT2 gene measured by RT-qPCR. Gene expression was normalized to the RPLP0 transcripts and levels were expressed as percent of the 293T control cells. The data shown represent the means and standard deviations of the results of two technical replicates. Black crosses indicate samples analyzed for protein expression. (B) ASCT2 proteins in normal or tumor tissues detected by immunohistochemistry. Represented are examples of high, moderate and negative ASCT2 expression in cancer samples. Samples correspond to those used in RT-qPCR (H2, TN4, Lum4 and N2 for breast tissues; Pan2, 4, 8 and N2 for pancreas samples and Rec3, 8, 2 and N1 for rectum tissues). Scale bars in 10X columns: 50 pm; scale bars in 20X columns: 20 pm; black squares: localization of 20X view.
Figure 3: Fusogenic capacity of WT and truncated Env towards ASCT2/1 receptor.
(A) Fusion assay in A23 cells transfected with expression vectors for WT/truncated envelopes and ASCT2/1 receptors. EV: Empty Vector. After staining with X-Gal, syncytia were revealed in blue. The area and the number of syncytia formed were quantified by Image J software. The results were presented as a mean of triplicate experiments. The asterisks indicate a significant difference (p < 0.05) between groups (Student’s t test). ** p < 0.01 ; **** p < 0.0001. (B) (Left) Fusion assay between A23 cells transfected with expression vectors for WT/truncated envelopes and 293TWT/293T KOASCT2 cells. Fusion was quantified by measuring absorbance at 570nm after addition of CPRG. The results were obtained from two separate experiments. Dash line: EV-related background level. (Right) Western Blotting analysis of endogenous ASCT2 expression in 293T KOASCT2 cells compared to the wild type 293T cells. y-Tubulin was used as a loading control.
Figure 4: Truncation at the cytoplasmic tail enhanced fusogenic capacity of retroviral envelopes in tumor cell lines.
Fusion experiments performed between A23 cells transfected with WT/truncated envelopes and A549 lung carcinoma cells or 22rv1 prostate carcinoma epithelial cells. White triangles showed examples of black large syncytia revealed after X-Gal staining. Scale bar: 500 pm.
Figure 5: VSV pseudotyping efficacy of WT or truncated ASCT2-targeted retroviral envelopes.
(A) Schematic representation of the protocol for pseudotyping VSVAG with retroviral envelopes (Created with BioRender.com). VSVAG vectors carrying heterologous envelopes were produced and used for transduction of target cells. Titers were analyzed 24h postransduction by flow cytometry based on GFP expression (Transduction Unit/mL) (B) Transduction of 293TWT cells with VSV pseudotypes carrying WT/truncated envelopes or no envelope. The data were shown as a mean of three assays and were tested for significance by Student’s t test (* p < 0.05; ** p < 0.01). (C) Transduction of 293TWT/293T KOASCT2 cells transiently transfected with EV/ASCT2, with VSV pseudotypes carrying the indicated truncated envelopes or the VSV-G envelope as a positive control. The average of two independent experiments was presented.
Figure 6: Characterization of ASCT2-targeted viruses.
(A) Schematic overview of the genomic organization of the wildtype and newly generated VSV. (B) Titers of the wild type and ASCT2-targeted VSV with or without ultracentrifugation (Plaque Forming Unit/mL). (C) Western Blotting analysis of ASCT2 expression in the established Chinese hamster ovary (CHO) cells. The protein sample of 293T cells served as highly ASCT2-expressed control. y-Tubulin was used as a loading control. (D) The established CHO cells were infected with VSV-Syn1A53, -SynOry1A25, -RD114A18 or VSV- G at an MOI of 0.1. CHOEV served as receptornegative cell line. Photos represent GFP- fluorescent images taken 48h postinfection for CHOEV and CHOASCT2 c9. White arrows: examples of large syncytia induced by infection. Scale bar, 200 pm. (E) Cell viability of the established CHO cells infected with the indicated viruses at an MOI of 0.1 , at 72hpi. The percentage of living cells was calculated in relation to mock-infected control culture, which was set to 100%. The results are an average of three technical replicates and were analyzed for significance by 2way ANOVA with Tukey’s multiple comparisons test (** p < 0.01 ; *** p < 0.001 ; **** p < 0.0001).
Figure 7: Infection and cytotoxicity induced by generated viruses on monolayer cancer cell cultures.
A549, MCF-7, 22rv1 and MIAPACA-2 cancer cell lines, and MR0015 patient-derived tumor cells were infected with VSV-Syn1A53, - SynOry1A25, -RD114A18 or VSV-G at an MOI of 1 (A) GFP-fluorescent and bright field (BF) images represent infection on A549 cells at 24 h postinfection. Large syncytia formation is indicated by white arrows (largest syncytia of VSV- SynOry1A25 are less fluorescent). Scale bar, 100 pm. (B) Cell viability after infection of the indicated cells, measured every 24h postinfection. Depicted is the percentage of living cells in relation to mock-treated control, which was set to 100%. The data shows an average of three biological and three technical replicates. 2way ANOVA with Tukey’s multiple comparisons test was used to analyze for significance (* p < 0.05; ** p < 0.01 ; *** p < 0.001; **** p < 0.0001).
Figure 8: Infection and killing on breast cancer-derived spheroid cultures.
Spheroids established from SUM52PE cells were infected with VSV-Syn1A53, - SynOry1A25, -RD114A18 or VSV-G at an MOI of 0.1. 125 nM Incucyte® Cytotox Red Dye that labels dead cells with red fluorescence were added at the same time as infection. (A) Bright field (2 left columns - BF), green (2 center columns - GFP) and red fluorescence (2 right columns - RFP) images of the infected spheres at 24 and 72h pi are shown. Scale bar, 500 pm. (B) Cell viability was determined everyday within 96 h after virus addition. The percentage of living cells was calculated in relation to mock culture at each time, which was set to 100%. Average values of two biological and three technical replicates are shown. The asterisks indicate a significant difference (**** p < 0.0001) between groups (2way ANOVA with Tukey’s multiple comparisons test).
Figure 9: Oncolytic activity of ASCT2-targeted viruses on pancreatic cancer organoid cultures.
Organoids were generated from pancreatic cancer cells isolated from patients and treated with the indicated amount of VSV-Syn1A53, -SynOry1A25, -RD114A18 or VSV-G. (A) Shown is bright field (top row - BF), GFP (center row), overlaid (bottom row - BF/GFP) images captured after 48h of infection. White arrows indicate bubble-like membrane blebs induced by fusion. Scale bar, 100 pm. (B) Cell killing was assessed by the measurement of cell viability at 48h and 96h post infection. The percentage of living cells was determined in relation to uninfected control, which was set to 100%. The data shows average values of two biological and two technical replicates.
Figure 10: Infection of retargeted viruses on precision-cut tumor slice cultures. (A) Immunohistochemistry analysis of ASCT2 protein expression in MR0015 and MR0347 PDX. Black squares indicate localization of 10X view. (B) Following vibratome sectioning of fresh PDX samples, tumor slices (300pm-thick) were treated with 4x106 PFUs of VSV-Syn1A53, - RD114A18 and VSV-G or 106 PFUs for VSV-SynOry1A25. Mock treated slices served as controls. GFP signal was monitored via a fluorescence microscope within 48h after virus treatment. Overlaid pictures (BF/GFP) at 48h post infection are shown.
Figure 11 : Infection of retargeted viruses on normal cells.
Granulocytes, monocytes and lymphocytes isolated from whole blood, or human placenta- derived villous cytotrophoblasts (VCTs) were treated with VSV-Syn1A53, -SynOry1A25, - RD114A18 or VSV-G at an MOI of 1. 48h after virus addition, the percentage of infected cells was determined by detection of GFP-positive cells in flow cytometry. Images represent the gating in flow cytometry for blood cell populations. Average values of two biological and three technical replicates are shown and tested for significance by 2way ANOVA with Tukey’s multiple comparisons test (* p < 0.05; **** p < 0.0001).
Figure 12: Alignment of the C-terminal regions of ASCT2-targeted retroviral envelopes and the corresponding truncations at intracytoplasmic tail.
Dot line indicates the position of the truncation sites, as determined from the alignment with Syn1A53. ED: Ectodomain (grey). TM: Transmembrane region (grey). CT: Cytoplasmic tail (bold). Amino acid positions are indicated on the right.
Figure 13: ASCT2 and ASCT1 transcriptional levels in tumor and normal cells.
(A) Quantification of ASCT2 and ASCT1 expression by RT-qPCR in tumor cells. Gene expression was normalized to the RPLP0 level. (B) RT-qPCR analysis of ASCT2 211 mRNA levels in the indicated cancer cells, in leukocytes isolated from whole blood and in placenta- derived villous cytotrophoblasts (VCTs). 293T cells were considered as the positive control for ASCT2 expression. Gene expression was normalized to the RPLP0 level. Figure 14: ASCT2 genomic locus with the position of the gRNAs used to knockout ASCT2 in 293T cells.
The ASCT2 gene is composed of eight exons and has two transcriptional variants that differ in their initiation site (NM_005628.2 (SEQ ID NO: 99) and NM_001145145.1 (SEQ ID NO: 100)) (Scalise M et al. 2018. The Human SLC1A5 (ASCT2) Amino Acid Transporter: From Function to Structure and Role in Cell Biology. Front Cell Dev Biol 6:96) (Yoo HC et al. 2020. A Variant of SLC1A5 Is a Mitochondrial Glutamine Transporter for Metabolic Reprogramming in Cancer Cells. Cell Metab 31 :267-283.e12). The mitochondrial variant lacks exon 1 and the majority of exon 2. The guideRNA 631fwd was chosen to target the common exon 3.
Figure 15: Fusogenic capacity of WT and truncated envelopes towards ASCT2 receptor in 293T cells.
Fusion assay between A23 cells transfected with WT/truncated envelopes and 293T KOASCT2 cells with or without ASCT2 transfected. Fusion was quantified by measuring absorbance at 570nm after addition of CPRG. The results were obtained from two separate experiments.
Figure 16: Construction of recombinant Measles virus (MV) Hemagglutinin (H) proteins to target ASCT2, and evaluation of their fusogenic function and expression level.
(A) Schematic representation of MV H protein bound to the RBD of Syn1 or to Suppressyn. RBD: Receptor Binding Domain, TM: transmembrane region, CT: intracytoplasmic region. The H protein ectodomain used is mutated on 4 residues (H*) to impair recognition of its native receptors and truncated by 18aa in its intracytoplasmic region. The Syn1 RBD/Suppressyn and the His tag were grafted to the H protein at the C-terminal position via a linker. (B) (upper) schematic representation of the fusion assay in 293T cells, that express endogenous levels of ASCT2, with expression vectors for MV H*-RBD/Supp + MV fusion protein (F). After X-Gal staining, syncytia were revealed in blue, (lower) bright field images representing fusion on 293T cells at 48hours post co-culture; EV: empty vector, for the determination of background fusion levels. Large syncytia formation was detected with MV H*-RBD Syn1 and MV H*-Suppressyn expression vectors, and not with EV vector. Scale bar: 200|jm. (C) Measurement of the expression of H-RBD Syn1 , and -Suppressyn proteins on the cell surface. 293T cells were transfected with expression vectors for the indicated envelopes. After 48 h, recombinant H proteins were detected by flow cytometry using an anti- His tag antibody.
Figure 17: MPZL1 and GLUT-1 are overexpressed in many tumor types as compared to healthy cells (TCGA/GTEX).
MPZL1 and GLUT1 are overexpressed in several primary solid tumors as compared to the related non tumoral tissue. Boxplots of normalized (TPM) and Iog2-transformed expression of MPZL1 (upper) and GLUT1 (lower). Filled pale grey boxes: expression in tumors (T) retrieved from TCGA (The Cancer Genome Atlas)(Recount2). Filled dark grey: basal expression in normal and adjacent tissues (N) from GTEx (Genotype-Tissue Expression)- Recount2 and TCGA, respectively. The number of samples analyzed in each group (T, N) is shown on the x axis. The (T,N) P-values are shown as asterisks above the boxplots: *P < 0.05; Mann-Whitney U-test. The names of the cohorts are given in the text.
Figure 18: Infection efficiency of VSV particles pseudotyped by Syn-Mab1.
(A) Results of pseudotyping assays with Syn-Mab1 (N=3); target cells: 293T; (* = p<0,05; Student T-Test). (B) Results of pseudotyping assays with Syn-Mab1 ; target cells: CHO empty vector and CHO expressing MPZL1 (N=1). ISFV: pseudotypes with the envelope of Isfahan virus, exhibiting high titer (positive control). EV: pseudotypes produced in the absence of envelope (background infection level).
Figure 19: Capacity of the HTLV-1 and HTLV-1 A8 envelopes to pseudotype and induce syncytia. (A) Results of the pseudotyping assays with HTLV-1 (N=2) and HTLV-1 A8 (N=2) envelopes; target cells: 293T. ISFV: pseudotypes with the envelope of Isfahan virus, exhibiting high titer (positive control). EV: pseudotypes produced in the absence of envelope (background infection level). (B) Microscopy images of syncytia induced by the interaction of envelopes with their receptor in a cell-cell fusion assay, oa (transfection control), o+a (background), SynA (positive control) (N=3). Objective x4. Syncytia are colored blue after X-gal coloration).
Figure 20: Infection, syncytia formation and cytotoxicity induced by IRPOVs in the canine DH82 cancer cell line.
Infection (MOI 1) of DH82 cells by the different IRPOVs and wild-type VSV-G virus. (A) Representative images, acquired by microscopy (white light and fluorescence) 24 hpi, x10 objective, scale: 100 pm. Arrows indicate syncytia. (B) Kinetics of the measurement of the amount of ATP released following lysis of DH82 cells with the CellTiter-Glo kit, indirectly reflecting cell proliferation during the three days following infection. Cell lysis was measured by luminescence. Results obtained in experimental triplicates and biological duplicates. Representation of means +/- standard error of the mean (SEM).
Figure 21 : Infection of a 3D canine tumor cell model by the IRPOVs.
Spheroids obtained in 72h by culturing REM 134 cells, under conditions of very low adhesion, followed by infection with the different modified and wild-type viruses at an MOI of 10. (A) Representative images taken at 24hpi and 72hpi by brightfield and fluorescence microscopy, x10 objective, scale: 100 pm. Incucyte Cytotox Red Dye marks dead cells (125 nM). Arrows point to cell fusion, characterized by the appearance of bubbles. (B) Kinetics of the amount of ATP released up to 8 days post-infection, following lysis of spheroids by CellTiter-Glo. Amount measured by luminescence. Results obtained in experimental triplicates and biological duplicates. Representation of means +/- standard error of the mean (SEM).
EXAMPLES
EXAMPLE No. 1 - IRPOV specifically directed against ASCT2
MATERIALS AND METHODS
RNAseq datasets collection
Uniformly processed RNAseq data from GTEx (Genotype-Tissue Expression project) for normal tissues (N: 11751 samples) and from TCGA (The Cancer Genome Atlas) for human tumor (T: 10187 samples) and non-tumor control adjacent tissues (C: 1745 samples) from a total of 33 different anatomic sites were downloaded from Recount2 resource (https://jhubiostatistics.shinyapps.io/recount/). Gene expression was quantified and reported in TPM unit (Transcripts Per Million of reads). Profile of ASCT2 expression was obtained using ENSEMBLJd ENSG00000105281 (Slc1a5).
The names of the cohorts are given in Table 1. Table 1 : Absolute and relative levels of mean ASCT2 expression in tumor-TCGA and normal-GTEx tissue samples. Samples with median values > 6 (Iog2 (TPM+1)) or with T/N median Iog2 fold change >1 are written in bold. _
Median value Median
(log2(TPM+1)) fold change
_ (Iog2)
System TCGA T N T/N cohorts
DIGESTIVE ESCA Esophageal Carcinoma 7.08 5.52 1.56
STAD Stomach 6.39 5.1 1.27
Adenocarcinoma COAD Colon Adenocarcinoma 7.44 4.93 2.5
READ Rectum Adenocarcinoma 7.58 na na
LIHC Liver Hepatocellular 3.02 1.83 1.19
Carcinoma CHOL Cholangiocarcinoma 6.03 na na
PAAD Pancreatic 6.19 4.07 2.12
Adenocarcinoma
URINARY KICH Kidney Chromophobe 3.83 4.38 -0.54
KIRC Kidney renal clear cell 5.46 4.38 1.09 carcinoma
KIRP Kidney renal papillary cell 5.22 4.38 0.84 carcinoma
BLCA Bladder Urothelial 7.43 7.19 0.24
Carcinoma
NERVOUS GBM Glioblastoma Multiforme 4.33 1.01 3.32
LGG Brain Lower Grade 3.51 1.01 2.5
Glioma
UVM Uveal Melanoma 4.91 na na
SKCM Skin Cutaneous 6.23 6.12 0.11
Melanoma
SARC Sarcoma 6.45 5.41 1.05
HNSC Head and Neck 7.4 na na
Squamous Cell
Carcinoma
RESPIRATORY LUAD Lung Adenocarcinoma 6.67 6.41 0.25
LUSC Lung Squamous Cell 7.57 6.41 1.16
Carcinoma
MESO Mesothelioma 5.79 na naitwo times
ENDOCRINE PCPG Pheochromocytoma and 3.84 3.74 0.1
Paraganglioma
ACC Adrenocortical Carcinoma 2.46 3.74 -1.28
THCA Thyroid Carcinoma 4.44 4.28 0.16
FEMALE BRCA Breast Invasive 6.86 6.47 0.39
Carcinoma OV Ovarian Serous 6.13 5.84 0.29
Cystadenocarcinoma UCEC Uterine Corpus 5.96 5.61 0.35
Endometrial Carcinoma UCS Uterine Carcinosarcoma 6.23 5.61 0.62
CESC Cervical Squamous Cell 7.55 5.96 1.6 Carcinoma
MALE PRAD Prostate Adenocarcinoma 7.67 6.8 0.87
TGCT Testicular Germ Cell 5.62 5.87 -0.26
Tumors
THYM Thymoma 4.93 na na
HEMATOLOGICAL DLBCL Diffuse Large B-cell 6.06 na na
Lymphoma
LAML Acute Myeloid Leukemia 5.6 7.91 -2.31
Cells
293T, BSR (subclone of BHK-21 cells), Vero NK, A549, MIAPACA2 and MCF-7 cells were all cultivated in DMEM (Sigma-Aldrich, Germany) supplemented with 10% FCS (Gibco, Germany) and 1% Penicilline-Streptomycine (Gibco, Germany). 22rv1 and SUM52PE cells (gift from Dr C. Nahmias, IGR) were cultivated in RPMI (Sigma-Aldrich, Germany) supplemented with 10% FCS and 1% Penicilline-Streptomycine. A23 Gala and A23 Galco cells stably expressing the Gala and Galco peptides of p-galactosidase (Bacquin A et al. 2017. A Cell Fusion-Based Screening Method Identifies Glycosylphosphatidylinositol- Anchored Protein Ly6e as the Receptor for Mouse Endogenous Retroviral Envelope Syncytin-A. J Virol 91 :e00832-17) and BSRT7 cells stably expressing T7 polymerase (gift from Dr Y. Gaudin, Gif sur Yvette, France) were grown in DMEM supplemented with 10% FCS and 1 mg/ml Geneticin. CHO-EV and CHO-ASCT2 cells were generated by stable integration of an empty vector (EV) and the human ASCT2-expressing vector, respectively, into Chinese hamster ovary CHO-K1 cells. The cells were cultivated in Ham’s F12 (Sigma- Aldrich, Germany) supplemented with 10% FCS and 400 pg/ml Hydromycine B. PBMCs (monocytes and lymphocytes) and granulocytes were isolated from whole blood by Ficoll density gradient centrifugation (Lymphocytes separation medium, Eurobio Scientific, France). Cells were maintained in RPMI supplemented with 10% FCS and 1% Penicilline- Streptomycine. Villous cytotrophoblasts (VCTs) isolated from human placenta were kindly provided by Dr S. Degrelle (Paris Cite University, France) and maintained in DMEM without phenol red (Gibco, Germany) supplemented with 10% FCS and 1% Glutamax (Gibco, Germany). Patient-derived cells developed from the MR0015 PDX sample (MATCH-R trial, IGR) were kindly given by Dr L. Friboulet (IGR) and cultivated in DMEM supplemented with 10% FCS and 1% Penicilline-Streptomycine.
Plasmids and cloning of truncated envelops
Based on sequence alignment to Syn1A53 (SEQ ID NOs: 85 and 86) (Lavillette D et al. 2002. The envelope glycoprotein of human endogenous retrovirus type W uses a divergent family of amino acid transporters/cell surface receptors. J Virol 76:6442-6452), truncated positions at the level of cytoplasmic tails of other retroviral envelopes were determined. Truncated sequences of viral envelopes were then amplified by PCR from phCMV expression plasmids containing the inserts of the wild-type envelopes: DasyEnv1.1 (SEQ ID NOs: 41 and 42) (Malicorne S et al. 2016. Genome- Wide Screening of Retroviral Envelope Genes in the Nine-Banded Armadillo (Dasypus novemcinctus, Xenarthra) Reveals an Unfixed Chimeric Endogenous Betaretrovirus Using the ASCT2 Receptor. J Virol 90:8132- 8149), Syn1 (SEQ ID NOs: 37 and 38), SynOryl (SEQ ID NOs: 39 and 40) (Heidmann O et al. 2009. Identification of an endogenous retroviral envelope gene with fusogenic activity and placenta-specific expression in the rabbit: a new “syncytin” in a third order of mammals. Retrovirology 6:107), EnvRD114 (SEQ ID NOs: 43 and 44) and MPMV (SEQ ID NOs: 45 and 46) (kindly provided by F.L. Cosset, Lyon), BaEV (SEQ ID NOs: 47 and 48) (kindly provided by Els Verhoeyen, Lyon) and SRV-2 (SEQ ID NOs: 51 and 52) (synthetized by Genecust, France) using specific primers (Table 2) and SeqAmp Polymerase (Takara, USA). Next, the truncated sequences were inserted into a new phCMV plasmid via Xhol/Mlul or Notl/Mlul restriction sites. Table 2: List of the primers used for cloning of truncated envelopes.
Primer name Sequence (SEQ ID NO)
Figure imgf000089_0001
Syn1-Xhol-F1 ATACATCTCGAGAACAACCAGGAGGAAAGT ( 101 )
Syn1del53-STOP-Mlul-R4 ATACATACGCGTCTATAGTTTTACAGCTTCGAT ( 102 )
DasyTAER-S2-Xhol ATACATCTCGAGTCCTCCTCCCATAACTGATA ( 103 )
DasyTAERdel28-STOP-MlulR8 ATACATACGCGTCTAGCCAAGAGCGGAGTCAAC ( 104 )
TAER S2 Xhol ATCACCTCGAGTGCTGGAATTGTTGTCATTGTTG ( 105 )
SynOry1del25-STOP-Mlul-R1 ATACATACGCGTCTAGGCTGCTAAATTATCTAC ( 106 )
RD114-S1-Xh0l ATACATCTCGAGGTCCCTGTACTAACCCAAAA ( 107 )
RD114del18-STOP-Mlu1 ATACATACGCGTCTAGGCATGTACAACATTAAG ( 108 )
MPMV-ATGKozak-Xhol-F1 ATACATCTCGAGGCCACCATGAACTTCAATTATCA ( 109 )
MPMVdel23-STOP-Mlul-R1 ATACATACGCGTCTAGGCCTGGATGCTCTCAAT ( 110 )
BaEV-ATGKozak-Xhol-F1 ATACATCTCGAGGCCACCATGGGATTCACAACAAA ( i l l )
BaEVdel18-STOP-Mlul-R1 ATACATACGCGTTCAAGCGTGTATTATGTTTAA ( 112 )
SRV-2-N0tl-F1 ATACATGCGGCCGCGTCTCCCAGAGATCACT ( 113 )
SRV-2del23-STOP-Mlul-R1 ATACATACGCGTTCAAGCTTGGATGGCATCCAT ( 114 )
Biological samples
All patient samples were obtained with written informed consent. Formalin-fixed, paraffin- embedded (FFPE) and frozen samples of tumor and normal tissues were collected from the Biological Resource Centre (BB-0033-00074) and the Department of Pathology and Laboratory Medicine of GRCC (Gustave Roussy Cancer Campus/Research Agreements RT09916 and RT14017).
RNA extraction and RT-qPCR
For tissue samples, OCT-frozen tumor and normal tissues were sectioned with a cryostat. Non-OCT frozen samples were disrupted with a mortar. Ten sections of 50 pm or 20-30 mg of tissue fragment were mechanically disrupted with glass beads, and RNA extraction was performed with RNeasy Isolation Kit (Qiagen, Germany) according to the manufacturer’s instructions. The same kit was used for RNA extraction of cells as well. After the treatment with DNase I (Invitrogen, USA), RNA quality and concentration were evaluated using a NanoDrop ND-1000 spectrophotometer (ThermoFischer Scientific, USA). Reverse transcription was performed with 1 pg RNA using Maxima reverse transcriptase and random hexamers (ThermoFischer Scientific, USA). qPCR experiments were run on an ABI Prism 7000 sequence detection system with SYBR green PCR master mix reagent (Qiagen, Germany) and specific primers: SLC1A5 gene: forward: TCGATTCGTTCCTGGATCT (SEQ ID NO: 115), reverse: ATGTTCATCCCCTCCACCT (SEQ ID NO: 116);
RPLPO gene: forward: GGCGACCTGGAAGTCCAACTA (SEQ ID NO: 117), reverse: CCATCAGCACCACAGCCTTC (SEQ ID NO: 118);
SLC1A4 gene: forward: CATCGCTGTCGCCTACTTT (SEQ ID NO: 119), reverse: CTCTTTGGGGACA-GGAGGAG (SEQ ID NO: 120).
Gene expression level was quantified using the AACT method and normalized to the level of housekeeping gene RPLPO.
Viral genome was extracted by Viral RNA mini kit (Qiagen, Germany) as per the manufacturer’s instructions. Reverse transcription was performed as above, and virus genes were amplified on cDNA by PCR using SeqAmp Polymerase (Takara, USA) and the following primers: Syn1A53 gene: forward: ATACATCTCGAGAACAACCAGGAGGAAAGT (SEQ ID NO: 121), reverse: ATACATACGCGTCTATA-GTTTTACAGCTTCGAT (SEQ ID NO: 122);
SynOry1A25 gene: forward: ATCACCTCGAGTGCTGGAA-TTGTTGTCATTGTTG (SEQ ID NO: 123), reverse: ATACATACGCGTCTAGGCTGCTAAATTATCTAC (SEQ ID NO: 124);
RD114A18 gene: forward: ATACATCTCGAGGTCCCTGTACTAACCCAAAA (SEQ ID NO: 125), reverse: ATACATACGCGTCTAGGCATGTACAACATTAAG (SEQ ID NO: 126); VSV-N gene: forward: ATACATCTCGAGATGTTCTTCCACATGTTC (SEQ ID NO: 127), reverse: ATACATACGGCTTCATTT-GTCAAATTCTGACTT (SEQ ID NO: 128).
Immunohistochemistry (IHC)
Tumor or normal tissues were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 4pm-thick sections. After heat-induced antigen retrieval (ER2 buffer pH9), the sections were incubated with antibody against ASCT2 (1 :200, clone D7C12 cat. no. 8057, Cell Signaling Technology, U.S.A.) for 30m at room temperature. Revelation was performed using a Bond Polymer Refine Detection kit (Leica Biosystems, #DS9800). A whole tumor or normal tissue section was digitized using a slide scanner (VS120, Olympus).
Protein extraction and Western Blot analysis
For protein extraction of CHO or 293T cell lines, cells were lysed in RIPA buffer (ThermoFischer Scientific, USA) supplemented with HaltTM Protease Inhibitor Cocktail (ThermoFischer Scientific, USA). Next, cellular protein samples were separated in SDS- PAGE gels (NuPAGETMNovex 10% Bis-Tris gels, ThermoFischer Scientific, USA) and transferred onto nitrocellulose membranes. The membranes were blocked in PBS containing 5% nonfat milk powder and 0.1% Tween for 1 h at room temperature and then incubated with primary antibodies at 4°C overnight. The antibodies used were anti-ASCT2 (1 :1000, cat. no. 8057, Cell Signaling Technology, U.S.A.) and anti-yTubulin (1 :1000, cat. n° T5326, Sigma- Aldrich, USA). Next day, membranes were incubated for 1h at room temperature with antirabbit IgG, HRP-linked antibody (1 :5000, cat. no° 7074, Cell Signaling Technology, USA) for detecting ASCT2 or anti-mouse HRP-conjugated secondary antibody (1 :5000, GE Healthcare, USA) for detecting yTubulin. Protein detection was performed by using enhanced chemiluminescence reagents (SuperSignal™ West Pico PLUS Chemiluminescent Substrate, ThermoFischer Scientific, USA) and iBright camera system (ThermoFischer Scientific, USA).
Establishment of 293T KOASCT2 cell line
The guideRNA 631 fwd (SEQ ID NOs: 129 and 130) targeting the exon 3 of the human SLC1A5 gene (SEQ ID NO: 1) was selected from the CRISPOR site (www.CRISPOR.fr) (Figure 13), and a lentiCRISPR vector containing the gRNA was established. The vector was stably transfected into 293T cells using Lipofectamine LTX (Invitrogen, USA) according to the protocol provided. The cells were amplified and then selected in the presence of 1 pg/mL puromycin. The silencing efficacy of the gRNA was confirmed by Western blotting on the resistant cell population or clones. DNA was also extracted from transfected 293T cells using DNA/RNA Kit (Qiagen, Germany), and the targeting region was amplified by PCR and subjected to sequencing to analyze for insertions/deletions.
Cell-cell fusion assay
This assay was previously established in the laboratory (Bacquin A et al. 2017. A Cell Fusion-Based Screening Method Identifies Glycosylphosphatidylinositol-Anchored Protein Ly6e as the Receptor for Mouse Endogenous Retroviral Envelope Syncytin-A. J Virol 91 :e00832-17). Briefly, it was performed in 48-well plates with the following cell combinations: A23-Gala with A23-Galco/293TWT/293T-KOASCT2 or tumor cell lines. 24h after seeding, A23-Gala cells were transfected with the expression vectors of envelopes, while A23 Galco, 293TWT, 293T-KOASCT2 and tumor cell lines were transfected with the expression vectors of Galco with or without ASCT2/ASCT 1 receptors co-transfected. On the next day, the cells were detached with trypsin and co-cultured in a 1 :1 ratio. 48h later, the fusogenicity between the cells was revealed either by X-gal staining or by a Chlorophenol red- -D- galactopyranoside (CPRG) assay.
X-gal staining allows visualization and counting of syncytia. Cells were fixed then stained with 100 pg/mL X-Gal solution (Euromedex, France). Syncytia turned blue thank to hydrolysis of the X-gal substrate by p-galactosidase formed by complementation of the gala and galco peptides. For automatic counting and calculation of pixel area of syncytia, images of X-Gal-stained cells were analyzed by Imaged software (Bacquin A et al. 2017. A Cell Fusion-Based Screening Method Identifies Glycosylphosphatidylinositol-Anchored Protein Ly6e as the Receptor for Mouse Endogenous Retroviral Envelope Syncytin-A. J Virol 91 :e00832-17). For the CPRG assay, cells were lysed in a mixture solution of 1 mM MgCh, 1 mM NP40 for 1 h at 4°C. The cell lysate was subsequently mixed with CPRG solution (1 mM MgCh, 1 mM NP40, 1 mM CPRG (Sigma Aldrich, USA)). B-galactosidase hydrolyzed CPRG, resulting in the release of red chlorophenol with an absorbance peak at 570 nm. The fusogenicity between the cells was then quantified by absorbance measurements at 570nm.
VSV Pseudotying assay
The production of VSVAG pseudotypes followed the protocol provided by Dr Y. Gaudin (Gif sur Yvette, France) (Figure 5A) (Ferlin A et al. 2014. Characterization of pH-sensitive molecular switches that trigger the structural transition of vesicular stomatitis virus glycoprotein from the postfusion state toward the prefusion state. J Virol 88:13396-13409). The viral vector employed was the VSVAG-G*/GFP (kindly provided by Dr Y. Gaudin, Gif sur Yvette, France), which contains a gene coding for GFP but no longer has the gene coding for glycoprotein G at the genome level. However, VSVAG-G*/GFP has the glycoprotein G on its surface, allowing one-cycle infection. 293T-KOASCT2 or BSR cells were seeded at a rate of 3.106 and 1.1.106 cells in a 6 cm dish, respectively. Next day, producing-virus cells were transfected with 2pg of the expression vectors for the envelopes using jetPrime (PolyPlus, France). The following day, transfected cells are infected with VSVAG-G*/GFP at a MOI (Multiplicity Of Infection') of 3 for 4h. The virus inoculum was then removed, and cells were washed four times and incubated in DMEM medium supplemented with 1 % SVF. Target cells were seeded into a single well of a 24-well plate on the same day. 24 hours later, cell supernatant containing the pseudotyped particles were harvested and filtered through a 0.22pm filter. For transduction, pseudotyped particles were serially diluted and a total of 300 pl of the dilutions was added per well of target cells. Transduction was then aided by centrifugation at 1200g for 2h at 25°C. After 24h, titers were determined by flow cytometry analysis based on the percentage of green fluorescent cells taken between 5% and 40%.
Generation of the viruses
For cloning into the VSV genome plasmid of Syn1A53 (SEQ ID NOs: 85 and 86), SynOry1A25 (SEQ ID NO: 87 and 88) or RD114A18 (SEQ ID NO: 91 and 92), the G coding sequence (SEQ ID NO: 3) in the plasmid MC11-VSV-eGFP (encoding the non-attenuated Indiana serotype; gift from Dr K. W. Peng (Mayo Clinic, USA)) (Ammayappan A et al. 2013. Neuroattenuation of vesicular stomatitis virus through picornaviral internal ribosome entry sites. J Virol 87:3217-3228), based on the plasmid pVSV-XN2 (SEQ ID NO: 2) (Lawson ND et al. 1995. Recombinant vesicular stomatitis viruses from DNA. Proc. Natl. Acad. Sci. U. S. A. 92:4477- 4481), was exchanged against that of the truncated envelope via Mlul/Avrll restriction sites.
To rescue VSV-Syn1A53, -SynOry1A25 or -RD114A18, the newly constructed plasmids of VSV-Env were employed in transfection experiments along with helper plasmids (encoding structural proteins and enzymes of VSV being under T7 promoter control and contain an IRES element) as described in Harty RN et al. (Vaccinia virus-free recovery of vesicular stomatitis virus. Journal of Molecular Microbiology and Biotechnology. 2001 Oct;3(4):513- 517. PMID: 11545270). The T7 RNA polymerase was expressed constitutively from BSR-T7 cells, allowing vaccinia virus-free recovery of VSV. The helper vectors containing an IRES element allows cap-independent translation of VSV proteins in T7 RNA polymerase- expressing cells. Briefly, BSR-T7 cells at approximately 80% confluency were transfected with 10pg of VSV-Env, 1-5pg of helper plasmids using Fugene6 (Promega, Germany). At 24 hours postransfection, the cells were trypsinized, pelleted for 10 minutes at 3000 rpm, and overlaid on permissive Vero-NK cells expressing endogenous ASCT2. From 48h of coculture, green fluorescent and/or syncytia formation was noticed. Virus-containing supernatant was then harvested, clarified, and stored at -80°C. To produce large amounts of infectious VSV, VSV-Env viruses were incubated on Vero-NK cells at a MOI of 0.00001. Supernatant was harvested at 3 days after infection, filtered using 0.22 pM filter and stored at -80°C. To concentrate the virus, supernatants were ultracentrifuged at 25,000rpm for 2h at 4°C (Beckman Coulter, USA). For VSV-Syn1A53, to investigate evolution of the envelope over time, eleven sequential passages in Vero NK cells were performed.
All viruses were handled under biosafety level 2 conditions (installation L2-0334, authorization by the Haut Conseil des Biotechnologies, France).
Viral titration
Vero E6 cells were seeded in 12-well plates (5x105 cells/well) and grown overnight in growth medium. Serial dilutions of infectious recombinant VSV were used to infect Vero E6 monolayers (200 pl/well). Plates were incubated for 1 h at 37 °C to allow viral adsorption. Then, 1ml/well of overlay containing DMEM and 0.6% agarose (Euromedex, France) (ratio 1 :1) was added to each well and plates were incubated at 37 °C, 5% CO2 for 72 h. Finally, the cells were fixed with 4% PFA and stained with 1% crystal violet solution (Sigma Aldrich, USA). The number of plaques in each well was determined, and VSV titer was calculated.
Cell viability
Monolayer cells
For analyzing the viability of virus-infected monolayer cells, A549, MCF-7, 22rv1, MIAPACA- 2 and patient-derived MR0015 cells were seeded at 1.9x104, 3.4x104, 3.4x104, 2.8x104, 3.4x104 per well in 96-well plates and infected at a MOI of 1. 2.4x104 CHO EV or CHO expressing ASCT2 cells were seeded in 96-well plates and infected at a MOI of 1 and 0.1. Cell viability of infected cells was determined using the CellTiter-Glo Luminescent Cell Viability assay (Promega, Germany) according to the manufacturer’s protocol.
SUM52PE spheroids
SUM52PE cells were seeded at 5000 cells per well in 96-well round bottom previously treated with PolyHEMA (Santa Cruz, USA) to avoid cell adherence, followed by centrifugation at 1200rpm for 5m to pellet cells (Rodrigues-Ferreira S et al. 2019. Improving breast cancer sensitivity to paclitaxel by increasing aneuploidy. Proc Natl Acad Sci U S A 116:23691-23697). The plate was then returned to culture at 37 °C. Spheroids were established after 72h of culture and then infected with viruses at 5000 and 500 PFUs per well (MOI of 1 and 0.1 , respectively). To monitor the live/dead status of spheroids, Incucyte® Cytotox Red Dye (Sartorius, Germany) that stains dead cells with red fluorescence was added simultaneously at 125nM. CellTiter-Glo 3D Luminescent Cell Viability assay (Promega, Germany) was used to determine cell viability of infected spheroids.
Patient-derived pancreatic cancer organoids
PGR4 organoids were established and kindly provided by Dr F. Jaulin (IGR). Patient-derived pancreatic cancer cells were cultivated in a Matrigel dome at 37 °C and were able to form organoids within 5 days. Next, well-established organoids were infected with VSV-Syn1A53, -SynOry1A25, -RD114A18 and wild-type viruses in the absence of Matrigel at 720000 PFUs, 840000 PFUs, 7200000 PFUs and 3600000 PFUs respectively. Cell viability of infected organoids was determined using the CellTiter-Glo 3D Luminescent Cell Viability assay (Promega, Germany).
Patient-derived xenograft model and precision-cut tumor slices culture
All animal procedures and studies were performed in accordance with the approved guidelines for animal experimentation by the ethics committee at University Paris Sud following EU regulation (APAFIS#2790-2015112015055793). MR0015 and MR347 xenografts established from tumor biopsies derived from patients in MATCH-R trial were collected from mice. A piece of tumor tissue (approximate dimensions 5 x 5 x 5 mm) was sampled using a sterile scalpel and submerged in a solution of 8% low gelling temperature Agarose (Sigma Aldrich, USA). The complex was chilled on ice until the complete gelling of Agarose, and then the Agarose-embedded tumor was sectioned into 300pm-thick slices using a vibrating-blade microtome (VT1200S, Leica, Germany). Each tissue slice was transferred to an insert (0.4 pm pore size, 12 mm diameter, Millicell®, Millipore, Ireland) that was placed in a well of a 6-well plate containing 1.1 ml of complete culture medium. The plate was kept in ice-cold condition until completion of the sectioning process. Next, 500ml viral supernatant containing 4x106 PFUs of viruses (except 106 PFUs for VSV-SynOry1A25) was added on top of the tumor slice. Within 48h of infection, evaluation of fluorescence was performed using a fluorescence microscope (Leica DMi8, Leica Microsystems, Germany). Infection with viruses was finally shut down by fixation of the tumor slices in PFA 4%.
Statistical analysis
Statistical significance was assessed as specified, using GraphPad Prism software (GraphPad Inc, La Jolla, CA)
RESULTS
ASCT2 expression in solid tumors vs normal tissues
In order to estimate ASCT2 expression levels in tumors, it was analyzed the RNAseq-based transcriptome of cancer samples retrieved from TCGA (The Cancer Genome Atlas) cohorts. ASCT2 expression levels observed in tumors were then compared to those of TCGA’s nontumor adjacent tissues (‘Control’) as well as non-diseased (‘Normal’) tissues from the GTEx (Genotype-Tissue Expression) project (lists in Table 1). As shown in Figure 1, evidence of high expressing cases (Iog2 (TPM+1)) median value >6) was highlighted for a series of solid tumors, as for instance the digestive carcinomas of esophagus, stomach, colon, rectum, bile duct and pancreas, the melanoma, the carcinoma of bladder, head and neck, lung and prostate, as well as the carcinoma of female (breast, ovary, uterus and cervix). Basal or significant levels of ASCT2 were detected in most of GTEx normal tissues (empty grey boxes), with a very low levels observed in liver and brain. Of note, it could be observed a high level of ASCT2 expression in TCGA normal bone marrow/whole blood at a level similar to that of some tumor samples. This was taken into account when evaluating the use of ASCT2 as a therapeutic target of oncolytic virotherapy (cf. Figure 11). Most of the TCGA’s control adjacent tissues (empty black boxes) displayed ASCT2 levels similar to those of GTEx tissues, with some discrepancies in cohorts such as colon or thyroid, reflecting a possible precancerous state in corresponding adjacent TCGA tissues.
Overall, to quantify ASCT2 activation in the different tumors, the fold change between the medians of tumor-TCGA samples and normal- GTEx samples was calculated for each individual cohort, based on TPM values (T/N median fold change in Table 1). Accordingly, a large proportion of solid tumors showed a significant enhanced expression of ASCT2, with a median fold change T/N > 2 for most digestive tumors (esophagus, stomach, colon, liver and pancreas), as well as for neural, sarcoma, lung squamous and cervical tumors, and a group of tumors displayed lower but significant ASCT2 up-regulation (kidney papillary, uterine and prostate carcinoma).
As noticed above, unlike the majority of solid tumors, hematological tumors LALM did not exhibit any ASCT2 activation.
Detection of ASCT2 expression at the RNA and protein levels in primary tumors
To investigate further the mRNA levels of ASCT2 in certain types of cancer, RT-qPCR analysis was performed on frozen tumors and normal tissues of breast, pancreas and rectum obtained from Biological Resources Centre of Gustave Roussy hospital (Figure 2A). In general, it was observed heterogeneity of SLC1A5 expression, with however high values in several tumor samples in comparison to normal tissues. Regarding the result of breast carcinoma, the SLC1A5 overexpression was found predominantly in triple negative tumors (4/6 samples). A difference in SLC1A5 expression between tumor and healthy tissues was clearly shown in pancreatic samples (7/9 tumors). The difference in SLC1A5 expression between tumor and normal tissues from rectum samples was not clearly observed. The analysis indicated however 3 of 8 tumors having higher SLC1A5 expression than normal tissues. These results suggest an up-regulation of ASCT2 in certain types of solid tumors.
To verify the protein expression of ASCT2, it was carried out immunohistochemistry analyses in some tissues used in RT-qPCR (Figure 2B). In breast, pancreas and rectum tumor samples, it was noticed the expression of ASCT2 at strong and moderate level. Regarding breast carcinoma, positive ASCT2 expression was found mostly in HER+ and triple negative tumors. Besides, some of tumor tissues showed no evidence of ASCT2 expression such as the samples of luminal breast cancer. The majority of normal tissues demonstrated weak ASCT2 protein level compared to positively expressed tumor samples. At the cell level, ASCT2 is located mainly at the plasma membrane, as expected for a membrane protein. These observations confirm the overexpression of ASCT2 in breast, pancreas and rectum tumors compared to normal tissues, which was shown in RT-qPCR analysis as well as in previous studies.
Truncation of retroviral envelopes results in improved fusion activity towards ASCT2
The human ASCT2 transporter is a receptor used by several retroviral envelopes some of which have little homology to each other (<30%) (Bernhardt S et al. 2017. Proteomic profiling of breast cancer metabolism identifies SHMT2 and ASCT2 as prognostic factors. Breast Cancer Res BCR 19:112) (Lavillette D et al. 2002. The envelope glycoprotein of human endogenous retrovirus type W uses a divergent family of amino acid transporters/cell surface receptors. J Virol 76:6442-6452). It was therefore sought to incorporate several of these envelopes independently into viral vectors to target the same ASCT2 receptor, in order to circumvent a possible anti-Env immunity that would be established upon successive administrations in vivo. The envelopes studied comprise human Syncytin-1 (SEQ ID NOs: 37 and 38) and rabbit Syncytin-Ory1 (SEQ ID NOs: 39 and 40) envelopes, Dasy-Env1 (SEQ ID NOs: 41 and 42) envelope from armadillo, feline D-type RD114 (SEQ ID NOs: 43 and 44) envelope, MPMV (SEQ ID NOs: 45 and 46) (Mason-Pfizer Monkey Virus - SRV-3), BaEV (SEQ ID NOs: 47 and 48) (Baboon Endogenous Virus) and SRV-2 (SEQ ID NOs: 51 and 52) envelopes. Based on sequence alignment with the C-terminal truncated Syn1 (Syn1A53; SEQ ID NOs: 85 and 86) (Lavillette D et al. 2002. The envelope glycoprotein of human endogenous retrovirus type W uses a divergent family of amino acid transporters/cell surface receptors. J Virol 76:6442-6452), truncations were performed at the same position in the cytoplasmic tail of other retroviral envelopes to optimize their infectivity (SynOry1A25 (SEQ ID NOs: 87 and 88), DasyEnvA28 (SEQ ID NOs: 89 and 90), RD114A18 (SEQ ID NOs: 91 and 92), MPMVA23 (SEQ ID NOs: 93 and 94), BaEVA18 (SEQ ID NOs: 95 and 96) and SRV-2A23 (SEQ ID NOs: 97 and 98)) (Figure 12). Transgenes derived from these envelopes, with or without C-terminal truncations, were cloned into expression vectors. These vectors were then tested for fusogenicity, infectivity, and specificity towards ASCT2, by performing cell-cell fusion and pseudotyping assays. Of note, the sodium-dependent neutral amino acid transporter type I (ASCT1/SLC1A4), which is related to ASCT2, is an auxiliary receptor for some envelopes, such as Syn1 (SEQ ID NOs: 37 and 38) and BaEV (SEQ ID NOs: 47 and 48) (Marin M et al. 2000. Sodium-Dependent Neutral Amino Acid Transporter Type 1 Is an Auxiliary Receptor for Baboon Endogenous Retrovirus. J Virol 74:8085-8093). It was therefore also wanted to know whether envelope interactions are specific for ASCT2 or also involve ASCT 1.
First, it was tested the fusogenic capacity of wild-type envelopes and of their truncated versions towards ASCT2/ASCT 1 receptors in hamster non permissive A23 cells. It was used a fusion assay previously developed in our lab, based on the alpha complementation (Bacquin A et al. 2017. A Cell Fusion-Based Screening Method Identifies Glycosylphosphatidylinositol-Anchored Protein Ly6e as the Receptor for Mouse Endogenous Retroviral Envelope Syncytin-A. J Virol 91 :e00832-17) (Figure 3A). The surface and the number of syncytia (p-gal+) formed per unit were quantified by Image J. In the presence of the ASCT2 receptor, all WT envelopes formed syncytia, except for BaEV (SEQ ID NOs: 47 and 48) and SRV-2 (SEQ ID NOs: 51 and 52) showing very few p-gal+ cells. The fusion activities of the DasyEnvA28 (SEQ ID NOs: 89 and 90), SynOry1A25 (SEQ ID NOs: 87 and 88), RD114A18 (SEQ ID NOs: 91 and 92), MPMVA23 (SEQ ID NOs: 93 and 94), and BaEVA18 (SEQ ID NOs: 95 and 96) truncations were significantly enhanced compared to their WT versions (p<0.01), with SynOry1A25 (SEQ ID NOs: 87 and 88) forming the largest surface of syncytia among the truncated envelopes. This was not observed with Synlwt (SEQ ID NOs: 37 and 38) and Syn1A53 (SEQ ID NOs: 85 and 86), whose fusogenicity was quite similar regarding both the surface and the number of syncytia. In the presence of the ASCT1 receptor, no syncytium was obtained with RD114 (SEQ ID NOs: 43 and 44), MPMV (SEQ ID NOs: 45 and 46) or SRV-2 (SEQ ID NOs: 51 and 52) while SynOry1A25 (SEQ ID NOs: 87 and 88) and BaEVA18 (SEQ ID NOs: 95 and 96) fused very slightly (Figure 3A). In contrast, it was observed more syncytia in the presence of Syn1 (SEQ ID NOs: 37 and 38), Dasy Env (SEQ ID NOs: 41 and 42) and their truncations (SEQ ID NOs: 85 and 86, and 89 and 90). For Syn1 (SEQ ID NO s: 37 and 38), the surface of syncytia obtained by interaction with ASCT 1 was however about 2-fold lower than with ASCT2. Conversely, both the area and number of syncytia formed by DasyEnv (SEQ ID NOs: 41 and 42) and DasyEnvA28 (SEQ ID NOs: 89 and 90) in the presence of ASCT 1 was 2-fold higher than with ASCT2.
The fusion activity of retroviral envelopes is dependent to ASCT2
293T cells naturally express a large amount of endogenous ASCT2 and very little of ASCT 1 (about 15-fold less) (Figure 13-A). To verify that the fusion generated by the different envelopes is ASCT2-dependent, 293T KOASCT2 cells were established using CRISPR-Cas9 (Figure 14), and the fusogenic capacity of the envelopes was compared in both 293TWT and 293TKOASCT2 cells (Figure 3B). This fusion assay, more sensitive than the previous one, allowed the use of a colorimetric method to quantify p-gal activity. DasyEnv (SEQ ID NOs: 41 and 42), Syn1 (SEQ ID NOs: 37 and 38), SynOryl (SEQ ID NOs: 39 and 40), RD114 (SEQ ID NOs: 43 and 44), MPMV (SEQ ID NOs: 45 and 46) and their truncations, as well as the two BaEVA18 (SEQ ID NOs: 95 and 96) and SRV-2A23 (SEQ ID NOs: 97 and 98) truncations induced fusion in 293TWT cells. As expected, except for DasyEnvA28 (SEQ ID NOs: 89 and 90), these envelopes were no longer able to induce fusion in 293T KOASCT2 cells. In addition, transfection of 293TKOASCT2 cells with the plasmid expressing ASCT2 restored the fusion of these envelopes (Figure 15). These results support the ASCT2- dependent fusion of the tested envelopes.
Taken together, the results of fusion assays in A23 and 293T cells (Figure 3A,B) indicate that the interaction of RD114, MPMV and SRV-2 wt/truncated is highly specific for ASCT2, whereas DasyEnv wt/truncated (SEQ ID NOs: 41 and 42/89 and 90) and to a much lesser extent Syn1 wt/truncated (SEQ ID NO: 37 and 38/85 and 86), SynOry1A25 (SEQ ID NOs: 87 and 88) and BaEVA18 (SEQ ID NOs: 95 and 96), use ASCT1 in addition to ASCT2 as an auxiliary receptor.
Fusion activity of retroviral envelopes in tumor cell lines expressing ASCT2
It was aimed to test the fusogenic capacity of the transgenes in tumor cell lines. It was therefore gathered a panel of tumoral cell lines, in which ASCT2/1 expression level was quantified by qRT-PCR (Figure 13-A). A549 lung cancer cells and 22rv1 prostate cancer cells, which express a significant level of the ASCT2, were chosen for the fusion assay (Figure 4). In the two cases, a significant fusion activity was observed for the truncated versions of all envelopes. An increase of blue syncytia compared to the WT versions was noticed, except for Syn1- (SEQ ID NOs: 37 and 38) as observed in the A23 fusion assay- and MPMV (SEQ ID NOs: 45 and 46) envelopes, whose WT (SEQ ID NO: 37 and 38, and 45 and 46) and truncated (SEQ ID NO: 85 and 86, and 93 and 94) versions seemed to induce similar levels of fusion. The variation of fusion capacity among the envelopes was comparable in the two cell lines. It was observed a slightly better fusion activity of the envelopes in 22rv1 cells in comparison to A549 cells. Overall, these results support the use of truncated envelopes for enhancing fusion activity in cancer cells which highly expressed ASCT2.
Pseudotyping of VSV vectors with the retroviral envelopes
To then investigate the infectious capacity of the WT or truncated envelopes, it was analyzed the transduction efficiency of VSV-AG-GFP pseudotypes (Figure 5A). Of note, for an optimal production of pseudotypes, it was used producing cells that do not fuse in the presence of the envelopes, i.e. 293T KOASCT2 or BSR cells. Firstly, VSVs pseudotypes were used to transduce the 293T cells that strongly express ASCT2 (Figure 5B). All envelopes tested are able to transduce 293T cells with a significantly higher viral titer than with the "no Env" condition, with the exception of BaEV WT (not shown) Interestingly, truncation drastically enhances the viral titer in almost all cases (VSV-Syn1A53: 3-fold (p<0.05); VSV- SynOry1A25: 10-fold (p<0.01); VSV-RD114A18: 1000-fold (p<0.05); VSV-MPMVA23: 50- fold; VSV-BaEVA18: 500-fold; VSV-SRV-2A23: 10-fold), except for VSV-DasyEnv. VSV- RD114A18 and VSV-MPMVA23 particles showed the highest titers among the tested envelopes. Secondly, ASCT2 deletion in 293T cells resulted in loss of transduction of all truncated envelopes studied (Figure 5C). Furthermore, restoration of ASCT2 expression by transfection into 293T KOASCT2 recovered the transduction of these pseudotypes. These data clearly demonstrate that cell entry of VSVs pseudotyped with the tested envelopes occurred through ASCT2 receptor.
Taken all the results of fusion and pseudotyping assays (Table 3), it was selected Syn1A53 (SEQ ID NOs: 85 and 86), SynOry1A25 (SEQ ID NOs: 87 and 88), RD114A18 (SEQ ID NOs: 91 and 92), MPMVA23 (SEQ ID NOs: 93 and 94) and BaEVA18 (SEQ ID NOs: 95 and 96) to tentatively generate replicative VSV production.
Table 3: Fusion capacity towards ASCT2/ASCT1 and pseudotyping efficiency of truncated retroviral envelopes used in this study. _
Fusion efficiency T _ _ _
ASCT1 i on sa u Cii o n GTTiGicriG
Figure imgf000096_0001
Dasy EnvA28 High High Moderate
Syn1A53 Moderate Moderate Moderate
SynOry1A25 High Low Moderate
RD114A18 Moderate No High
MPMVA23 High No High
BaEVA18 High Moderate Moderate
SRVA23 Low No Moderate
Generation and basic characterization of ASCT2-targeted VSV-Env replicative viruses
To retarget the virus towards cells expressing ASCT2, the open reading frame (ORF) of VSV-G (SEQ ID NO: 3) was replaced with the truncated gene of retroviral envelopes within the VSV full-length expression vector (Figure 6A). It was successfully generated recombinant VSVAG-Syn1A53, -SynOry1A25 and -RD114A18 replicative viruses by the reverse genetic system, using the BSR-T7 producing cells and the Vero NK permissive cells as target. VSV-G replicative virus was obtained in parallel.
After rescuing the panel of recombinant viruses, the composition of each virus was verified. It was extracted the viral RNA, performed RT-PCR to amplify the envelope genes as well as VSV-N gene. The Syn1A53 (SEQ ID NOs: 85 and 86), SynOry1A25 (SEQ ID NOs: 87 and 88) and RD114A18 (SEQ ID NOs: 91 and 92) genes were detected in each corresponding virus, and all viruses contained the VSV-N gene. For VSV-Syn1A53 viruses, it was initially performed 11 sequential passages. To characterize the evolution of the Syn1A53 envelope (SEQ ID NOs: 85 and 86) during passages, it was extracted the RNA of Vero NK rescue cells at passage 3, 5, 8, 11 and performed RT-PCR to amplify the Syn1A53 envelope gene, which was further sequenced. No mutations were observed in the sequences between passage 3 and passage 11. Regarding the production of viruses, it was noticed different titers obtained between viruses (Figure 6B), with the best titer obtained for the wt VSV-G (5.108 PFU/rnL), followed by VSV-RD114A18, VSV-Syn1A53 and VSV-SynOry1A25, respectively. It was also succeeded to concentrate the viruses at least 6 times by ultracentrifugation (Figure 6B).
To address usage of ASCT2 as entry receptor by the newly established viruses, non permissive CHO hamster cells stably expressing high levels of ASCT2 (Figure 6C) were infected with VSV-Syn1A53, -SynOry1A25 and -RD114A18 viruses with an MOI of 0.1. As shown in Figure 6D for the CHO-ASCT2-c9 clone, at 48h post infection, CHO cells stably expressing ASCT2 showed green fluorescence, indicative of successful infection, while CHO cells expressing empty vector showed no fluorescence. Syncytia formation (red arrow heads) was pronounced in cells infected with the recombinant viruses, but not detected in cells infected with the native VSV. Of note, syncytium formation hampered the use of cytometric analyses to quantify infection at this stage. To determine the relationship between ASCT2 receptor expression and the cytotoxic activity of the viruses, it was also performed infection on CHO cell lines stably expressing progressive levels of ASCT2, and the cytotoxic activity of the viruses was analyzed in cell killing assays using an ATP kit (Figure 6E). At 72h post infection, the recombinant viruses showed very low level of fluorescence and no cytotoxic effect on the CHO pop cells with the lowest ASCT2 expression level. On the other hand, infections resulted in a dramatic killing of the two CHO cell lines c9 and c10, which exhibit high level of ASCT2 expression (p<0.0001). The cytotoxicity effect is greatest in CHO ASCT2 c10 cells that express the strongest level of ASCT2. Especially, VSV-Syn1A53 killed nearly 100% of the c10 cells within 72 hpi. Of note, there was no difference in terms of cell viability between the CHO cell lines infected with the VSV-G viruses and those treated with Mock. These results revealed that the tropism of VSV-Env was targeted to cell lines expressing ASCT2 receptor and that their cytotoxic activity appeared to correlate with the density of the receptor expressed in the target cells.
Infection and killing capacities of rVSV in tumor cells using 2D monolayer cultures
Next, it was wanted to assess the oncolytic activity of the recombinant viruses on a panel of cancer cells expressing ASCT2 (Figure 13-B). Photos in Figure 7A show infection of A549 lung cancer cells at the MOI of 1. At 24h pi, it was already noticed the signs of infection with the VSV-Env compared to untreated control, revealed by green fluorescence. Furthermore, syncytia formation was detected in cells infected by VSV-Syn1A53, -SynOry1A25 and - RD114A18 viruses, and not in cells infected by VSV-G viruses. This led to formation of cell aggregates that detach from the surface plate (Figure 7A - Bright Field). The most fusion- competent virus is VSV-SynOry1A25, whose large syncytia formed displayed a reduced green fluorescence intensity, most probably because of the fusion between GFP+ infected and GFP- uninfected cells. As expected, VSV-G infected cells displayed a typical cytotoxic phenotype, with rounded cells finally detaching from the plate. Next, the oncolytic activity of the viruses was analyzed in killing assays in a panel of selected tumor cell types expressing ASCT2, including the A549 lung cancer, the MCF-7 breast cancer, the 22rv1 prostate cancer, and the MIAPACA2 pancreas cancer cell lines, as well as in primary tumor cells isolated from a bladder patient-derived xenograft (MR0015, MatchR study, IGR) (Recondo G et al. 2020. Feasibility and first reports of the MATCH-R repeated biopsy trial at Gustave Roussy. NPJ Precis Oncol 4:27) (Figure 7B). In killing kinetics using an MOI of 1 , all of the tested viruses killed nearly 90-100% of the A549, 22rv1 , MIAPACA2 and MR0015 PDX cells within 72h pi. In MCF-7 cells, cell viability after 72h of infection with VSV-Syn1A53, - SynOry1A25 and -RD114A18 was reduced to approximately 10% of the mock condition, and reached a significantly lower level than that obtained with the wild type VSV-G (p<0.05). Regarding the A549 cells, it was noticed a variation of the killing capability by the tested viruses within 48hpi. Strong syncytia formation induced by VSV-SynOry1A25 resulted in the fastest cytotoxicity speed compared to other viruses, followed by VSV-Syn1A53. However, all viruses killed almost all of cells after 72h of infection. Regarding the 22rv1 and MIAPACA2 cells, initially, VSV-Syn1A53, -SynOry1A25 as well as the WT VSV were likely to kill cells more efficiently than VSV-RD114A18, at 72hpi all conditions showed nearly no living cells. Concerning the primary tumor cells from MR0015 PDX, the killing kinetic of the three recombinant viruses was identical within 72hpi. After 48h of infection, the WT VSV showed a greater cytotoxicity than other viruses (p<0.01) but at 72hpi, the cell viability with the four viruses dropped nearly to the same level (<10%). In conclusion, despite the heterogeneity in killing profile observed between different cancer cells, VSV-Syn1A53, -SynOry1A25 and - RD114A18 viruses demonstrated strong oncolytic activity against tumor cell lines and primary cancer cells, with remarkably equivalent efficacy to VSV WT.
Oncolytic activity of recombinant viruses on 3D breast cancer spheroids
To evaluate the efficacy of the established viruses in a system that better mimics tumor complexity, it was also performed 3D cell culture models. First, spheroids were generated from the breast carcinoma cell line SUM52PE. When the shape of spheroids was well established, the tested viruses were added into the spheroid cultures at an MOI of 1 and 0.1. As shown in Figure 8A for the infection at an MOI 0.1 , green fluorescent signal was detected as early as 24 hpi for all viruses. Interestingly, syncytia formation induced by VSV-Syn1A53, -SynOry1A25 and -RD114A18 led to the disruption of the spheroid structure, especially at 72hpi (Bright Field). This phenomenon was not observed in the spheroid infected with the WT VSV, in which green fluorescent was however greater. It was also monitored the live/dead status of spheroids over time, by incubating infected spheroids with Incucyte® Cytotox Red Dye that labels dead cells with red fluorescence. At 24hpi, very low red signals were noticed for the three recombinant viruses, and a stronger intensity of fluorescence for the WT virus. Yet, after 72h of infection, the red fluorescence with the three recombinant viruses was much more intense, indicating a diminution in cell viability within the spheroids. Cell killing was quantified as well using the ATP kit (Figure 8B). Overall, within 96hpi, the spheroids treated with Mock showed a stable living state, while the percentage of cell viability -expressed relative to mock condition - fell dramatically in spheroids infected with all viruses. Cell viability was reduced to 50% earlier before 48 hpi in VSV-Syn1A53 and WT viruses infected cells, while VSV-SynOry1A25 and -RD114A18 required more than 48h to kill 50% of cells. Notably, the VSV-Syn1A53, -RD114A18 and WT VSV viruses killed 100% of cells at 96hpi. The infection with VSV-SynOry1A25 resulted in a reduction in cell numbers, but there still remained about 30% of living cells at 96hpi. Among the viruses, cytotoxicity was most powerful with VSV-Syn1A53 and the WT viruses, which seemed to share a similar killing kinetic over time. These results revealed for the first time the antitumor effect of the recombinant viruses on 3D culture models.
Infection and killing of patient-derived pancreatic cancer organoids
The anti-tumor potency of the ASCT2-targeted viruses was secondly assessed on a 3D culture of primary pancreatic cancer cells (PGR4). These cells positively express ASCT2 (Figure 13-B) and are able to form organoids in a Matrigel dome. After 48h of infection, all retargeted viruses were able to infect PGR4 organoids and to induce syncytia formation (Figure 9A). In fact, fusion between tumor cells led to impaired organoid structure with soap bubble-like membrane blebs not observed in organoids infected with the wildtype VSV (BF/GFP). In addition, at 48 hpi, organoids treated with all of the recombinant viruses showed a significant decrease of viability as compared with the untreated organoids (30%- 60% living cells) (Figure 9B). At 72hpi, it was noticed a slightly lower percentage of living cells in comparison to 48 hpi (20%-40% living cells). These results support the oncolytic potency of ASCT2-targeted viruses against patient-derived cancer cells on a 3D model. Infection of ASCT2-targeted OVs in PDX slices culture
To evaluate the infection of the established viruses in 3D patient-derived tumors, it was next employed a tumor slice culturing system. Indeed, fresh biopsy samples of patient-derived xenografts (IGR) were collected and accurately sectioned into slices of 300pm by a vibratome. The tumor slices were then treated with viruses within 48h, and the infection was monitored by green fluorescence. Mock treated slices served as control. In this study, it was employed established xenografts in NOD scid gamma (NSG) mice, originating from patients of MATCH-R study (IGR) (Recondo G et al. 2020. Feasibility and first reports of the MATCH- R repeated biopsy trial at Gustave Roussy. NPJ Precis Oncol 4:27). Two PDX models were used, namely the above-mentioned bladder MR0015 and a lung adenocarcinoma MR0347. The positivity for ASCT2 expression in the tumors was confirmed by RT-qPCR and IHC (Figure 10A). Regarding the infection of MR0015 slices, at 48hpi, GFP fluorescence was observed in slices treated with all of the tested viruses (Figure 10B). The fluorescence intensity induced by VSV-Syn1A53, -RD114A18 and the WT VSV viruses was the strongest. Identical observation was made for the MR0347 slices, with VSV-SynOry1A25 showing a very slight signal. (Figure 10B). In fact, because of low titer of VSV-SynOry1A25 stock obtained, the number of VSV-SynOry1A25 viruses added to each slice was less than other viruses. Moreover, as noticed for the A549 cells (Figure 7A), the strong fusion activity of VSV-SynOry1A25 is associated with a low GFP signal. Of note, all mock treated controls remained negative for GFP signal. Overall, for the two PDX, it was noticed a raise of fluorescence intensity at 48hpi as compared to 24hpi, indicative of virus propagation within tumor slices (data not shown). These results demonstrate that patient-derived xenograft tumors were susceptible to the retargeted viruses, and the precision-cut tumor slice culture model is easily amenable to study virus efficacy in tumor samples obtained following surgically excision in the future.
ASCT2-targeted OVs do not infect healthy cells
In the last step, it was aimed to verify the safety of the oncolytic viruses towards healthy cells. As expected from the native VSV properties, the newly established viruses should be highly susceptible to host IFN responses on normal cells and no longer have tropism towards these cells. To investigate the safety at the systemic level, it was first performed infection at a MOI of 1 in blood cell populations, including PBMCs (monocytes and lymphocytes), and granulocytes isolated from whole blood (Figure 11). At 48hpi, the cells were subjected to flow cytometric analysis to determine the percentage of infected cells. The WT VSV demonstrated a notable infection of the granulocyte population, compared to the recombinant VSV-Syn1A53, -SynOry1A25 and -RD114A18 viruses (10.2% of cells infected by VSV-G, p<0.0001), whose infection rate remained at a background level. For the monocyte and lymphocyte populations of PBMCs, the percentage of cells infected with the viruses or with untreated condition was not different. As ASCT2 is known to be abundantly expressed in villous cytotrophoblasts (VCT) of human placenta as well, the tropism of the retargeted viruses was also verified on primary cytotrophoblasts isolated from human placenta and differentiating into syncytia upon cultures (Figure 13-B). VCT cells were subjected to infection with an MOI of 1 of each virus type. At 48hpi, no infection was noticed in all tested recombinant viruses, as assessed by GFP fluorescence examination and flow cytometry analysis (Figure 11). A slightly but significantly infection was observed with the wildtype virus VSV-G compared to the others (2% of cells infected by VSV-G, p<0.05). As a control, infection performed in parallel with the tested viruses on 293T cells led to significant infection (data not shown). Taken together these data revealed the lack of detectable infection with ASCT2-targeted OVs in healthy cells expressing significant levels of ASCT2. In particular, the retargeting to ASCT2 suppresses the tropism of wild-type VSV towards granulocytes and villous cytotrophoblasts of human placenta. DISCUSSION
In this study, it was provided a large scale pan-cancer analysis of high throughput RNAseq data (TCGA, GTex) demonstrating that ASCT2 is highly expressed in tumors of the digestive and female (breast, ovary, uterus and cervix) origin, as well as in skin, bladder, head and neck, lung and prostate cancers. Of interest for specific targeted therapy, in some of these cancers, notably esophageal, stomach, colon, pancreas, lung and cervical carcinoma, ASCT2 expression is largely upregulated as compared to normal non-cancerous tissues. These results support the use of ASCT2 as a therapeutic target for anticancer treatments. It was thus developed here oncolytic VSVs armed with heterologous and engineered retroviral envelopes (termed VSV-Syn1A53, -SynOry1A25, -RD114A18) to target ASCT2 and efficiently induce fusion between tumor cells. The infection and killing potency of three recombinant VSVs were demonstrated in different cancer models, allowing sequential administrations of oncolytic viruses to avoid humoral immunity against viruses.
Seven retroviral envelopes were screened for their function towards ASCT2 target, including fusion activity and transduction efficacy into cells expressing ASCT2. Truncation at the level of cytoplasmic tail was also screened. It was here noticed a hyperfusogenic phenotype as well as an improvement of infection efficacy in VSV pseudotypes, for the majority of truncated envelopes. In the case of Syn1 (SEQ ID NOs: 37 and 38) and SRV-2 (SEQ ID NOs: 51 and 52), pseudotyping function was optimized by truncation, while fusion was just slightly increased and insignificantly different compared to the full-length envelope. The optimized functions of truncated envelopes could also be conferred by removal of a putative C-terminal R peptide region, which plays a role in fusion inhibition.
The choice of envelopes investigated for oncolytic viruses was also based on their limited tropism towards ASCT1 auxiliary receptor to minimize their unexpected targeting. Unlike ASCT2, involvement of ASCT1 in cancer was not well documented. In tumor cells, it was found expression of ASCT1 at least 5-fold lower than ASCT2. In agreement with previous studies, it was demonstrated an absence of interaction of RD114 (SEQ ID NOs: 43 and 44), MPMV (SEQ ID NOs: 45 and 46) and SRV-2 (SEQ ID NOs: 51 and 52) envelopes with ASCT1 and a moderate binding to ASCT1 of Syn1 (SEQ ID NOs: 37 and 38) and BaEV (SEQ ID NOs: 47 and 48) envelopes (Lavillette D et al. 2002. The envelope glycoprotein of human endogenous retrovirus type W uses a divergent family of amino acid transporters/cell surface receptors. J Virol 76:6442-6452) (Marin M et al. 2000. Sodium-Dependent Neutral Amino Acid Transporter Type 1 Is an Auxiliary Receptor for Baboon Endogenous Retrovirus. J Virol 74:8085-8093). In addition to the tropism to ASCT2, here it was also noticed a mild interaction of SynOryl (SEQ ID NOs: 39 and 40) with ASCT1. In contrast, DasyEnv (SEQ ID NOs: 41 and 42) was found to strongly interact with ASCT1, with a tropism at least equivalent to ASCT2.
Interestingly, it was noticed different cytotoxic efficacies between the ASCT2-targeting viruses, although depending on cell types. On the monolayer A549 cells, VSV-SynOry1A25 was the most efficient in tumor cell lysis within 48h after infection. Nevertheless, on MCF-7, 22rv1, MIAPACA-2 cells and patient-derived MR0015 cells, infection of VSV-SynOry1A25 demonstrated a comparable killing kinetic to that of VSV-Syn1A53. Whatever the cells tested, the cytotoxicity induced by the recombinant viruses was similar to that of the wild type VSV after 72h of infection or even outperformed it in MCF-7 cells. Hyperfusogenic phenotype induced by VSV-SynOry1A25 may be advantageous for fast cytotoxicity in A549 cells. Indeed, cell-cell fusion could confer to virus a bystander effect, meaning that infected cells could fuse to their adjacent uninfected counterpart, allowing to boost virus propagation and potentially to enhance killing potency (Krabbe T, Altomonte J. 2018. Fusogenic Viruses in Oncolytic Immunotherapy. 7. Cancers 10:216) (Burton C, Bartee E. 2019. Syncytia Formation in Oncolytic Virotherapy. Mol Ther - Oncolytics 15:131-139). The situation was different in SUM52PE spheroid cultures, in which VSV-SynOry1A25 viruses were able to induce cytotoxicity but killed approximately 70% of the cells within 96h, whereas VSV- Syn1A53 and in a lesser extent, VSV-RD114A18, turned out to be the most powerful with almost 100% cell death after 72h of infection. Especially, VSV-Syn1A53 shared the same killing profile with VSV-G. Of note, among the three established recombinant viruses, virus yield obtained with VSV-SynOry1A25 was often lower than with the two others (Figure 6B), indicating a moderate or delayed virus production. In the SUM52PE culture, the treatment might require more time to achieve the complete cell fusion-enhanced cytotoxicity of SynOryl A25 viruses. Titration of virus yield over time after virus addition would be necessary to monitor the virus production activity within the 2D and 3D cultures.
In addition to be highly effective, the recombinant OVs which have been engineered are original, compared with the OVs which are currently in pre-clinical development. First, they are redirected toward a successful tumor marker. The targeted tropism of recombinant viruses, in combination with the IFN-dependent infection of the VSV vector, lead to an enhanced tumor-specificity for a better safety. Secondly, they are modified to be highly fusogenic, for improved tumor toxicity and immune stimulatory properties. Although OV either retargeted (VSV-LCMV, VSV-Echi9 (Muik A et al. 2014. Re-engineering vesicular stomatitis virus to abrogate neurotoxicity, circumvent humoral immunity, and enhance oncolytic potency. Cancer Res 74:3567-3578) (Ammayappan A et al. 2013. Characteristics of oncolytic vesicular stomatitis virus displaying tumor-targeting ligands. J Virol 87:13543- 13555)), or "armed" with a fusogenic transgene (HSV-GALV-R; VSV-p14 (Thomas S et al. 2019. Development of a new fusion-enhanced oncolytic immunotherapy platform based on herpes simplex virus type 1. J Immunother Cancer 7:214) (Le Boeuf F et al. 2017. Reovirus FAST Protein Enhances Vesicular Stomatitis Virus Oncolytic Virotherapy in Primary and Metastatic Tumor Models. Mol Ther Oncolytics 6:80-89)) exist, ASCT2-directed VSV are the first ones that combine the two advantageous properties. Moreover, the panel of recombinant VSV are able to overcome the major immune obstacles that systemic injections of OV usually face. Indeed, because the envelopes carried by the pseudotyped VSV display little homology (pairwise identity less than 48%) immune response established against one envelope would not target others during successive injections, allowing an efficient administration protocol.
In conclusion, the demonstration of the oncolytic potency of ASCT2-targeted viruses in emerging cancer models supports the use of these viruses as an anticancer treatment.
EXAMPLE No. 2 - Alternative IRPOV specifically directed against ASCT2
MATERIALS AND METHODS
Cloning
For recombinant H constructs, a RBD fragment derived from Syn1 (SEQ ID NOs: 150 and 151) or Suppressyn (SEQ ID NOs: 152 and 153) were first amplified by PCR using:
■ SuW-F1-Agel: ATACATACCGGTGCACCCCCTCCATGC (SEQ ID NO: 158)
■ SuW-R2-Notl: ATACATGCGGCCGCGCTAGAGGTGCCATGTAC (SEQ ID NO: 159)
■ Suppressynhomo-F1-Agel: ATACATACCGGTGCCCCTCCGAGCTGCC (SEQ ID NO: 160)
■ Suppressynhomo-R1-Notl: ATACATGCGGCCGCTAGTTTTTGTATAAAGGAATG (SEQ ID NO: 161)
The PCR products were then introduced into plasmid pCG-H*mutA18 (Hemagglutinin H protein with four points mutations to prevent binding to MV receptors and a C-term truncation by 18aa; donated by Dr. Buchholz, Paul- Ehrlich-lnstitut, Germany) (Funke et al., Targeted cell entry of lentiviral vectors. Mol Ther. 2008 Aug; 16(8): 1427-36) via the Agel/Notl restriction sites. Cell-cell fusion assay based on a-complementation of p-galactosidase
The assay was performed in 48-well plates with 293T cells. Cells were seeded on day 1 and transfected on day 2 with expression vectors for Gala or Galco peptides, with or without the expression vectors for MV F protein, H*-RBD-Syn1 , H*-Suppressyn or no Env (empty vector, ED). The H and F expression vectors were transfected at a 1 :3 ratio. On day 3, cells were co-cultured in a ratio of 1:1. After 48 h, the fusogenicity of the cells was revealed by X-gal which enables syncytia to be visualized in blue. Cells are fixed with a fixation solution (0.16% glutaraldehyde, 1% formaldehyde) and then stained with a solution of 4 M ferro-cyanide, 4 M ferri-cyanide, 2 M MgCh and 66.6 pg/mL X-gal. Fused cells appear blue due to hydrolysis of the X-gal substrate by p-galactosidase formed by complementation of gala and galco peptides. Syncytia formed in wells were observed under white-light microscopy.
Protein expression measurement
2.105293T cells were seeded in a 24-well plate. On day 2, cells were transfected with 200 ng of envelope expression vectors using Fugene6 (Promega). 48h after transfection, the cells were harvested and blocked in PBS with 2% BSA for 15 minutes at 37°C. Cells were then incubated with the anti-His tag antibody (Penta His AF488 Conjugate, 1/100, ref 1019199, Qiagen) for 45 minutes at 4°C. Washes were performed 3 times before cells were run in flow cytometry.
RESULTS
Here it was tested a second strategy for retargeting VSV to the ASCT2 receptor, this time based on the design of chimeric glycoproteins M/F glycoproteins of measles virus. To this end, the Receptor Binding Domain of Syncytin-1 (aa 21-144; SEQ ID NOs: 150 and 151) which was shown to interact with ASCT2 was grafted to the hemagglutinin H protein. Suppressyn (SEQ ID NOs: 152 and 153), a soluble protein derived from an endogenous retrovirus envelope and capable of binding to ASCT2, was also grafted to protein H to target the same receptor (Figure 16A). An H mutated variant with inability to recognize the native receptors CD46 and SLAM, thanks to four mutations in its extracellular ectodomain (Funke et al., Targeted cell entry of lentiviral vectors. Mol Ther. 2008 Aug;16(8):1427-36), was used.
By performing fusion assays in 293T cells, that express endogenous ASCT2, the ability of the recombinant H constructs to induce cell-cell fusion were studied, in the presence of the fusion (F) protein of measles virus (Figure 16B). As expected, the mutated H* linked to RBD Syn1 (SEQ ID NOs: 154 and 155) or to Suppressyn (SEQ ID NOs: 156 and 157) induced fusion with ASCT2 expressing 293T cells, as indicated by the presence of numerous blue syncytia detected in 293T cells transfected with expression vectors for these proteins, with no or very few blue cells in 293T cells transfected with a vector without envelope (EV). H constructs feature a His tag at the extracellular C-terminus (Figure 16A), allowing verification of protein expression at the membrane using an anti-His antibody. Flow cytometry analysis of HIS tag expression showed a high expression of H*-RBD Syn1 (80%) and a moderate expression (25%) of the H*-suppressyn protein (Figure 16C).
These experiments showed that it is possible to integrate the binding domain (RBD) or a fragment of an envelope protein targeting ASCT2 into the H mutated protein that remains functional in fusion assays.
EXAMPLE No. 3 - IRPOV specifically directed against MPZL1 or GLUTI
MATERIALS AND METHODS
Generation of the viruses For cloning into the VSV genome plasmid of Syn-Mab1 (receptor: MPZL1) (SEQ ID NOs: 146 and 147) or HTLV-1 Env (receptor: GLUT1) (SEQ ID NO: 148 and 149), the G coding sequence (SEQ ID NO: 3) in the plasmid MC11-VSV-eGFP (encoding the non-attenuated Indiana serotype; gift from Dr K. W. Peng (Mayo Clinic, USA)) (Ammayappan A et al. 2013. Neuroattenuation of vesicular stomatitis virus through picornaviral internal ribosome entry sites. J Virol 87:3217-3228), based on the plasmid pVSV-XN2 (SEQ ID NO: 2) (Lawson ND et al. 1995. Recombinant vesicular stomatitis viruses from DNA. Proc. Natl. Acad. Sci. U. S. A. 92:4477- 4481), is exchanged.
To rescue VSV-Syn-Mab1 or -HTLV-1 Env, the newly constructed plasmids of VSV-Env are employed in transfection experiments along with helper plasmids (encoding structural proteins and enzymes of VSV being under T7 promoter control and contain an IRES element) as described in Harty RN et al. (Vaccinia virus-free recovery of vesicular stomatitis virus. Journal of Molecular Microbiology and Biotechnology. 2001 Oct;3(4):513-517. PMID: 11545270). The T7 RNA polymerase is expressed constitutively from BSR-T7 cells, allowing vaccinia virus-free recovery of VSV. The helper vectors containing an IRES element allows cap-independent translation of VSV proteins in T7 RNA polymerase-expressing cells. Briefly, BSR-T7 cells at approximately 80% confluency are transfected with 10pg of VSV-Env, 1-5pg of helper plasmids using Fugene6 (Promega, Germany). At 24 hours post-transfection, the cells are trypsinized, pelleted for 10 minutes at 3000 rpm, and overlay on permissive Vero- NK cells or 293T cells, expressing endogenous levels of MPZL1 and Glutl . From 48h of coculture, green fluorescent and/or syncytia formation is noticed. Virus-containing supernatant is then harvested, clarified, and stored at -80°C. To produce large amounts of infectious VSV, VSV-Env viruses are incubated on Vero-NK or 293T cells at a MOI of 0.00001. Supernatant is harvested at 3 days after infection, filtered using 0.22 pM filter and stored at - 80°C. To concentrate the virus, supernatants are ultracentrifuged at 25,000rpm for 2h at 4°C (Beckman Coulter, USA).
All viruses are handled under biosafety level 2 conditions (installation L2-0334, authorization by the Haut Conseil des Biotechnologies, France).
Viral titration
Vero E6 cells are seeded in 12-well plates (5x105 cells/well) and grown overnight in growth medium. Serial dilutions of infectious recombinant VSV are used to infect Vero E6 monolayers (200 pl/well). Plates are incubated for 1 h at 37 °C to allow viral adsorption. Then, 1ml/well of overlay containing DMEM and 0.6% agarose (Euromedex, France) (ratio 1 :1) is added to each well and plates are incubated at 37 °C, 5% CO2 for 72 h. Finally, the cells are fixed with 4% PFA and stained with 1% crystal violet solution (Sigma Aldrich, USA). The number of plaques in each well is determined, and VSV titer is calculated.
RESULTS
MPZL1 and GLUT-1 are overexpressed in many tumor types as compared to healthy cells (TCGA/GTEX)
MPZL1 and GLUT1 expression levels were analyzed in the RNAseq-based transcriptome of cancer samples retrieved from TCGA (The Cancer Genome Atlas) cohorts, and compared expression levels observed in tumors to those of TCGA’s non-tumor adjacent tissues (‘Control’) as well as non-diseased (‘Normal’) tissues from the GTEx (Genotype-Tissue Expression) project, (http://gepia2.cancer-pku.en/#index).
Figure 17 shows the solid tumors which displayed a significant enhanced expression of MPZL1 and/or GLUT1, as compared to the normal tissue.
ACC, Adrenocortical carcinoma; BRCA, Breast invasive carcinoma; CESC, Cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, Cholangiocarcinoma; COAD, Colon adenocarcinoma; DLBC, Lymphoid Neoplasm Diffuse Large B-cell Lymphoma; ESCA, Esophageal carcinoma; GBM, Glioblastoma multiforme; HNSC, Head and Neck squamous cell carcinoma; KIRC, Kidney renal clear cell carcinoma; LGG, Brain Lower Grade Glioma; LIHC, Liver hepatocellular carcinoma; LUAD, Lung adenocarcinoma; LUSC, Lung squamous cell carcinoma; OV, Ovarian serous cystadenocarcinoma; PAAD, Pancreatic adenocarcinoma; READ, Rectum adenocarcinoma; STAD, Stomach adenocarcinoma; TGCT, Testicular Germ Cell Tumors; THYM, Thymoma; UCS, Uterine Carcinosarcoma.
Pseudotyping of VSV vectors with the Syn-Mab1 retroviral envelope
To test the infectious capacity of Syn-Mab1 in the VSV virus, the capacity of Syn-Mab1 to pseudotype G-defective VSV-GFP (VSV-AG-GFP) particles was tested (of. Materials & Methods of Example No. 1). Infectivity of pseudotypes was evaluated by flow cytometry in 293T target cells, expressing endogenous levels of MPZL1 , and in CHO hamster target cells, in which we transiently overexpressed the MPZL1 receptor. The titer measured for pseudotypes produced in the absence of envelope (“EV”) represents residual infection by the helper pseudotype VSVAG-G*/GFP. Isfahan virus (ISFV) Env is a positive control, being an envelope that pseudotypes with a high titer. It was observed that Syn-Mab1 -pseudotyped particles were able to infect 293T cells at a titer that was 3 logs higher than the background (EV) and 3.6 times higher than that of ISFV Env (Figure 18A). To validate the tropism of the Syn-Mab1 envelope for its receptor MPZL1, CHO cells expressing low levels of endogenous MPZL1 or CHO cells transiently overexpressing MPZL1 from lizard were infected. As expected, pseudotypes carrying the Syn-Mab1 envelope infected CHO-MPZL1 cells slightly better than CHO EVs (Figure 18B).
Pseudotyping of VSV vectors with the HTLV-I retroviral envelope and fusion activity of HTLV-1 truncated retroviral envelope in tumor cell lines expressing ASCT2
To evaluate the virotherapeutic capacity of HTLV-1 Env in the VSV virus, it was tested: i. the capacity of HTLV-I Env to pseudotype G-defective VSV-GFP (VSV-AG-GFP) particles (cf Materials & Methods of Example No. 1) using 293T target cells expressing endogenous levels of Glut-1 , and ii. the ability of HTLV-1 Env to induce syncytia formation, using cell-cell fusion assay (of. Materials & Methods of Example No. 1).
The HTLV-1 wild-type envelope and the HTLV-1 Env protein with a 8 aa truncation at the C- terminal level (HTLV-1A8) (Kim et al., J Virol. 2003 Jan;77(2):963-9) were tested. As illustrated in Figure 19A, HTLV-1 and HTLV-1 A8 envelopes were able to infect 293T cells, with a titer 4 to 40 times higher than the background (EV). Interestingly, the 8 aa truncation of the HTLV-1 WT increased the viral titer by 10 times compared to the titer obtained with HTLV-1 WT. As ilustrated Figure 19B, the truncated HTLV-1 A8 envelope was able to induce syncytia formation (colored blue), unlike the HTLV-1 WT envelope, indicating that the truncation improved the fusogenic capacity of the envelope.
Generation of the viruses
Pseudotyped VSV-Syn-Mab1 and VSV-HTLV-1 Env are produced and functional.
EXAMPLE No. 4 - IRPOVs specifically directed against ASCT2 have cytotoxic activity against canine 2D and 3D tumoral models
IRPOVs infect, and induce syncytia formation and lysis of canine 2D cancer cells
To characterize the infectious and oncolytic activities of the IRPOV viruses directed against ASCT2 in canine cells, the DH82 line (macrophages derived from a canine histiocytic sarcoma) (donation C.Puff, Hanover) was infected at an MOI of 1 and cell death were measured up to 72 hpi by a viability test measuring ATP (CellTiter-Glo). Infection was visualized by brightfield and fluorescent microscopy (GFP) at 24h (Figure 20A), 48h and 72h post-infection.
Infection with VSV-G led to a characteristic cytopathic phenotype, with rounded cells detaching from the surface, as early as 24 hpi in DH82 cells. The VSV-SynOry1A25 (see Example No. 7) and VSV-RD114A18 (see Example No. 7) viruses, and to a lesser extent VSV-Syn1A53 (see Example No. 7) displayed a hyperfusogenic phenotype (Figure 20A; arrows). The reduced fluorescence intensity of syncytia induced by the most fusogenic VSV- SynOry1A25 even confirmed fusion between GFP+ and GFP' cells.
Infection with the four viruses led to a drastic reduction in their metabolic activity, with cell viability reduced to approximately 5% of the uninfected condition at 72 hpi (Figure 20B).
IRPOVs infect and induce lysis of canine 3D cancer cells
In order to study a model representative of the structural complexity of tumors, canine REM 134 cells (mammary carcinoma) were cultured under conditions of very low adhesion, allowing spheroids to form within 72 hours. The spheroids were infected with the IRPOVs and VSV-G at an MOI of 10, and the kinetics of viral multiplication and their oncolytic activity were monitored for several days. As shown in Figure 21A, GFP+ infection was visible as early as 24 hpi for all four viruses, although VSV-G showed a higher intensity. The fusogenic phenotype of VSV-Syn1A53, VSV-SynOry1A25 and VSV-RD114A18 was manifested at 72 hpi by the appearance of characteristic bullae, indicated by the arrows.
Viability was then quantified by lysis of REM 134 spheroids using CellTiter-Glo (Figure 21 B). The amount of ATP was virtually stable in uninfected spheroids for 8 days after infection. In contrast, the metabolic activity of spheroids infected with the four viruses decreased steadily up to 8 dpi (days post-infection), with a more marked decrease up to 4 dpi. This effect was corroborated by the mortality observed by Incucyte Cytotox Red Dye labelling in Figure 21A. It is interesting to note a difference in kinetics, with a significant reduction in the amount of ATP measured as early as 24 hpi for VSV-SynOry1A25 and VSV-G, compared with 48 hpi for VSV-Syn1A53 and 72 hpi for VSV-RD114A18.

Claims

1. Viral particle for its use in the prevention and/or the treatment of a tumor, said viral particle being an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV), the IRPOV genome of which encodes an IRPOV specifically directed against a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2” (ASCT2), the “Myelin protein zero-like 1" (MPZL1), or the “ubiquitous vertebrate glucose transporter 1" (GLUT1), said IRPOV genome comprising an “Infectious and Replicative Oncolytic Virus" (IROV) genome comprising:
■ an inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome; and
■ a nucleic acid sequence encoding a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein, said retroviral envelope glycoprotein or a fragment thereof:
- being capable of allowing the production of an IRPOV;
- having retained its native targeting capacity for said host cell surface protein; and
- having retained its native infectious activity permitting to said IRPOV to entry into a host cell expressing said host cell surface protein.
2. Viral particle for its use according to claim 1, wherein said retroviral envelope glycoprotein or a fragment thereof has retained its native capacity to fuse the plasma membranes of two or more adjacent host cells leading to the formation of a syncytium.
3. Viral particle for its use according to claim 1 or 2, wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
4. Viral particle for its use according to any one of claims 1 to 3, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof.
5. Viral particle for its use according to any one of claims 1 to 4, wherein said retroviral envelope glycoprotein or a fragment thereof has a native targeting capacity for ASCT2, in particular said retroviral envelope glycoprotein or a fragment thereof comprising the conserved “S-[D/N]-[G/R]-XIGX2X3X4X5X6X7” (SEQ ID NOs: 131 to 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N, ■ Xs = any amino acid or no amino acid,
■ Xs = any amino acid, and
■ X? = any amino acid.
6. Viral particle for its use according to any one of claims 1 to 4, wherein said retroviral envelope glycoprotein or a fragment thereof has a native targeting capacity for MPZL1, in particular said retroviral envelope glycoprotein or a fragment thereof is Syn-Mab1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 146, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 147.
7. Viral particle for its use according to any one of claims 1 to 4, wherein said retroviral envelope glycoprotein or a fragment thereof has a native targeting capacity for GLLIT1, in particular said retroviral envelope glycoprotein or a fragment thereof is HTLV-1 Env, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 148, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 149.
8. Viral particle for its use according to any one of claims 1 to 7, said tumor being one of the following group: digestive tumors, prostate tumors, brain tumors, head and neck tumors, lung tumors and women's cancers.
9. Use of a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2” (ASCT2) or the “Myelin protein zero-like 1" (MPZL1), for producing an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV) specifically directed against said host cell surface protein from an “Infectious and Replicative Oncolytic Virus” (IROV), the native envelope glycoprotein of said IROV being inactivated in said IRPOV and said retroviral envelope glycoprotein or a fragment thereof when present in said IRPOV:
■ is capable of allowing the production of an IRPOV;
■ has retained its native targeting capacity for said host cell surface protein; and
■ has retained its native infectious activity permitting to said IRPOV to entry into a host cell expressing said host cell surface protein.
10. IRPOV genome encoding an IRPOV specifically directed against a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2” (ASCT2) or the “Myelin protein zero-like 1" (MPZL1), said IRPOV genome comprising an IROV genome comprising:
■ an inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome; and ■ a nucleic acid sequence encoding a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein, said retroviral envelope glycoprotein or a fragment thereof:
- being capable of allowing the production of an IRPOV;
- having retained its native targeting capacity for said host cell surface protein; and
- having retained its native infectious activity permitting to said IRPOV to entry into a host cell expressing said host cell surface protein.
11. IRPOV genome according to claim 10, wherein said retroviral envelope glycoprotein or a fragment thereof has retained its native capacity to fuse the plasma membranes of two or more adjacent host cells leading to the formation of a syncytium.
12. IRPOV genome according to claim 10 or 11 , wherein said retroviral envelope glycoprotein or a fragment thereof has a C-terminal truncated intracytoplasmic tail.
13. IRPOV genome according to any one of claims 10 to 12, wherein said retroviral envelope glycoprotein or a fragment thereof is an endogenous retroviral envelope glycoprotein or a fragment thereof.
14. IRPOV genome according to any one of claims 10 to 13, wherein said retroviral envelope glycoprotein or a fragment thereof has a native targeting capacity for ASCT2, in particular said retroviral envelope glycoprotein or a fragment thereof comprising the conserved “S-[D/N]-[G/R]-XIGX2X3X4X5X6X7” (SEQ ID NOs: 131 to 134) ASCT2 binding motif responsible for the native targeting capacity for said host cell surface protein wherein:
■ Xi = G or R or no amino acid,
■ X2 = V or P,
■ X3 = any amino acid,
■ X4 = D or N,
■ X5 = any amino acid or no amino acid,
■ Xe = any amino acid, and
■ X7 = any amino acid.
15. IRPOV genome according to any one of claims 10 to 14, wherein said retroviral envelope glycoprotein or a fragment thereof has a native targeting capacity for MPZL1, in particular said retroviral envelope glycoprotein or a fragment thereof is Syn-Mab1, the nucleic acid of which has at least 90% of identity with the sequence SEQ ID NO: 146, or the amino acid sequence of which has at least 90% of identity with the sequence SEQ ID NO: 147.
16. Plasmid comprising the IRPOV genome according to any one of claims 10 to 15 and means for expressing it.
17. Use of the IRPOV genome according to any one of claims 10 to 15 or the plasmid according to claim 16 for producing a viral particle, said viral particle being an IRPOV, the IRPOV genome of which comprises the IRPOV genome according to any one of claims 10 to 15.
18. Method of production of a viral particle, said viral particle being an IRPOV, the IRPOV genome of which comprises the IRPOV genome according to any one of claims 10 to 15, said method comprising at least the steps of: a. co-transfection of an animal eukaryotic cell, in particular said animal eukaryotic cell constitutively expressing the T7 RNA polymerase, with: i. a plasmid according to claim 16; and ii. helper plasmids encoding for structural proteins and enzymes of IRPOV and the means for expressing it, in particular said structural proteins and enzymes of IRPOV being under T7 promoter control and contain an IRES element allowing cap-independent translation of IRPOV proteins, to obtain a transfected animal eukaryotic cell; b. co-culturing said transfected eukaryotic animal cell with Vero-NK cells expressing a host cell surface protein, to enable the production of a viral particle, said host cell surface protein being a tumor marker and said tumor marker being the ASCT2 or MPZL1 ; and c. harvesting and purifying said viral particle.
19. Viral particle, said viral particle being an IRPOV, the IRPOV genome of which comprises the IRPOV genome according to any one of claims 10 to 15.
20. Pharmaceutical composition comprising a viral particle according to claim 19 and at least a pharmaceutically acceptable vehicle.
21. Viral particle for its use as a drug, said viral particle being an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV), the IRPOV genome of which encodes an IRPOV specifically directed against a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2” (ASCT2), the “Myelin protein zero-like 1" (MPZL1), or the “ubiquitous vertebrate glucose transporter 1" (GLUT1), said IRPOV genome comprising an “Infectious and Replicative Oncolytic Virus" (IROV) genome comprising:
■ an inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome; and
■ a nucleic acid sequence encoding a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein, said retroviral envelope glycoprotein or a fragment thereof: - being capable of allowing the production of an IRPOV;
- having retained its native targeting capacity for said host cell surface protein; and
- having retained its native infectious activity permitting to said IRPOV to entry into a host cell expressing said host cell surface protein, and at least a pharmaceutically acceptable vehicle.
22. Kit-of-parts comprising at least two viral particles for the separate or sequential administration of said at least two viral particles, each viral particle comprising a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for a host cell surface protein distinct from the other, said viral particle being an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV), the IRPOV genome of which encodes an IRPOV specifically directed against a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2” (ASCT2), the “Myelin protein zero-like 1" (MPZL1), or the “ubiquitous vertebrate glucose transporter 1" (GLLIT1), said IRPOV genome comprising an “Infectious and Replicative Oncolytic Virus" (IROV) genome comprising:
■ an inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome; and
■ a nucleic acid sequence encoding a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein, said retroviral envelope glycoprotein or a fragment thereof:
- being capable of allowing the production of an IRPOV;
- having retained its native targeting capacity for said host cell surface protein; and
- having retained its native infectious activity permitting to said IRPOV to entry into a host cell expressing said host cell surface protein.
23. Kit-of-parts comprising at least two viral particles for the separate or sequential administration of said at least two viral particles, each viral particle comprising a retroviral envelope glycoprotein distinct from the other or a fragment thereof distinct from the other having a native targeting capacity for the same host cell surface protein, said viral particle being an “Infectious, Replicative and Pseudotyped Oncolytic Virus" (IRPOV), the IRPOV genome of which encodes an IRPOV specifically directed against a host cell surface protein, said host cell surface protein being a tumor marker and said tumor marker being the “Alanine, Serine, Cysteine Transporter 2” (ASCT2), the “Myelin protein zero-like 1" (MPZL1), or the “ubiquitous vertebrate glucose transporter 1" (GLLIT1), said IRPOV genome comprising an “Infectious and Replicative Oncolytic Virus" (IROV) genome comprising:
■ an inactivated nucleic acid sequence encoding the native envelope glycoprotein of the IROV encoded by said IROV genome; and
■ a nucleic acid sequence encoding a retroviral envelope glycoprotein or a fragment thereof having a native targeting capacity for said host cell surface protein, said retroviral envelope glycoprotein or a fragment thereof:
- being capable of allowing the production of an IRPOV;
- having retained its native targeting capacity for said host cell surface protein; and - having retained its native infectious activity permitting to said IRPOV to entry into a host cell expressing said host cell surface protein.
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