Methods for selectively modulating activity of distinct subtypes of cells

Abstract

To provide methods for selectively modulating the activity of distinct subtypes of cells.SOLUTION: The invention relates to a pseudotyped retrovirus-like particle or retroviral vector comprising both engineered envelope glycoproteins derived from a virus of the Paramyxoviridae family, one fused to a cell-targeting domain and the other fused to a functional domain. The invention also relates to the use of the pseudotyped retrovirus-like particle or retroviral vector that selectively modulates a specific subset of cells, in particular activities of specific immune cells. The pseudotyped retrovirus-like particle or retroviral vector is particularly useful for gene therapy, immunotherapy and / or vaccination.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to methods for selectively modulating the activity of different subtypes of cells. [Background technology]

[0002] Cells of the immune system are involved in many types of pathologies. Therefore, enhancing or reducing the activity of immune system cells is the focus of many therapeutic strategies. The expression of certain cell surface proteins can distinguish between many different types of immune cells, each with distinct functions. To date, no technology is available that allows for the selective activation or deactivation of distinct cell subtypes, especially in vivo.

[0003] The use of cytokines to induce or promote the development of desired immune responses is an attractive approach in cancer immunotherapy. Cytokines are typically used as non-specific adjuvants to support other immunotherapies or to be administered in addition to chemotherapy. However, to date, cytokine-based treatments have only rarely been used due to unresolved systemic toxicity. Moreover, cytokines are usually applied systemically, thereby hitting all cell types that express relevant cytokine receptors. Therefore, fine-tuned modulation at disease-related sites is not possible.

[0004] To overcome this drawback, cytokines have been fused with anti-tumor antibodies or linked to microparticles or nanoparticles carrying anti-tumor antibodies, thereby targeting cytokines to tumors and reducing systemic side effects. However, these fusion proteins or cytokine-antibody-loaded particles have low stability and are rapidly cleared in vivo, resulting in relatively low cytokine concentrations at disease sites. In addition, cytokine release by cytokine-antibody-loaded particles is pH-dependent. Furthermore, cytokines (e.g., IL-2) delivered to tumors by these previous methods nonselectively stimulate a wide variety of different immune cells, including both immune effector cells and immunosuppressive cells, within the complex tumor microenvironment, limiting their application in cancer treatment.

[0005] Regarding gene therapy, important target cells, such as resting human T cells, B cells, and HSCs (hematopoietic stem cells), are difficult to transduce with lentiviral vectors. Conventional VSV-LV-mediated transduction occurs only when T cells are activated. Current gene therapy trials activate T cells via their cognate antigen receptors, which typically induces phenotypic and functional changes in T cells, ultimately resulting in shortened T cell survival and reduced antitumor efficacy in vivo.

[0006] Several chimeric lentiviral vectors have been created to facilitate gene transfer into resting T cells. For example, Verhoeyen et al. (Blood, 2003, Vol. 101, No. 6) described an HIV-derived vector pseudotyped with two envelope glycoproteins: a chimeric MLV (murine leukemia virus) envelope glycoprotein (gp) fused to IL-7 at the N-terminus and a VSV-G (vesicular stomatitis virus) glycoprotein. These IL-7 vector particles efficiently transduce resting T cells and induce T cell activation, but transduce CD4+ and CD8+ T cells equally well. Therefore, these IL-7 vector particles cannot distinguish between different T cell subsets. Furthermore, the fact that a non-targeting envelope gp, such as VSV-G gp, is required may lead to transduction of unwanted hematopoietic or endothelial cells in some cases. One exception is lentiviral vectors pseudotyped with measles virus (MeV) glycoprotein (MV-LV). MV-LV can mediate transduction of resting T lymphocytes without a change in the G0 / G1a cell cycle state. Zhou et al. (J Immunol, 2015, Vol. 195, No. 5) also disclosed that CD4-targeting LV can transduce freshly isolated resting T cells, but with extremely low efficiency (less than 10%) even at high particle doses.

[0007] Finally, current strategies for generating less differentiated tumor-specific T cells for adoptive T cell therapy rely on optimizing T cell stimulation and culture protocols. For example, SCM cells (stem memory T cells), or T CM To generate T cells (central memory T cells), a combination of IL-15 and IL-7 or IL-21 and IL-7 is used to stimulate and expand T cells. This culture system is quite expensive because it requires constant cytokine supplementation (every two days). Several cell sorting steps are then typically performed to obtain the desired cell type. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Verhoeyen et al. (Blood, 2003, Vol. 101, No. 6) [Non-patent document 2] Zhou et al. (J Immunol, 2015, Vol. 195, No. 5) [Non-patent document 3] Needleman and Wunsch (1970), J. Mol. Biol., 48:443-453 [Non-patent document 4] Funke et al., Molecular therapy, 2008 Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there remains a need to provide methods for selectively and efficiently modulating the activity of different subtypes of immune cells. [Means for solving the problem]

[0010] The present inventors have surprisingly found that it is possible to selectively modulate the activity of different subtypes of immune cells by using pseudotyped retrovirus-like particles or retroviral vectors (e.g., lentivirus-like particles (VLPs) or lentiviral vectors (LVs)) that carry both a cell-specific targeting domain (e.g., specific for CD4+ T cells or CD8+ T cells) and a functional domain, such as a cytokine, each fused to a glycoprotein of a virus from the Paramyxoviridae family. The retroviral vectors also specifically and efficiently deliver packaged genes to target cells.

[0011] Indeed, it was unexpectedly found that combining functional domains with cell-specific targeting domains on pseudotyped retrovirus-like particles or retroviral vectors significantly improved the selective modulation of activity of target immune cell subtypes compared to the corresponding particles or vectors containing only the cell-specific targeting domain.

[0012] For example, the present inventors have demonstrated that T cell targeting particles presenting stimulatory cytokines selectively activate target T cell subsets in mixed cell type cultures and in vivo in a human blood system mouse model, and also deliver packaged genes to target T cell subtypes without the need for stimulatory culture conditions. Measles virus (MeV) glycoprotein-based CD4 targeting particles presenting IL-7 (interleukin 7) (4 H / IL7 H -VLP) indeed specifically activates and promotes the survival of cultured primary CD4+ cells, while IL7-presenting CD8-targeting particles (8) based on NiV (Nipah virus) glycoproteins G / IL7 G -VLP) specifically activates and promotes the survival of CD8+ T cells. H / IL7 H 4-LV efficiently and specifically deliver the GFP transgene to CD4+ T cells in a cell mixture in a dose-dependent manner. Compared to the parental CD4-targeting LV (which does not co-present IL-7), 4 H / IL7 H -LV is more effective in selectively delivering the therapeutic ErbB2CAR transgene to resting CD4+ T cells. Similar, exclusive gene transduction and stimulation of CD8+ T cells was also achieved using CD8 / IL-7 co-presenting NiV glycoprotein-pseudotyped lentiviral vectors (8 G / IL7 G -LV). G / IL7 G-LV has been shown to be effective in selectively activating CD8+ T cells and targeting gene delivery. G -LV (see examples).

[0013] The retrovirus-like particles and retroviral vectors according to the present invention comprise: The advantage of increasing the local concentration of cytokines at the cellular target site, thereby improving the efficiency of cytokine therapy and preventing its severe side effects; the advantage of providing a more stable and constant stimulation of target cells compared to soluble cytokines, thereby improving the efficiency of cytokine therapy; The advantage of this is that it allows the combination of different cytokines and different targeting domains on a single particle or retroviral vector, resulting in a very flexible system; The advantage of being able to couple cell stimulation with cell-specific gene transfer, either in vitro or in vivo, resulting in more efficient transduction of specific resting T cell subsets; The advantage of being able to couple gene transfer with induction of a T cell phenotype in vitro or in vivo through interaction with cytokines displayed on the particle or vector surface, in a manner that controls the differentiation of transduced cells. It offers many advantages, such as: the advantage of enabling the delivery of biological materials and / or therapeutic agents, including but not limited to genes, mRNA, shRNA, microRNA, peptides, proteins, protein fragments, and combinations thereof, to specific cells or subsets of cells, in vitro or in vivo; The advantage of allowing for easier cell manufacturing processes (e.g., T-cell or hematopoietic cell manufacturing processes) for adoptive therapy; Advantages of using retrovirus-like particles or retroviral vectors pseudotyped with glycoproteins derived from Nipah virus to prevent resistance problems in vivo due to low levels of pre-existing antibodies in humans. Present the following.

[0014] Therefore, the present invention provides a) at least one cell-targeting fusion protein comprising: (i) a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family, preferably lacking at least a portion of the cytoplasmic region of said envelope glycoprotein G or envelope glycoprotein H, and preferably being at least partially unable to bind to at least one natural receptor or transmembrane domain of said envelope glycoprotein G or envelope glycoprotein H; and (ii) at least one cell-targeting domain; b) at least one modulating fusion protein comprising (i) a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family, preferably lacking at least a portion of the cytoplasmic domain of said envelope glycoprotein G or envelope glycoprotein H, and preferably at least partially unable to bind to at least one natural receptor or transmembrane domain of said envelope glycoprotein G or envelope glycoprotein H, and (ii) at least one functional domain; and c) at least one glycoprotein derived from the envelope glycoprotein F of a virus of the Paramyxoviridae family, preferably lacking at least a portion of the cytoplasmic domain of said envelope glycoprotein F; Including, the cell-targeting fusion protein and / or the modulating fusion protein comprises a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family; It relates to pseudotyped retrovirus-like particles or retroviral vectors.

[0015] The virus of the Paramyxoviridae family may be a virus of the genus Morbillivirus, for example, selected from the group consisting of measles virus (MeV), canine distemper virus, whale morbillivirus, peste des petit ruminants virus, porcine distemper virus, and rinderpest virus, or a virus of the genus Henipavirus, for example, selected from the group consisting of Nipah virus (NiV), Sheeda virus, and Hendra virus.

[0016] The proteins of a) and / or b) preferably comprise at least two mutations compared to the sequence of said envelope glycoprotein G or envelope glycoprotein H, which mutations result in at least partial inability of said envelope glycoprotein G or envelope glycoprotein H to bind to at least one natural receptor.

[0017] The cell targeting domain can be selected from the group consisting of a DARPin, an scFv, a targeting peptide, and combinations thereof, and / or the functional domain can be selected from the group consisting of a cytokine, a growth factor, a hormone, a neurotransmitter, an apoptotic ligand, and combinations thereof.

[0018] The target cells may be selected from the group consisting of hematopoietic cells (including T cells, B cells, monocytes, Th1 cells, Th2 cells, Treg cells, mast cells, dendritic cells (DCs), natural killer (NK) cells, natural killer T (NKT) cells, macrophages, hematopoietic stem cells, precursor T cells and / or precursor B cells, erythroblasts, platelets and / or neutrophils), stromal cells, endothelial cells, liver cells, muscle cells, cells of the nervous system, diseased cells, and combinations thereof.

[0019] The present invention also relates to the use of a pseudotyped retrovirus-like particle or retroviral vector as defined above to selectively modulate the activity of a target cell and / or to selectively transduce, for example, a target cell as defined above.

[0020] The present invention also relates to a method for selectively modulating the activity of and / or transducing a target cell, the method comprising the step of contacting a pseudotyped retrovirus-like particle or a retroviral vector as defined above with a cell comprising said target cell, e.g. a target cell as defined above.

[0021] The present invention also relates to a pseudotyped retrovirus-like particle or a retroviral vector as defined above for use as a medicament, preferably in immunotherapy, gene therapy and / or vaccination, for example in the prevention and / or treatment of immune diseases (e.g. autoimmune diseases), cancer, genetic diseases, allergic diseases, inflammatory diseases, infectious diseases, metabolic diseases, neurological diseases (e.g. neuronal atrophy, Parkinson's disease, Huntington's disease, Alzheimer's disease), muscular diseases, and combinations thereof.

[0022] The present invention also provides a nucleic acid comprising a sequence encoding a cell-targeting fusion protein and / or a sequence encoding a modulating fusion protein, the cell-targeting fusion protein comprises (i) an envelope glycoprotein G or envelope glycoprotein H, or a protein derived from a transmembrane domain, of a virus of the Paramyxoviridae family, and (ii) at least one cell-targeting domain; the modulating fusion protein comprises (i) a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family, or a transmembrane domain, and (ii) at least one functional domain; It also relates to nucleic acids.

[0023] The present invention also relates to a vector comprising a nucleic acid as defined above.

[0024] The present invention also provides a method for producing a pseudotyped retrovirus-like particle or retroviral vector as defined above, comprising injecting into a packaging cell line: (i) at least one nucleic acid encoding a cell-targeting fusion protein comprising (i) an envelope glycoprotein G or an envelope glycoprotein H, or a protein derived from a transmembrane domain, of a virus of the Paramyxoviridae family, and (ii) at least one cell-targeting domain; (ii) at least one nucleic acid encoding a modulating fusion protein comprising (i) an envelope glycoprotein G or envelope glycoprotein H, or a protein derived from a transmembrane domain, of a virus of the Paramyxoviridae family, and (ii) at least one functional domain; (iii) at least one nucleic acid encoding a glycoprotein derived from the envelope glycoprotein F of a virus of the Paramyxoviridae family; (iv) at least one vector comprising a nucleic acid encoding a core protein from said retrovirus; and (v) optionally, at least one vector comprising a genome derived from a packaging-competent retrovirus; co-transfecting The cell-targeting fusion protein and / or the modulating fusion protein comprises a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family. It also relates to methods. DETAILED DESCRIPTION OF THE INVENTION

[0025] Pseudotyped retrovirus-like particles or retroviral vectors As used herein, the term "retrovirus-like particle" refers to a particle that contains retroviral proteins Gag, Pol, and Env (envelope), but does not contain genetic information derived from a retrovirus.

[0026] The Gag, Pol, and Env proteins are derived from the retrovirus and are provided in trans by the packaging cell line.

[0027] As used herein, a "retroviral vector" comprises Gag, Pol, and Env (envelope) proteins and an RNA molecule. The RNA molecule does not contain the gag, env, or pol genes, but does contain the psi element and LTRs required for efficient packaging of the RNA molecule into the resulting particle. The RNA molecule may further comprise a gene of interest under the control of an appropriate promoter, such that the gene is expressed upon integration into the genome of a host or target cell.

[0028] Retrovirus-like particles or retroviral vectors are virus-like particles or viral vectors, respectively, whose core proteins, i.e., those encoded by the Gag and Pol genes, are derived from a retrovirus.

[0029] As used herein, the term "retrovirus" refers to a virus whose genome consists of an RNA molecule and contains the enzyme reverse transcriptase.

[0030] Retroviruses are members of the family Retroviridae. Retroviruses can be of the genus Oncovirus, Lentivirus, or Spumavirus.

[0031] The oncovirus can be an alpharetrovirus, a betaretrovirus, a deltaretrovirus, an epsilonretrovirus, or a gammaretrovirus.

[0032] When the retrovirus is an oncovirus, the retrovirus can be MLV (murine leukemia virus), ASV (avian sarcoma virus), feline leukemia virus, bovine leukemia virus, RSV (Rous sarcoma virus), MPMV (Mason-Pfizer monkey virus), HTLV I (human T-cell leukemia virus I), or HTLV II (human T-cell leukemia virus II).

[0033] When the retrovirus is a lentivirus, said retrovirus may be HIV (human immunodeficiency virus), preferably HIV-1 or HIV-2, SIV (simian immunodeficiency virus), EIAV (equine infectious anemia virus), FIV (feline immunodeficiency virus), or CAEV (caprine arthritis-encephalitis virus).

[0034] When the retrovirus is a spumavirus, said retrovirus may be HFV (human foamy virus).

[0035] For example, the retroviral vector can be a lentiviral vector, an alpharetroviral vector, a murine retroviral vector, or an FIV vector.

[0036] The genomes of such retroviruses are readily available in gene databases.

[0037] In a preferred embodiment, the retrovirus is a lentivirus, more preferably an HIV such as HIV-1 or HIV-2.

[0038] Thus, the present invention preferably relates to a lentivirus-like particle (LVP) or lentiviral vector (LV), preferably an LVP or LV derived from HIV.

[0039] The term "pseudotyped" in the expression "pseudotyped retrovirus-like particle or retroviral vector" herein means that the retrovirus-like particle or retroviral vector carries an envelope glycoprotein that is derived from at least another virus and / or is an engineered envelope glycoprotein, e.g., a chimeric envelope glycoprotein and / or a mutated envelope glycoprotein, in addition to the retrovirus.

[0040] The retrovirus-like particles or retroviral vectors according to the invention are pseudotyped with engineered glycoproteins derived from the glycoproteins of, for example, a virus of the Paramyxoviridae family, preferably a virus of the Morbillivirus or Henipavirus genera, as described in more detail below.

[0041] Paramyxoviridae envelope glycoproteins The modified envelope glycoproteins used to pseudotype the retrovirus-like particles or retroviral vectors according to the invention are derived from viruses of the Paramyxoviridae family.

[0042] The virus of the Paramyxoviridae family is preferably a virus of the genus Morbillivirus or Henipavirus.

[0043] Morbillivirus and Henipavirus viruses use two glycoproteins to enter target cells: the attachment protein (called glycoprotein G in Henipaviruses or glycoprotein H in Morbilliviruses) and glycoprotein F (also called the fusion protein or F protein). The F protein mediates the fusion of the viral membrane with the host cell membrane. Glycoprotein G / H recognizes receptors on the target membrane and assists the F protein in its membrane fusion function. Both glycoprotein G / H and glycoprotein F are used in modified forms to pseudotype the retrovirus-like particles or retroviral vectors according to the invention.

[0044] The Morbillivirus virus is, for example, selected from the group consisting of measles virus, canine distemper virus, whale morbillivirus, peste des petit ruminants virus, porcine distemper virus, and rinderpest virus.

[0045] A preferred Morbillivirus is the measles virus (MeV).

[0046] The Henipavirus virus is, for example, selected from the group consisting of Nipah virus, Sheeda virus, and Hendra virus.

[0047] A preferred virus of the Henipavirus genus is Nipah virus (NiV).

[0048] In a preferred embodiment, the modified envelope glycoproteins are derived from envelope glycoprotein H and envelope glycoprotein F of measles virus or envelope glycoprotein G and envelope glycoprotein F of Nipah virus.

[0049] An example of the sequence of the envelope glycoprotein G of Nipah virus is the sequence of SEQ ID NO:9.

[0050] An example of the sequence of the envelope glycoprotein F of Nipah virus is the sequence of SEQ ID NO: 11.

[0051] An example of the sequence of the envelope glycoprotein H of the measles virus (called glycoprotein H) is the sequence SEQ ID NO:10.

[0052] An example of the sequence of the envelope glycoprotein F of the measles virus is the sequence SEQ ID NO:12.

[0053] Proteins derived from envelope glycoprotein G / H A pseudotyped retrovirus-like particle or retroviral vector according to the invention comprises at least one cell-targeting fusion protein and at least one modulating fusion protein, wherein the cell-targeting fusion protein and / or the modulating fusion protein comprises a first protein derived from envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family.

[0054] The envelope glycoprotein G or envelope glycoprotein H is as defined above in the section entitled "Envelope glycoproteins of Paramyxoviridae."

[0055] The Paramyxoviridae virus is as defined above in the section entitled "Pseudotyped Retrovirus-Like Particles or Retroviral Vectors."

[0056] As used herein, the expression "a protein derived from envelope glycoprotein G or envelope glycoprotein H" means that the protein contains at least one modification by comparison with the sequence of envelope glycoprotein G or envelope glycoprotein H.

[0057] A preferred envelope glycoprotein G is the envelope glycoprotein G of Nipah virus, also referred to as NiV envelope glycoprotein G, NiV G glycoprotein, or NiV-G.

[0058] A preferred envelope glycoprotein H is the envelope glycoprotein H of the measles virus, also called MeV glycoprotein H, MeV hemagglutinin, or MV-H.

[0059] The envelope glycoprotein G or envelope glycoprotein H is the envelope glycoprotein of a virus that is a wild-type or vaccine strain, or a mutant thereof, provided that the mutant retains the ability of the wild-type or vaccine strain glycoprotein to recognize receptors on target membranes and assist the F protein in its membrane fusion function.

[0060] The reference sequence for NiV envelope glycoprotein G is the sequence of SEQ ID NO:9.

[0061] The NiV envelope glycoprotein G can be a protein having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9. For example, the NiV envelope glycoprotein can be a protein having the sequence of SEQ ID NO: 9.

[0062] The reference sequence for MeV hemagglutinin is the sequence of SEQ ID NO:10.

[0063] The MeV hemagglutinin can be a MeV hemagglutinin of a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10. For example, the MeV hemagglutinin can be a MeV hemagglutinin of the sequence of SEQ ID NO: 10.

[0064] In a preferred embodiment, the protein derived from envelope glycoprotein G lacks at least a portion of the cytoplasmic region of said envelope glycoprotein G.

[0065] A protein lacking at least a portion of the cytoplasmic region of the envelope glycoprotein G is also referred to as a protein truncated within its cytoplasmic region.

[0066] As used herein, the expressions "a protein lacking x amino acids in its cytoplasmic region," "a protein truncated by x amino acids in its cytoplasmic region," and "Δcx protein" are synonymous and can be used interchangeably.

[0067] The use of an envelope glycoprotein G truncated within its cytoplasmic domain significantly improves its incorporation into retrovirus-like particles and retroviral vectors, thereby allowing the production of pseudotyped retrovirus-like particles or retroviral vectors with high titers and production yields.

[0068] The cytoplasmic domain of the envelope glycoprotein G is located at the N-terminus.

[0069] Therefore, when determining the location of the cleaved portion of the Δcx glycoprotein using the uncleaved glycoprotein G as a reference, counting begins at the second amino acid residue at the N-terminus of envelope glycoprotein G, i.e., omitting the first methionine residue.

[0070] For example, a protein lacking the X amino acid in the cytoplasmic domain of glycoprotein G, sequence SEQ ID NO: Z, differs from glycoprotein G in that it lacks amino acids 2-1+X of sequence SEQ ID NO: Z.

[0071] The location of the cytoplasmic region within the envelope glycoprotein G sequence can be readily determined by one skilled in the art.

[0072] The cytoplasmic region of the MeV H glycoprotein consists of amino acids 1 to 34 of the sequence of SEQ ID NO:10, for example.

[0073] The cytoplasmic region of NiV G glycoprotein consists of, for example, amino acids 1 to 45 of the sequence of SEQ ID NO:9.

[0074] For example, a protein derived from envelope glycoprotein G or envelope glycoprotein H may lack at least 10 amino acids, at least 15 amino acids, at least 18 amino acids, or at least 20 amino acids within the cytoplasmic region.

[0075] For example, a protein derived from the envelope glycoprotein H of the measles virus may lack at least 10 amino acids, at least 15 amino acids, at least 18 amino acids, at least 20 amino acids, or at least 24 amino acids within the cytoplasmic region. In preferred embodiments, the protein derived from the envelope glycoprotein H of the measles virus lacks 15, 18, 20, or 24 amino acids.

[0076] For example, the protein derived from the envelope glycoprotein G of Nipah virus may lack at least 10 amino acids, at least 15 amino acids, at least 18 amino acids, at least 20 amino acids, at least 25 amino acids, or at least 30 amino acids within the cytoplasmic region. In a preferred embodiment, the protein derived from the envelope glycoprotein G of Nipah virus lacks 34 amino acids.

[0077] In a preferred embodiment, the protein derived from envelope glycoprotein G or envelope glycoprotein H is at least partially incapable of binding to at least one natural receptor of said envelope glycoprotein G or envelope glycoprotein H.

[0078] The at least partial inability of the envelope glycoprotein G or envelope glycoprotein H to bind to at least one natural receptor can be obtained by at least one mutation introduced into the sequence of the envelope glycoprotein G or envelope glycoprotein H.

[0079] For example, a protein derived from envelope glycoprotein G or envelope glycoprotein H may contain at least one point mutation, preferably at least two point mutations, compared to the sequence of said envelope glycoprotein.

[0080] Point mutations can be deletions, additions, or substitutions of amino acids.

[0081] In a preferred embodiment, the point mutation is a substitution.

[0082] In a preferred embodiment, the protein derived from envelope glycoprotein G or envelope glycoprotein H is not capable of binding to at least one natural receptor of said envelope glycoprotein G or envelope glycoprotein H, i.e. is not fully capable of binding to it.

[0083] The inability of proteins derived from envelope glycoprotein G or envelope glycoprotein H to bind to their natural receptors greatly increases cell targeting efficiency.

[0084] The ability of envelope glycoprotein G to bind to at least one natural receptor can be assessed by any method known to those skilled in the art.

[0085] The natural NiV receptors are the ephrin B2 and ephrin B3 receptors for NiV.

[0086] Thus, proteins derived from NiV envelope glycoprotein G are preferably at least partially incapable of binding to NiV ephrin B2 receptor and / or ephrin B3 receptor, preferably to both NiV ephrin B2 receptor and ephrin B3 receptor.

[0087] For example, the envelope glycoprotein NiV-G may contain at least two or at least three point mutations selected from the group consisting of E501A, W504A, Q530A, and E533A compared to the sequence of SEQ ID NO:9.

[0088] In a preferred embodiment, the glycoprotein NiV-G comprises or consists of the point mutations E501A, W504A, Q530A, and E533A, thus resulting in the inability of NiV to bind to the ephrin B2 and ephrin B3 receptors.

[0089] The natural MeV receptors are SLAM, nectin-4, and CD46.

[0090] Thus, proteins derived from MeV envelope glycoprotein H are preferably at least partially incapable of binding to SLAM, nectin 4, and / or CD46, preferably at least to both SLAM and CD46.

[0091] For example, the envelope glycoprotein MV-H may contain at least two or at least three point mutations selected from the group consisting of Y481A, R533A, S548L, and F549S compared to the sequence of SEQ ID NO:10.

[0092] In a preferred embodiment, the envelope glycoprotein MV-H comprises or consists of the point mutations Y481A, R533A, S548L and F549S, thus resulting in the inability to bind to SLAM and CD46.

[0093] Thus, a protein derived from envelope glycoprotein G or envelope glycoprotein H preferably lacks at least one part of the cytoplasmic region of said envelope glycoprotein G or envelope glycoprotein H and / or is at least partially incapable of binding to at least one natural receptor for said envelope glycoprotein G or envelope glycoprotein H.

[0094] In a more preferred embodiment, either the protein derived from envelope glycoprotein G or envelope glycoprotein H lacks at least one portion of the cytoplasmic region of said envelope glycoprotein G or envelope glycoprotein H and is at least partially incapable of binding to at least one natural receptor for said envelope glycoprotein G or envelope glycoprotein H.

[0095] As used herein, a "sequence at least x% identical to a reference sequence" means that the sequence is identical to the reference sequence or differs from the reference sequence by up to 100-x amino acid changes for every 100 amino acids in the reference sequence.

[0096] Alignment and percent identity determination can be performed manually or automatically, for example, using the Needle program, which is based on the algorithm by Needleman and Wunsch, as described in Needleman and Wunsch (1970), J. Mol. Biol., 48:443-453, using, for example, the following parameters for comparison of polypeptide sequences: comparison matrix: BLOSUM62, gap opening penalty: 10 and gap extension penalty: 0.5, end gap penalty: false, end gap opening penalty=10, end gap extension penalty=0.5; and for comparison of polynucleotide sequences: comparison matrix: DNAFULL, gap opening penalty=10, gap extension penalty=0.5, end gap penalty: false, end gap opening penalty=10, end gap extension penalty=0.5.

[0097] As defined herein, an amino acid sequence that is "at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical" to a reference sequence may include mutations, such as deletions, insertions, and / or substitutions, compared to the reference sequence.

[0098] In the case of substitutions, the substitutions preferably correspond to conservative substitutions as indicated below in Table 1. In preferred embodiments, sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence differ from the reference sequence only by conservative substitutions.

[0099] [Table 1]

[0100] In another preferred embodiment, an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence corresponds to a naturally occurring allelic variant of the reference sequence.

[0101] Glycoprotein derived from envelope glycoprotein F The pseudotyped retrovirus-like particle or retroviral vector according to the invention also comprises at least one glycoprotein derived from the envelope glycoprotein F of a virus of the Paramyxoviridae family, preferably a glycoprotein lacking at least a portion of the cytoplasmic domain of said envelope glycoprotein F.

[0102] Envelope glycoprotein F is as defined above in the section entitled "Envelope glycoproteins of Paramyxoviridae."

[0103] The Paramyxoviridae virus is as defined above in the section entitled "Pseudotyped Retrovirus-Like Particles or Retroviral Vectors."

[0104] As used herein, the expression "a protein derived from envelope glycoprotein F" means that the protein comprises at least one modification by comparison with the sequence of envelope glycoprotein F.

[0105] A preferred envelope glycoprotein F is the envelope glycoprotein F of Nipah virus, also referred to as NiV envelope glycoprotein F, NiV F glycoprotein, or NiV-F.

[0106] Another preferred envelope glycoprotein F is the envelope glycoprotein F of the measles virus, also referred to as MeV glycoprotein F or MeV-F.

[0107] The envelope glycoprotein F is a wild-type or vaccine strain envelope glycoprotein or a mutant thereof, provided that the mutant retains the ability of the wild-type or vaccine strain glycoprotein F to mediate fusion of the viral membrane with the target or host cell membrane.

[0108] The reference sequence for NiV envelope glycoprotein F is the sequence of SEQ ID NO: 11. NiV envelope glycoprotein F can be a protein having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11. For example, NiV envelope glycoprotein F can be a protein having the sequence of SEQ ID NO: 11.

[0109] The reference sequence for MeV envelope glycoprotein F is the sequence of SEQ ID NO:12.

[0110] MeV envelope glycoprotein F can be a protein having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12. For example, MeV envelope glycoprotein F can be a protein having the sequence of SEQ ID NO: 12.

[0111] In a preferred embodiment, the protein derived from envelope glycoprotein F lacks at least a portion of the cytoplasmic region of said envelope glycoprotein F.

[0112] A protein lacking at least a portion of the cytoplasmic region of said envelope glycoprotein F is also referred to as a protein truncated within its cytoplasmic region.

[0113] As used herein, the expressions "a protein lacking x amino acids in its cytoplasmic region," "a protein truncated by x amino acids in its cytoplasmic region," and "Δcx protein" are synonymous and can be used interchangeably.

[0114] The use of envelope glycoprotein F truncated within its cytoplasmic domain significantly improves its incorporation into retrovirus-like particles and retroviral vectors, thereby allowing the production of pseudotyped retrovirus-like particles or retroviral vectors with high titers and production yields.

[0115] The cytoplasmic domain of envelope glycoprotein F is located at the C-terminus.

[0116] Therefore, when determining the location of the cleaved portion of Δcx glycoprotein using uncleaved glycoprotein F as a reference, counting begins from the C-terminus of glycoprotein F.

[0117] For example, compared to glycoprotein F, which consists of n amino acids and has the sequence of SEQ ID NO: Z, a glycoprotein lacking X amino acids in its cytoplasmic region differs from glycoprotein F in that it lacks amino acids n-x+1 to n of the sequence of SEQ ID NO: Z.

[0118] The location of the cytoplasmic region within the envelope glycoprotein F sequence can be readily determined by one skilled in the art.

[0119] The cytoplasmic region of MeV glycoprotein F consists of, for example, amino acids 518 to 550 (33 amino acids) of the sequence of SEQ ID NO:12.

[0120] The cytoplasmic region of NiV glycoprotein F consists of, for example, amino acids 519 to 546 (28 amino acids) of the sequence of SEQ ID NO:11.

[0121] For example, a protein derived from envelope glycoprotein F may lack at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, or at least 30 amino acids within the cytoplasmic region.

[0122] For example, a protein derived from the envelope glycoprotein F of measles virus may lack 30 amino acids in the cytoplasmic region compared to the envelope glycoprotein F of said measles virus. For example, the protein derived from the envelope glycoprotein F of measles virus and lacking 30 amino acids in the cytoplasmic region comprises or consists of the sequence of SEQ ID NO: 15.

[0123] For example, a protein derived from Nipah virus envelope glycoprotein F may lack 22 amino acids in the cytoplasmic region compared to said Nipah virus envelope glycoprotein F. For example, said protein derived from Nipah virus envelope glycoprotein F and lacking 22 amino acids in the cytoplasmic region comprises or consists of the sequence of SEQ ID NO: 16.

[0124] target cell A target cell is a cell of interest whose activity is to be modulated and / or which is transduced with at least one gene of interest.

[0125] Target cells express specific cell surface receptors on their surface, which allows for specific targeting of these cells but not cells that do not express said cell surface receptors.

[0126] As used herein, the terms "target cells" and "target subset of cells" are synonymous and can be used interchangeably.

[0127] The target cells may be selected from the group consisting of hematopoietic cells, stromal cells, endothelial cells, hepatic cells, muscle cells (eg, cardiac cells), nervous system cells, and / or diseased cells.

[0128] Neural cells are, for example, neurons and / or glial cells.

[0129] As used herein, "hematopoietic cells" refers to cells of the hematopoietic system.

[0130] Preferred target cells are hematopoietic cells.

[0131] The hematopoietic cells can be selected from the group consisting of T cells, B cells, monocytes, Th1 cells, Th2 cells, Treg cells, mast cells, dendritic cells (DCs), natural killer (NK) cells, natural killer T (NKT) cells, macrophages, hematopoietic stem cells, precursor T cells and / or precursor B cells, erythroblasts, platelets, neutrophils, and combinations thereof.

[0132] More preferred target hematopoietic cells are T cells, more preferably CD8+ T cells or CD4+ T cells.

[0133] A diseased cell can be a tumor cell, a tumor stem cell, a cell that lacks a particular functional gene, overexpresses a particular gene, and / or expresses a truncated or mutated form of a particular gene, an infected cell, and / or a dysfunctional cell.

[0134] Cell-specific targeting domain The cell-specific targeting domain (also referred to as the "cell targeting domain") enables the pseudotyped retrovirus-like particle or retroviral vector according to the invention to specifically bind to a surface receptor selectively expressed by the target cell.

[0135] The target cells may be as defined in the section of the same name above.

[0136] The cell-targeting domain of the targeting fusion protein is preferably selected from the group consisting of a DARPin, an scFv, a targeting peptide, and combinations thereof.

[0137] The term "DARPin" refers to designed ankyrin repeat proteins.

[0138] The term scFv refers to a single chain variable fragment of an antibody.

[0139] For example, the cell targeting domain is specific for CD3, CD8, CD4, a cancer cell marker, CD11b, CD19, CD62L, CD56, Glut-1 (glucose transporter), CD19, CD22, CD20, CCR5, or CXCR4.

[0140] Preferred cell targeting domains are DARPins specific for CD4 or scFvs specific for CD8.

[0141] Functional domains The functional domain is capable of modulating the activity of a target cell.

[0142] As used herein, "modulating the activity of a target cell" means activating or inhibiting, inducing a phenotypic change (e.g., maturation and / or differentiation), inducing proliferation, and / or inducing apoptosis of said target cell. Modulating the activity of a target cell includes, for example, enhancing or suppressing immunity, promoting a cell-type specific immune response.

[0143] The functional domain of the modulating fusion protein is preferably a receptor ligand.

[0144] As used herein, "receptor ligand" preferably refers to a molecule, preferably a protein, that is normally released by cells and that alters the physiological state of cells that express the cognate receptor on their cell surface. Receptor ligands are, for example, selected from the group consisting of cytokines, growth factors, hormones, neurotransmitters, apoptotic ligands, chemokines, glucose transporters, and combinations thereof.

[0145] For example, the cytokine may be selected from the group consisting of an interleukin (IL), a TNF (tumor necrosis factor), or an interferon.

[0146] The interleukin can be, for example, IL-2, IL-3, IL-6, IL-7, IL15, IL21, IL-17, or IL-12.

[0147] For example, the apoptosis ligand can be FAS ligand, CD40 ligand, or TNF alpha.

[0148] For example, the chemokine can be CXCL4, CCL5, or CXCL10.

[0149] For example, the growth factor can be GM-SCF (granulocyte-macrophage colony-stimulating factor), stem cell factor (SCF), thrombopoietin (TPO), or Flt-3 ligand.

[0150] For example, the hormone can be insulin, growth hormone, or a hormone peptide, such as vasopressin. For example, the neurotransmitter can be a neuropeptide selected from insulin, glucagon, calcitonin, neurotensin, or bradykinin.

[0151] A preferred functional domain is a cytokine, preferably an interleukin, more preferably IL-7.

[0152] Cell-Targeting and Modulating Fusion Proteins The retrovirus-like particles and retroviral vectors according to the present invention comprise two fusion proteins: one cell-targeting fusion protein and one modulating fusion protein.

[0153] The cell-targeting fusion protein comprises (i) a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family, or a transmembrane domain, and (ii) at least one cell-targeting domain.

[0154] The modulating fusion protein comprises (i) a protein derived from envelope glycoprotein G or envelope glycoprotein H, or a transmembrane domain, of a virus of the Paramyxoviridae family, and (ii) at least one functional domain.

[0155] Those skilled in the art will clearly understand that cell-targeting fusion proteins and modulating fusion proteins are two different types of fusion proteins, and therefore the cell-targeting domain and the functional domain are different.

[0156] The transmembrane domain can be any naturally occurring or non-naturally occurring transmembrane domain.

[0157] The transmembrane domain may be a receptor transmembrane domain, a transmembrane protein, preferably a viral transmembrane protein, a fragment of a transmembrane protein, a transmembrane peptide, or a variant thereof, such as a genetically engineered receptor transmembrane domain, a genetically engineered transmembrane protein, a fragment of a genetically engineered transmembrane protein, or a genetically engineered transmembrane peptide.

[0158] Examples of transmembrane domains are the transmembrane domain (TMD) of the platelet-derived growth factor receptor (PDGFR), the transmembrane domain of CD34, or the VSVG glycoprotein transmembrane domain.

[0159] The C-terminus of a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family is preferably fused directly or indirectly (e.g., via a linker) to the N-terminus of a cell targeting domain or functional domain.

[0160] The C-terminus of the transmembrane domain is preferably fused directly or indirectly (eg, via a linker) to the N-terminus of the cell targeting domain or functional domain.

[0161] The protein derived from envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family, the glycoprotein derived from envelope glycoprotein F, the cell targeting domain, and the functional domain are as defined above.

[0162] When both are present in a pseudotyped retrovirus-like particle or retroviral vector, the protein derived from the envelope glycoprotein G or envelope glycoprotein H of a Paramyxoviridae virus in the cell-targeting fusion protein and the protein derived from the modulating fusion protein can be derived from the same or different Paramyxoviridae viruses.

[0163] When the protein derived from envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family in the cell-targeting fusion protein and the protein derived from the modulating fusion protein are derived from viruses of different Paramyxoviridae families, the proteins are preferably derived from viruses of the same genus, and more preferably from viruses of the same species.

[0164] The proteins derived from envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family in the cell-targeting fusion protein and these proteins in the modulating fusion protein are preferably identical. Thus, in preferred embodiments, the cell-targeting fusion protein differs from the modulating fusion protein by its targeting domain, rather than by a functional domain, and perhaps by at least one linker and / or at least one tag, if present.

[0165] The two proteins of the fusion protein can be linked together with a linker. Any suitable linker known to those skilled in the art can be used.

[0166] For example, the linker can be (G4S)3, G4S, a factor Xa cleavage site, or a helical linker (eg, HL3, HL7, . . . ).

[0167] Fusion proteins can be tagged, for example, to facilitate their purification and / or detection.

[0168] If present, the tag is preferably placed at the C-terminus of the fusion protein, i.e., fused to the N-terminus of the cell targeting domain and / or functional domain. Any suitable tag known to those skilled in the art can be used.

[0169] For example, the tag can be a His tag, an RGS-His tag such as RGSH6, an HA tag, or a c-myc tag.

[0170] Specific pseudotyped retrovirus-like particles or retroviral vectors The present invention particularly a) at least one cell-targeting fusion protein comprising (i) an envelope glycoprotein G or envelope glycoprotein H, or a protein derived from a transmembrane domain, of a virus of the Paramyxoviridae family, and (ii) at least one cell-targeting domain; b) at least one modulating fusion protein comprising (i) an envelope glycoprotein G or envelope glycoprotein H, or a protein derived from a transmembrane domain, of a virus of the Paramyxoviridae family, and (ii) at least one functional domain; and c) at least one glycoprotein derived from the envelope glycoprotein F of a virus of the Paramyxoviridae family Including, the cell-targeting fusion protein and / or the modulating fusion protein comprises a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family; It relates to pseudotyped retrovirus-like particles or retroviral vectors.

[0171] Thus, the pseudotyped retrovirus-like particle or retroviral vector comprises at least one fusion protein comprising a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family, wherein the fusion protein is a cell-targeting fusion protein or a modulating fusion protein. In one embodiment, the pseudotyped retrovirus-like particle or retroviral vector comprises two fusion proteins, each comprising a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family, wherein one fusion protein is a cell-targeting fusion protein and the second fusion protein is a modulating fusion protein.

[0172] In one embodiment, the pseudotyped retrovirus-like particle or retroviral vector comprises: a) at least one cell-targeting fusion protein comprising (i) a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family, and (ii) at least one cell-targeting domain; b) at least one modulating fusion protein comprising (i) a transmembrane domain and (ii) at least one functional domain; and c) at least one glycoprotein derived from the envelope glycoprotein F of a virus of the Paramyxoviridae family Includes.

[0173] In one embodiment, the pseudotyped retrovirus-like particle or retroviral vector comprises: a) at least one cell-targeting fusion protein comprising (i) a transmembrane domain and (ii) at least one cell-targeting domain; b) at least one modulating fusion protein comprising (i) a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family, and (ii) at least one functional domain; and c) at least one glycoprotein derived from the envelope glycoprotein F of a virus of the Paramyxoviridae family Includes.

[0174] In one embodiment, the pseudotyped retrovirus-like particle or retroviral vector comprises: a) at least one cell-targeting fusion protein comprising (i) a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family, and (ii) at least one cell-targeting domain; b) at least one modulating fusion protein comprising (i) a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family, and (ii) at least one functional domain; and c) at least one glycoprotein derived from the envelope glycoprotein F of a virus of the Paramyxoviridae family Includes.

[0175] The proteins derived from envelope glycoprotein G of a virus of the Paramyxoviridae family in the cell-targeting fusion protein, these proteins in the modulating fusion protein, and the glycoprotein derived from envelope glycoprotein F may be derived from viruses of the same Paramyxoviridae family or from viruses of different Paramyxoviridae families.

[0176] When the proteins derived from envelope glycoprotein G of a virus of the Paramyxoviridae family in the cell-targeting fusion protein, these proteins in the modulating fusion protein, and / or the glycoprotein derived from envelope glycoprotein F of a virus of the Paramyxoviridae family are derived from viruses of different Paramyxoviridae families, these proteins are preferably derived from viruses of the same genus, and more preferably from viruses of the same species.

[0177] In one embodiment, the proteins in the cell-targeting fusion protein derived from envelope glycoprotein G of a virus from the Paramyxoviridae family, these proteins in the modulating fusion protein, and the glycoprotein derived from envelope glycoprotein F are derived from the same virus from the Paramyxoviridae family.

[0178] In another embodiment, the protein derived from envelope glycoprotein G of a Paramyxoviridae virus and the glycoprotein derived from envelope glycoprotein F of a Paramyxoviridae virus in the cell-targeting fusion protein are derived from the same Paramyxoviridae virus, while the modulating fusion protein comprises (i) a transmembrane domain, e.g., the transmembrane domain (TMD) of platelet-derived growth factor receptor (PDGFR), and (ii) at least one functional domain.

[0179] In another embodiment, the protein derived from envelope glycoprotein G of a Paramyxoviridae virus and the glycoprotein derived from envelope glycoprotein F of a Paramyxoviridae virus in the modulating fusion protein are derived from the same Paramyxoviridae virus, while the cell-targeting fusion protein comprises (i) a transmembrane domain, e.g., the transmembrane domain (TMD) of platelet-derived growth factor receptor (PDGFR), and (ii) at least one cell-targeting domain.

[0180] The different components of the pseudotyped retrovirus-like particle or retroviral vector, in particular the cell-targeting fusion protein, the modulating fusion protein, the glycoprotein derived from the envelope glycoprotein F of a virus of the Paramyxoviridae family, and the transmembrane domain, are as defined above.

[0181] A preferred pseudotyped retrovirus-like particle or retroviral vector is a) at least one cell-targeting fusion protein comprising: (i) a protein derived from the envelope glycoprotein G of a virus of the Paramyxoviridae family, the protein lacking at least a portion of the cytoplasmic region of said envelope glycoprotein G and at least partially not capable of binding to at least one natural receptor or transmembrane domain of said envelope glycoprotein G; and (ii) at least one cell-targeting domain; b) at least one modulating fusion protein comprising: (i) a protein derived from the envelope glycoprotein G of a virus of the Paramyxoviridae family, the protein lacking at least a portion of the cytoplasmic domain of said envelope glycoprotein G and at least partially unable to bind to at least one natural receptor or transmembrane domain of said envelope glycoprotein G; and (ii) at least one functional domain; and c) at least one glycoprotein derived from the envelope glycoprotein F of a virus of the Paramyxoviridae family, lacking at least a portion of the cytoplasmic domain of said envelope glycoprotein F; Including, In this case, the cell-targeting fusion protein and / or the modulating fusion protein comprises a protein derived from envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family.

[0182] Examples of pseudotyped retrovirus-like particles or retroviral vectors are shown in Table 2 below.

[0183] [Table 2]

[0184] In a preferred embodiment, the pseudotyped retrovirus-like particle or retroviral vector is obtainable or obtained by the production methods described herein below in the section entitled "Methods for producing pseudotyped retrovirus-like particles or retroviral vectors."

[0185] Nucleic acids and vectors The present invention also relates to nucleic acids encoding cell-targeting and / or modulating fusion proteins used to pseudotype the retrovirus-like particles or retroviral vectors according to the invention.

[0186] Thus, the present invention particularly relates to a nucleic acid comprising a sequence encoding a cell-targeting fusion protein and / or a sequence encoding a modulating fusion protein, the cell-targeting fusion protein comprises (i) a protein derived from the envelope glycoprotein G of a virus of the Paramyxoviridae family, preferably lacking at least a portion of the cytoplasmic domain of said envelope glycoprotein G, and preferably being at least partially unable to bind to at least one natural receptor or transmembrane domain of said envelope glycoprotein G, and (ii) at least one cell-targeting domain, the modulating fusion protein comprises (i) a protein derived from the envelope glycoprotein G of a virus of the Paramyxoviridae family, preferably lacking at least a portion of the cytoplasmic domain of said envelope glycoprotein G, and preferably at least partially unable to bind to at least one natural receptor or transmembrane domain of said envelope glycoprotein G, and (ii) at least one functional domain; Concerning nucleic acids.

[0187] When the nucleic acid comprises a sequence encoding a cell-targeting fusion protein and a sequence encoding a modulating fusion protein, the cell-targeting fusion protein and / or the modulating fusion protein comprises a protein derived from envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family.

[0188] The nucleic acid is preferably (i) a sequence encoding a protein derived from the envelope glycoprotein of a virus of the Paramyxoviridae family, which preferably lacks at least a portion of the cytoplasmic domain of said envelope glycoprotein G and which preferably is at least partially incapable of binding to at least one natural receptor of said envelope glycoprotein G; (ii) a sequence encoding one cell targeting domain or one functional domain; (iii) optionally a linker sequence between sequence (i) and sequence (ii); (iv) optionally, and preferably at the 3' end of sequence (ii), a sequence encoding a tag; comprising or consisting of In this case, sequence (i) and sequence (ii) are fused in frame.

[0189] The linker sequence encodes a linker as defined above, for example, (G4S)3.

[0190] Sequence (i) is located 5' of the nucleic acid compared to sequence (ii), which is located 3' of the nucleic acid.

[0191] In one embodiment, the nucleic acid comprises or consists of a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:13, and / or comprises or consists of a sequence that encodes a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:14.

[0192] In a preferred embodiment, the nucleic acid comprises or consists of a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:13, and / or comprises or consists of a sequence encoding a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:14.

[0193] The definition of percentage sequence identity is provided above.

[0194] A nucleic acid sequence that is "at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical" to a reference sequence can contain mutations, such as deletions, insertions, and / or substitutions, compared to the reference sequence.

[0195] In the case of substitutions, the substitutions preferably correspond to those which result in silent or conservative substitutions in the translated amino acid sequence, for example, compared to the reference sequences indicated in Table 1 above.

[0196] In preferred embodiments, a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence differs from the reference sequence only by substitutions that result in silent and / or conservative substitutions.

[0197] The present invention also relates to a nucleic acid encoding a glycoprotein derived from the envelope glycoprotein F of a virus of the Paramyxoviridae family, preferably lacking at least a part of the cytoplasmic domain of said envelope glycoprotein F.

[0198] The glycoprotein derived from the envelope glycoprotein F of a virus of the Paramyxoviridae family is as defined above.

[0199] The nucleic acid is preferably an isolated nucleic acid.

[0200] As used herein, the term "nucleic acid" refers to the phosphate polymeric forms of ribonucleosides (also referred to as "RNA molecules"), deoxyribonucleosides (also referred to as "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single- or double-stranded form.

[0201] The term "isolated," when referring to a biological component (such as a nucleic acid, vector, or protein), refers to a biological component that has been substantially separated or purified from other biological components in an organism's cells or the organism itself, including other chromosomal and extrachromosomal DNA and RNA, proteins, cells, and subcellular organelles, as they naturally occur. An "isolated nucleic acid" or "isolated vector" includes nucleic acid molecules that have been purified by standard purification techniques. These terms also encompass nucleic acids and vectors prepared by amplification and / or cloning, as well as chemically synthesized nucleic acids and vectors.

[0202] The nucleic acids according to the invention are preferably cloned into a vector.

[0203] Thus, the term "vector" has a different meaning than "retroviral vector."

[0204] As used herein, the term "vector" means a nucleic acid vector.

[0205] A vector generally contains an origin of replication, a multiple cloning site, and a selectable marker.

[0206] The vector preferably comprises an expression cassette, ie a nucleic acid according to the invention placed under the control of at least one expression signal allowing its expression.

[0207] The expression signals are in particular selected from promoters, terminators, enhancers and combinations thereof.

[0208] Suitable promoters, terminators and enhancers are well known to those skilled in the art.

[0209] Examples of vectors are eg plasmids.

[0210] As used herein, "plasmid" means a double-stranded circular DNA. A plasmid may contain a marker gene that allows for selection of cells containing the plasmid, an origin of replication that allows the cell to replicate the plasmid, and / or a multiple cloning site that allows for insertion of a nucleic acid according to the invention.

[0211] Therefore, the present invention also relates to a vector comprising a nucleic acid as defined above.

[0212] The vector is preferably an isolated vector.

[0213] Vectors are a first nucleic acid, (i) a) a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family, preferably lacking at least a portion of the cytoplasmic region of said envelope glycoprotein G or envelope glycoprotein H, and preferably at least partially unable to bind to at least one natural receptor for said envelope glycoprotein G or envelope glycoprotein H, or b) a sequence encoding a transmembrane domain; (ii) a sequence encoding one cell targeting domain; and (iii) optionally a linker sequence between sequence (i) and sequence (ii); (iv) optionally, and preferably at the 3' end of sequence (ii), a sequence encoding a tag; a first nucleic acid comprising or consisting of a sequence (i) and a sequence (ii) fused in frame; a second nucleic acid, (i) a) a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family, preferably lacking at least a portion of the cytoplasmic region of said envelope glycoprotein G or envelope glycoprotein H, and preferably at least partially unable to bind to at least one natural receptor for said envelope glycoprotein G or envelope glycoprotein H, or b) a sequence encoding a transmembrane domain; (ii) a sequence encoding one functional domain; and (iii) optionally a linker sequence between sequence (i) and sequence (ii); (iv) optionally, and preferably at the 3' end of sequence (ii), a sequence encoding a tag; a second nucleic acid comprising or consisting of the sequence (i) and the sequence (ii) fused in frame; and In this case, the first nucleic acid and / or the second nucleic acid comprises a sequence encoding a protein derived from the envelope glycoprotein G or the envelope glycoprotein H of a virus of the Paramyxoviridae family.

[0214] Thus, the first nucleic acid encodes a cell-targeting fusion protein and the second nucleic acid encodes a modulating fusion protein.

[0215] Gene of interest When a pseudotyped retroviral vector is used, at least one gene of interest may be present within the retroviral-derived genome of said retroviral vector.

[0216] The gene of interest may encode a therapeutic protein, an apoptotic protein, a chimeric antigen receptor, a cell surface receptor, an antibody, an antibody fragment, an shRNA, an antigen, a cytokine, a microRNA, a CRISPR (clustered, regularly interspaced, short palindromic repeat) / CAS element (e.g., CAS9 and / or guide RNA, particularly for disruption or correction of a specific gene), other nuclease systems such as zinc finger nucleases, an S / MAR (Scaffold / Matrix Attachment Region) episome, a ligand, and / or a receptor.

[0217] The retroviral vector inserts the S / MAR episome into the cell nucleus, and then the retroviral vector is replicated by the host cell DNA.

[0218] Examples of genes of interest include globin genes, hematopoietic growth factor genes (e.g., the erythropoietin (EPO) gene), interleukin genes (e.g., the interleukin 1 gene, the interleukin 2 gene, the interleukin 3 gene, the interleukin 6 gene, or the interleukin 12 gene, among others), colony-stimulating factor genes (such as the granulocyte colony-stimulating factor gene, the granulocyte / macrophage colony-stimulating factor gene, or the stem cell colony-stimulating factor gene), the platelet-specific integrin αIIbβ gene, multidrug resistance genes, the gp91 gene or the gp47 gene that are deficient in patients with chronic granulomatous disease (CGD), and antibodies that make cells resistant to infection by pathogens. These include viral genes (such as human immunodeficiency virus), genes encoding blood clotting factors VIII or IX that are mutated in hemophilia, genes encoding ligands involved in T cell-mediated immune responses (such as T cell antigen receptors and chimeric antigen receptors (CARs)), B cell antigen receptors (immunoglobulins, neutralizing antibodies against HIV, hepatitis C, hepatitis B, and / or other infectious diseases), gamma chain genes common to interleukin receptors, TNF genes, gamma interferon genes, CTLA4 genes, Melana, etc., genes expressed in tumor cells, MAGE genes (such as MAGE-1 and MAGE-3), P198 genes, P1A genes, and gp100 genes.

[0219] Methods for producing pseudotyped retrovirus-like particles or retroviral vectors The present invention also provides a method for producing a pseudotyped retrovirus-like particle or retroviral vector as defined above, comprising injecting into a packaging cell line: (i) at least one nucleic acid encoding a cell-targeting fusion protein; (ii) at least one nucleic acid encoding a modulating fusion protein; (iii) at least one nucleic acid encoding a glycoprotein derived from the envelope glycoprotein F of a virus of the Paramyxoviridae family, preferably lacking at least a portion of the cytoplasmic region of said envelope glycoprotein F; (iv) at least one vector comprising a nucleic acid encoding a core protein from said retrovirus; and (v) optionally, at least one vector comprising a genome derived from a packaging-competent retrovirus; and co-transfected with This also relates to a method comprising the step of obtaining co-transfected cells.

[0220] The cell-targeting fusion protein and / or the modulating fusion protein comprises a protein derived from envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family.

[0221] At least one vector containing a genome derived from a packaging-competent retrovirus is required solely to generate a retroviral vector.

[0222] Any suitable packaging cell line known to those skilled in the art may be used.

[0223] As used herein, "packaging cell line" refers to a cell line capable of expressing the different components of the pseudotyped retrovirus-like particles or retroviral vectors of the invention.

[0224] Nucleic acids and vectors encoding the different components of the pseudotyped retrovirus-like particles or retroviral vectors of the invention can be integrated into the genome of a packaging cell line, for example, in the case of murine leukemia virus-based vectors.

[0225] The packaging cell line is preferably compatible with expression of the lentiviral Gag and Pol genes.

[0226] For example, the packaging cell line can be selected from the group consisting of 293T cells, insect cells, TE 671 cells, and HT1080 cells.

[0227] The term "transfection" refers to the introduction of at least one foreign nucleic acid or foreign vector (e.g., DNA, cDNA, or RNA) into a cell, such that the host cell expresses the protein encoded by the nucleic acid or vector. The foreign nucleic acid or foreign vector may include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by the genetic machinery of the cell.

[0228] A vector containing the Psi sequence necessary for encapsidation is called a Psi-positive vector, whereas a vector not containing the Psi sequence is called a Psi-negative vector.

[0229] Only vectors containing a genome derived from a packaging-competent retrovirus are Psi-positive vectors. Other nucleic acids and vectors used in co-transfection are Psi-negative.

[0230] The nucleic acid encoding the cell-targeting fusion protein, the nucleic acid encoding the modulating fusion protein, and the nucleic acid encoding the glycoprotein derived from envelope glycoprotein F of a Paramyxoviridae virus are as defined above. The nucleic acids can be provided in the form of two or three separate vectors: for example, two separate vectors, such as a first vector comprising or consisting of a nucleic acid encoding the cell-targeting fusion protein, and a second vector comprising or consisting of a nucleic acid encoding the modulating fusion protein and a glycoprotein derived from envelope glycoprotein F of a Paramyxoviridae virus; or three separate vectors, a first vector comprising or consisting of a nucleic acid encoding the cell-targeting fusion protein, a second vector comprising or consisting of a nucleic acid encoding the modulating fusion protein, and a third vector comprising or consisting of a nucleic acid encoding a glycoprotein derived from envelope glycoprotein F of a Paramyxoviridae virus. Alternatively, the nucleic acid encoding the cell-targeting fusion protein, the nucleic acid encoding the modulating fusion protein, and the nucleic acid encoding the glycoprotein derived from envelope glycoprotein F of a virus of the Paramyxoviridae family can be provided by a single vector comprising or consisting of these three nucleic acids.

[0231] Vectors containing nucleic acid encoding the core protein from the retrovirus are also used.

[0232] As used herein, "core protein from a retrovirus" refers to the proteins encoded by the gag and pol genes. The gag gene encodes a polyprotein that is further processed by the retroviral protease into structural proteins, including core. The pol gene encodes, among other things, the retroviral protease, reverse transcriptase, and integrase.

[0233] Thus, a nucleic acid encoding a core protein from a retrovirus includes the gag and pol genes of said retrovirus.

[0234] The core protein from at least one retrovirus can be modified, for example, compared to the corresponding core protein from a wild-type retrovirus.

[0235] In one embodiment, the core protein from at least one retrovirus is modified by at least one amino acid mutation, such as an amino acid deletion, insertion or substitution.

[0236] In a preferred embodiment, the at least one modified core protein is a defective integrase. An example of a defective integrase carries a D116A mutation.

[0237] Thus, the nucleic acid encoding the core protein can comprise at least one mutation in the pol gene and / or at least one mutation in the pol gene, which can be a nucleotide deletion, insertion, or substitution.

[0238] In a preferred embodiment, the nucleic acid encoding the core protein contains at least one mutation in the pol gene, thereby encoding a defective integrase.

[0239] Retroviral vectors containing defective integrase are called integration-defective retroviral vectors, and they allow the transient delivery of encapsidated RNA molecules encoding genes of interest.

[0240] A preferred integration-defective retroviral vector is IDLV (integration-defective lentiviral vector).

[0241] The origin of the gag and pol genes gives the retrovirus-like particle or retroviral vector its name. For example, the phrase "HIV-1-derived retrovirus-like particle or retroviral vector" typically indicates that the gag and pol genes are the HIV-1 gag and pol genes or modified gag and pol genes derived from HIV-1.

[0242] Vectors containing genomes derived from packaging-competent retroviruses can also be used for co-transfection, particularly for the generation of retroviral vectors.

[0243] As used herein, a "vector containing a genome derived from a packaging-competent retrovirus" refers to a vector containing retroviral nucleic acid sequences known as "cis-acting" sequences. These vectors include long terminal repeats (LTRs) or modified LTRs lacking, for example, at least a portion of the U3 region, for control of transcription and integration, Psi sequences required for encapsidation, and primer binding site (PBS) and polypurine tract (PPT) sequences required for reverse transcription of the retroviral genome.

[0244] Retroviral vectors generated using vectors containing an LTR lacking at least a portion of the U3 region are, for example, self-inactivating (SIN-LTR) vectors.

[0245] In one embodiment, the vector comprising a genome derived from said packaging-competent retrovirus further comprises a gene of interest comprising a CRISPR / CAS element and / or an S / MAR episome.

[0246] When using the CRISPR / CAS system, a vector containing a genome derived from a packaging-competent retrovirus typically contains a gene encoding the endonuclease CAS (e.g., CAS9), a DNA sequence corresponding to a guide RNA (gRNA) specific to the gene of interest to be targeted, and optionally, a sequence for gene correction.

[0247] The retroviral-derived genome is preferably replication-deficient in the absence of any trans-correction functions. A replication-competent genome will further comprise the retroviral genes gag, pol, and env. In a replication-deficient genome, the viral genes gag, pol, and env are deleted. Assembly of a retrovirus-like particle or retroviral vector of the invention is achieved by providing, in trans, another vector encoding gag and pol but lacking the "cis" sequences (such as vector (iv)), and at least another vector or nucleic acid encoding a pseudotyped envelope glycoprotein (such as nucleic acids (i), (ii), and (iii) or a vector containing said nucleic acid). Expression of these vectors or nucleic acids allows encapsidation of the gene of interest, excluding genes required for viral genome replication and formation of complete viral particles.

[0248] The method for producing a pseudotyped retrovirus-like particle or retroviral vector as defined above comprises the steps of: b) culturing the co-transfected cells for a period of time sufficient to allow expression of the protein encoded by the nucleic acid and vector; c) allowing the encoded proteins to form retrovirus-like particles or retroviral vectors; It may further include.

[0249] Modulating the activity of and / or transducing specific cells or cell subtypes Another object of the present invention is the use, preferably the in vitro or ex vivo use, of a pseudotyped retrovirus-like particle or a retroviral vector as defined above to selectively modulate the activity of and optionally transduce target cells.

[0250] Yet another object of the present invention is a method, preferably an in vitro or ex vivo method, for selectively modulating the activity of and optionally transducing target cells, comprising the step of contacting a pseudotyped retrovirus-like particle or a retroviral vector as defined above with cells, including said target cells.

[0251] The term "selectively" in the phrase "selectively modulating the activity of target cells" means that essentially only the target cells have their activity modulated by the pseudotyped retrovirus-like particle or retroviral vector, e.g., the cells whose activity is modulated include less than 10%, e.g., less than 5% or less than 1% of non-target cells.

[0252] The term "selectively" in the phrase "selectively transducing target cells" means that essentially only target cells are transduced, e.g., the transduced cells comprise less than 10%, e.g., less than 5% or less than 1%, of the non-target cells that are transduced.

[0253] The phrase "modulating the activity of a target cell" is as defined above in the section entitled "functional domain."

[0254] As used herein, the terms "transducing" or "transducing" refer to the ability of a retrovirus-like particle or retroviral vector, upon binding to a target cell, to deliver biological material to the membrane or cytoplasm of the target cell. After delivery, the biological material may translocate to other compartments of the cell.

[0255] The expression "biological material" as used herein relates to one or more compounds susceptible to modifying the structure and / or function of a cell. In the context of the present invention, biological material is preferably nucleic acid comprising one or more genes of interest, which may be comprised in the genome of a retroviral vector, as explained above.

[0256] Conditions for carrying out cell transduction are well known to those skilled in the art and typically involve incubating the cells to be transduced, preferably cultured in, for example, retronectin-coated flasks, plates or dishes, and optionally pre-stimulated with a cytokine cocktail, with the pseudotyped retroviral vector, preferably at an MOI (multiplicity of infection) of between 0.5 and 100.

[0257] The target cells are in particular as defined above in the section of the same name.

[0258] The pseudotyped retrovirus-like particle or retroviral vector can be contacted with cells, including target cells, in a culture medium.

[0259] Those skilled in the art know how to culture a given cell in an appropriate medium.

[0260] Thus, pseudotyped retrovirus-like particles or retroviral vectors can be used in vitro or in vivo to alter defined cellular functions, thereby providing new tools for basic research.

[0261] Pharmaceutical Composition The present invention also relates to a pharmaceutical composition comprising at least one pseudotyped retrovirus-like particle or retroviral vector as defined above.

[0262] The amount of pseudotyped retrovirus-like particle or retroviral vector used in a pharmaceutical composition will depend, for example, on immunogenicity, the condition of the subject to which it is intended to be administered (e.g., weight, age, sex, health status, concomitant treatments, if any, and frequency of treatment), mode of administration, type of formulation, and / or number of target cells.

[0263] Pharmaceutical compositions preferably comprise at least one pseudotyped retrovirus-like particle or retroviral vector and at least one pharmaceutically acceptable carrier.

[0264] The term "pharmaceutically acceptable carrier," as used herein, is intended to encompass any carrier, which does not interfere with the effectiveness of the biological activity of the active ingredient and, preferably, is not toxic to the host to which it is administered.

[0265] Those skilled in the art are familiar with pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention. For example, pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-α-tocopherol polyethylene glycol 1000 succinate, Tweens, or other similar polymeric delivery matrices, surfactants used in pharmaceutical dosage forms, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, and potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, and magnesium silicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and wool fat. Cyclodextrins such as α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, or chemically modified derivatives such as hydroxyalkyl cyclodextrins, including 2-hydroxypropyl-β-cyclodextrin and 3-hydroxypropyl-β-cyclodextrin, or other solubilizing derivatives may also be advantageously used to enhance delivery of the compositions according to the invention.

[0266] The pharmaceutical composition comprises 10 6 IU~10 12 IU of pseudotyped retroviral vector, preferably 10 7 IU~10 9 IU, more preferably 10 7 IU~10 8 It may contain IU.

[0267] The term "IU" or "infectious unit" as used herein refers to the quantity of infectious vector particles determined by titration on a cell line and expressed as IU / ml.

[0268] Administration can be achieved by a single dose or multiple doses of a pharmaceutical composition according to the present invention, said multiple doses being injected simultaneously or separately over a period of time.

[0269] In one embodiment, the pharmaceutical composition is provided in a unit dosage form to facilitate accurate administration. The term "unit dosage form" refers to a physically discrete unit suitable as a unitary dosage for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with a suitable pharmaceutical excipient. Typical unit dosage forms include prefilled, premeasured ampoules or syringes of a liquid composition. In such compositions, the pseudotyped retrovirus-like particle or retroviral vector is usually the minor component, with the remainder being various vehicles or carriers and processing aids useful for forming the desired dosage form.

[0270] The present invention further provides a kit comprising a pharmaceutical composition comprising a pseudotyped retrovirus-like particle or retroviral vector as defined above, and instructions regarding the mode of administration, which may indicate, for example, the medical indication, the route of administration, the dosage, and / or the patient population to be treated.

[0271] Subject to treatment The subject to be treated can be a mammal, for example, a human or non-human mammal.

[0272] A human is also referred to as an "individual" or a "patient."

[0273] The human can be of any age, eg, infant, child, juvenile, adult, elderly, and of any gender.

[0274] The non-human mammal is preferably a rodent (eg, a mouse, rat, or rabbit), a feline (eg, a cat), a canine (eg, a dog), or a primate (eg, a chimpanzee).

[0275] The subject to be treated is preferably a human.

[0276] Prevention and / or treatment of diseases The present invention is particularly useful for the prevention and / or treatment of diseases that can be prevented and / or cured by selectively modulating the activity of specific cells or subsets of cells, and optionally by selectively transducing said specific cells or subsets of cells.

[0277] Preferably, the disease involves a particular cell or subset of cells.

[0278] Thus, the subject being treated may suffer from, or may be likely to suffer from, a disease that may be prevented and / or cured by selectively modulating the activity of, and optionally transducing, specific cells or subsets of cells.

[0279] The disease may be selected from the group consisting of immune diseases (e.g., autoimmune diseases), cancer, genetic diseases, allergic diseases, inflammatory diseases, infectious diseases (particularly bacterial and / or viral infections), metabolic diseases, neurodegenerative diseases (e.g., neuronal atrophy, Parkinson's disease, Huntington's disease, Alzheimer's disease), muscle diseases, and combinations thereof.

[0280] As used herein, the term "autoimmune disease" refers to a disease caused by an overactive immune response in the body, usually against substances and / or tissues present in the body. Thus, by specifically targeting immune cells involved in this overactive immune response, the pseudotyped retrovirus-like particles and retroviral vectors of the present invention are useful tools for the prevention and / or treatment of autoimmune diseases.

[0281] Autoimmune diseases include, in particular, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, atopic allergy, autoimmune aplastic anemia, autoimmune cardiomyopathy, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, Autoimmune uveitis, Baro's disease, Baro's concentric sclerosis, Behçet's syndrome, Burger's disease, Bickerstaff's encephalitis, Blau syndrome, bullous pemphigoid, Castleman's disease, celiac disease, chronic inflammatory demyelinating polyneuropathy, chronic relapsing multifocal osteomyelitis, Churg-Strauss syndrome, cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, complement component 2 deficiency, cranial arteritis, CREST syndrome, Crohn's disease, Cushing's syndrome, cutaneous leukocytoclastic vasculitis, Degos' disease, Dercum's disease, dermatitis herpetiformis, dermatomyositis, type 1 diabetes, diffuse cutaneous systemic sclerosis, dress Ler's syndrome, discoid lupus erythematosus, eczema, enthesitis-related arthritis, eosinophilic fasciitis, eosinophilic gastroenteritis, epidermolysis bullosa acquisita, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibrodysplasia ossificans progressiva, idiopathic pulmonary fibrosis, gastritis, gastrointestinal pemphigoid, giant cell arteritis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic inflammatory demyelinating disease, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura Plaque disease, IgA nephropathy, inclusion body myositis, inflammatory demyelinating polyneuropathy, interstitial cystitis, juvenile idiopathic arthritis, juvenile rheumatoid arthritis, Kawasaki disease, Lambert-Eaton myasthenic syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, linear IgA disease (LAD), Lou Gehrig's disease, lupoid hepatitis, systemic lupus erythematosus, Majeed syndrome, Meniere's disease, microscopic polyangiitis, Miller-Fisher syndrome, mixed connective tissue disease, morphea, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, neuromyelitis optica, neuromyotonia, ocular cicatricial pemphigoid,Opsoclonus-myoclonus syndrome, Ordo's thyroiditis, relapsing rheumatoid arthritis, paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsonage-Turner syndrome, pars planitis, pemphigus, pemphigus vulgaris, pernicious anemia, perivenous encephalomyelitis, POEMS syndrome, polyarteritis nodosa, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary biliary cirrhosis, progressive inflammatory neuropathies, psoriasis, psoriatic arthritis, pyoderma gangrenosum, pure red cell aplasia, Rasmussen's encephalitis, Raynaud's phenomenon, relapsing polychondritis , Reiter's syndrome, restless legs syndrome, retroperitoneal fibrosis, rheumatoid arthritis, rheumatic fever, sarcoidosis, Schmidt's syndrome, Schnitzler's syndrome, scleritis, scleroderma, Sjogren's syndrome, spondyloarthritis, Still's disease, stiff-man syndrome, subacute bacterial endocarditis, Susac's syndrome, Sweet's syndrome, Sydenham's chorea, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis, Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, unclassifiable connective tissue disease, unclassifiable spondyloarthritis, vasculitis, vitiligo, and Wegener's granulomatosis.

[0282] The term "cancer" as used herein encompasses any type of cancer, such as glioblastoma, neuroblastoma, B-cell lymphoma, T-cell lymphoma, breast cancer, hepatocellular carcinoma, cancer arising from hematopoietic cells, including leukemia, particularly T-cell based leukemias such as B-CLL (B-cell chronic lymphocytic leukemia), CML (chronic myeloid leukemia), or ATL (acute T-cell leukemia), ALL (acute lymphoblastic leukemia), AML (acute myeloid leukemia), and / or melanoma.

[0283] Therapeutic applications The present invention also relates to a pseudotyped retrovirus-like particle or a retroviral vector as defined above for use as a medicament, preferably in immunotherapy, gene therapy and / or vaccination.

[0284] Gene therapy is a therapy that uses a gene as a medicine, which can be obtained, for example, by delivering a gene into a cell of interest and / or correcting a gene of interest at an endogenous site. In gene therapy, a nucleic acid containing a gene of interest is delivered to a patient's cells, resulting in the expression of a protein encoded by the gene of interest and / or the correction of the gene of interest, thereby enabling the prevention and / or treatment of a disease.

[0285] The gene of interest may be present within an RNA molecule contained within a retroviral vector according to the present invention.

[0286] Gene correction can be performed using the CRISPR / CAS system.

[0287] Immune therapy, also called immunotherapy, is a treatment based on modulating (eg, stimulating or inhibiting) the activity of the immune system to prevent and / or treat disease.

[0288] In the context of the present invention, immunotherapy consists of modulating the activity of only specific target immune cells and, optionally, selectively transducing said target immune cells by using a retrovirus-like particle or retroviral vector according to the invention. For example, a retroviral vector can be used to activate B cells, such as to differentiate B cells in plasma cells, and, optionally, to transduce B cells with nucleic acids encoding ectopic antibodies against an infectious agent (e.g., against HIV, HCV, or HBC).

[0289] Immunotherapy also includes T cell therapy, in which T cells, in addition to being modulated in their function, can also be made more permissive to gene transfer, for example, by T cell receptor (CAR) gene transfer.

[0290] Adoptive T cell therapy is a treatment in which T cells are infused into a subject in need thereof. In the context of the present invention, the activity of said T cells can be modulated prior to infusion by using a retrovirus-like particle or retroviral vector according to the invention.

[0291] In the context of the present invention, vaccination consists of displaying specific viral epitopes on the surface of retrovirus-like particles or retroviral vectors that are targeted and simultaneously activate antigen-presenting cells (e.g., macrophages), which then present the epitopes to the immune system (T cells and B cells).

[0292] The present invention also relates to a pseudotyped retrovirus-like particle or retroviral vector as defined above for use as a medicament, wherein the particle or vector selectively modulates the activity of, and optionally selectively transduces, a target cell.

[0293] The present invention particularly relates to a pseudotyped retrovirus-like particle or retroviral vector for use as defined above in the prevention and / or treatment of a disease as defined above in the section entitled "Prevention and / or Treatment of Diseases".

[0294] The disease is, for example, an immune disease (e.g., an autoimmune disease), a cancer, a genetic disease, an allergic disease, an inflammatory disease, an infectious disease (particularly a bacterial and / or viral infection), a metabolic disease, a neurological disease (neuronal atrophy, Alzheimer's disease, Parkinson's disease, Huntington's disease, etc.), a muscle disease, or a combination thereof.

[0295] In one embodiment, pseudotyped retrovirus-like particles or retroviral vectors are used to improve DC (dendritic cell) vaccines, for example, by co-presenting a DC cell-specific ligand (such as CD11b) and GM-SCF (granulocyte-macrophage colony-stimulating factor) on the surface of the vector for protein or gene delivery.

[0296] In an advantageous embodiment, T cell activation is coupled with T cell-specific gene transfer, which significantly enhances the efficiency of gene transfer into resting T cells, e.g., in T cell-based gene therapy. Efficient in vivo gene transfer into T cell subsets is a revolution in the fields of gene therapy and cell therapy, as it allows for the omission of ex vivo culture and transduction steps, which induce high costs in clinical applications. Furthermore, leaving cells in their normal microenvironment in vivo allows them to preserve their phenotype and persist within the patient for a long period of time (e.g., tumor-specific CD8 cytotoxic cells).

[0297] In another embodiment, the present invention can be used to enhance selective gene delivery to resting B lymphocytes in B cell-based gene therapy, such as immunotherapy, by enabling B cells to produce neutralizing antibodies against infectious agents or to secrete recombinant proteins that are tolerated by the immune system because they can act as tolerogenic cells.

[0298] Furthermore, the presentation of one or more cytokines on the surface of pseudotyped retrovirus-like particles or retroviral vectors can induce T cell subsets to differentiate into phenotypes such as TSCM or TCM, which can persist for long periods in vivo.

[0299] In another embodiment, the present invention can be used for the in vivo expansion of (autologous) anti-cancer natural killer cells for cancer treatment.

[0300] In one embodiment, pseudotyped retrovirus-like particles or retroviral vectors are used to induce apoptosis in defined cell subsets by co-presenting an apoptotic ligand with a targeting domain specific for tumor cells or immune cells.

[0301] The pseudotyped retrovirus-like particle or retroviral vector may be provided in the form of a pharmaceutical composition.

[0302] The pharmaceutical composition is preferably as defined in the section of the same name above.

[0303] The present invention also relates to a method for treating a subject in need thereof, comprising the step of administering a therapeutically effective amount of a pseudotyped retrovirus-like particle or retroviral vector as defined above, preferably in the framework of immunotherapy, gene therapy and / or vaccination.

[0304] The present invention also relates to a method for treating a subject in need thereof, comprising administering a therapeutically effective amount of a pseudotyped retrovirus-like particle or retroviral vector as defined above, which selectively modulates the activity of, and optionally transduces, target cells.

[0305] The present invention also relates to a method for preventing and / or treating a disease, said method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a pseudotyped retrovirus-like particle or retroviral vector as defined above, said disease being preferably as defined above.

[0306] Any suitable method of administration known to those skilled in the art can be used. In particular, the pseudotyped retrovirus-like particles or retroviral vectors according to the invention can be administered by oral route, parenteral route (preferably by intravenous injection), intrathecal route, in particular intrafemoral (such as intramedullary) or intrabrachial route injection, and / or local intratumoral injection.

[0307] If the parenteral route is chosen, the pseudotyped retrovirus-like particles or retroviral vectors may be in the form of an injectable solution or suspension, conditioned, for example, in ampoules or flasks.

[0308] The pseudotyped retrovirus-like particles or retroviral vectors are preferably used or administered in therapeutically effective amounts.

[0309] A "therapeutically effective amount" refers to the quantity of pseudotyped retrovirus-like particle or retroviral vector that confers a therapeutic effect on the treated subject. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject indicates or feels an effect). As known to those skilled in the art, the effective dose will vary depending on the route of administration, the height and / or weight of the subject, as well as the possible use of other drugs.

[0310] As used herein, the term "comprising" encompasses the term "consisting of."

[0311] The invention will be further illustrated with reference to the following examples and figures.

[0312] All references cited herein, including journal articles or abstracts, published or unpublished patent applications, issued patents, or any other references, including all data, tables, figures, and text presented in the cited references, are incorporated herein by reference in their entirety. [Sequence List Free Text]

[0313] SEQ ID NO: 1 corresponds to the nucleic acid sequence encoding a truncated mutant (Y481A, R533A, S548L, F549S) MVH protein (HcDelta18) fused to the designed ankyrin repeat protein (DARPin) 29.2 specific for human CD4. SEQ ID NO:2 corresponds to the amino acid sequence encoded by the sequence of SEQ ID NO:1. SEQ ID NO: 3 corresponds to the nucleic acid sequence encoding the truncated mutant (E501A, W504, Q530A, E533A) NiVG protein (GcΔ34) fused to a single-chain antibody directed against human CD8 (scFvC8-Vh1) linked with an additional (G4S)3 linker. SEQ ID NO:4 corresponds to the amino acid sequence encoded by the sequence of SEQ ID NO:3. SEQ ID NO: 5 corresponds to the nucleic acid sequence encoding the truncated mutant (E501A, W504, Q530A, E533A) NiVG protein (GcΔ34) fused to IL-7. SEQ ID NO:6 corresponds to the amino acid sequence encoded by the sequence of SEQ ID NO:5. SEQ ID NO: 7 corresponds to the nucleic acid sequence encoding the truncated mutant (E501A, W504, Q530A, E533A) NiVG protein (GcΔ34) fused to a designed ankyrin repeat protein (DARPin) directed against human EpCAM. SEQ ID NO:8 corresponds to the amino acid sequence encoded by the sequence of SEQ ID NO:7. SEQ ID NO: 9 corresponds to the full-length amino acid sequence of the envelope glycoprotein G of Nipah virus. SEQ ID NO: 10 corresponds to the full-length amino acid sequence of the envelope glycoprotein H of the measles virus. SEQ ID NO: 11 corresponds to the full-length amino acid sequence of envelope glycoprotein F of Nipah virus. SEQ ID NO: 12 corresponds to the full-length amino acid sequence of envelope glycoprotein F of measles virus. SEQ ID NO: 13 corresponds to the nucleic acid sequence encoding a truncated mutant MeV H protein (HcΔ15) fused to IL-7. SEQ ID NO:14 corresponds to the amino acid sequence encoded by the sequence of SEQ ID NO:13. SEQ ID NO: 15 corresponds to the amino acid sequence of the measles virus F protein truncated by 30 amino acids in the cytoplasmic tail (FcΔ30). SEQ ID NO: 16 corresponds to the amino acid sequence of the Nipah virus F protein truncated by 22 amino acids in the cytoplasmic tail (FcΔ22). [Brief explanation of the drawings]

[0314] [Figure 1] Figure 1 shows efficient and stable transfer of the ErbB2-CAR gene into resting CD4+ T cells by 4H / IL7H-LV. To evaluate whether 4H / IL7H-LV can efficiently transfer a therapeutic gene (ErbB2-CAR, a CAR specific for ErbB2) into resting T cells, freshly isolated CD3+ T cells were transduced with 4H-LV or 4H / IL7H-LV. Cells transduced with VSV-LV or left untransduced (ut) were used as negative controls. After 3 days, half of the transduced cells were analyzed by FACS to determine the percentage of ErbB2-CAR+ cells among CD4- or CD8-gated cells. To confirm stable ErbB2-CAR expression, the other half of the transduced cells were further cultured for another 3 days in the presence of anti-CD3 / anti-CD28 / IL-2 stimulation after a washing step. The percentages of CD4+ / ErbB2+ and CD8+ / ErbB2+ cells were then determined by FACS. [Figure 2]Mutations in NiV envelope glycoprotein G that eliminate recognition of the native receptor are shown. Binding of ephrinB2 (A) and ephrinB3 (B) to NiV-G mutants is shown. 293T cells were transfected with mock, a plasmid encoding GcΔ34His, or different GcΔ34EpCAM mutants: non-mutant (GcΔ34EpCAM), E533A (GcΔ34EpCAMmut1), Q530A + E533A (GcΔ34EpCAMmut2.1), E501A + W504A (GcΔ34EpCAMmut2.2), or E501A + W504A + Q530A + E533A (GcΔ34EpCAMmut4) and incubated with 1 μg / ml of recombinant Fc-ephrinB2 or recombinant Fc-ephrinB3. The amount of receptor binding by the different mutants is shown as MFI values. Statistical analysis is compared to the non-mutated GΔ34-DARPin-Ac1 (n=3; mean ± standard deviation (SD) shown; *: P<0.1; **: P<0.01; ***: P<0.001 by unpaired t-test). [Figure 3] Figure 1 shows selective activation of CD4+ T cells by 4H / IL7H-VLP. Freshly isolated CD3+ T cells were either left untransduced (ut) or transduced with different VLPs pseudotyped with CD4-DARPin-Hmut (4H-VLP), CD4-DARPin / IL7-Hmut (4H / IL7H-VLP), or IL7-Hnse (Hnse / IL7H-VLP). Three days later, cells were stained with CD8, CD4, and CD71 antibodies. CD71 expression in CD4- or CD8-gated cells is shown. [Figure 4]

[0033] Figure 1 shows that functionally presented IL7 on targeting VLPs promotes T cell survival. Forward / side scatter profiles for adult resting CD3+ T cells incubated for 6 days with virus-like particles (VLPs) pseudotyped with CD4-targeting MV-H (4H-VLP), CD8-targeting MV-H (8H-VLP), CD4-targeting MV-H presenting IL7 (4H / IL7H-VLP), or CD8-targeting MV-H presenting IL7 (8H / IL7H-VLP). Cells cultured in medium alone (untransduced; ut) or in the presence of 15 ng / ml of IL7 were used as controls. The percentage of viable cells is indicated within each dot blot. [Figure 5]

[0043] Figure 1 shows that functionally presented IL7 on CD4-targeted LV promotes T cell survival. Forward / side scatter profiles for adult resting CD3+ T cells incubated for 6 days with lentiviral vectors (LV) pseudotyped with VSVG (VSV-LV), MV-Hmut (Hnse-LV), CD4-targeted MV-H (4H-LV), or CD4-targeted MV-H presenting IL7 (4H / IL7H-LV). Cells cultured in medium alone (untransduced; ut) or in the presence of 15 ng / ml of IL7 were used as controls. The percentage of viable cells is indicated within each dot blot. [Figure 6]Figure 1 shows selective activation of CD4+ T cells by 4H / IL7H-LV. Freshly isolated CD3+ T cells were either left untransduced (ut) or transduced with different types of LV pseudotyped with CD4-DARPin-Hmut (4HΔ18-LV), Hnse (Hnse-LV), or VSVG (VSVG-LV). IL7 was presented on two different forms of Hmut: a C-truncated form with 20 amino acids (HΔ20) or a C-truncated form with 15 amino acids (HΔ15). Two types of 4H / IL7H-LV were therefore generated and transduced into resting T cells. Three days later, cells were stained with CD3, CD8, CD69, and CD71 antibodies. Expression of activation markers, CD69 and CD71, in CD8+ and CD8- (CD4+) cells is shown. [Figure 7]

[0023] Figure 1 shows a schematic representation of pseudotyped lentiviral vectors (LVs). 4H-LV is a CD4-targeting LV pseudotyped with the MeV glycoprotein. 4H / IL7H-LV is a CD4-targeting LV pseudotyped with the MV glycoprotein, displaying IL7. The envelope glycoprotein H (MV-H) is truncated in its cytoplasmic tail by, for example, 15, 18, or 20 amino acids, and at least two of four point mutations (Y481A, R533A, S548L, and F549S) are introduced to block recognition by its natural receptors, SLAM and CD46, resulting in the envelope glycoprotein Hmut. A targeting domain (e.g., CD4-DARPin) and / or a cytokine (e.g., IL7) are then fused to the mutant H (Hmut) with or without a linker. The MV envelope glycoprotein F is truncated by 30 amino acids in its cytoplasmic tail (MV-FΔ30). [Figure 8]Figure 1 shows a schematic representation of pseudotyped virus-like particles (VLPs). 8G-VLP is a CD8-targeting VLP pseudotyped with the NiV glycoprotein. 8G / IL7G-VLP is a CD8-targeting VLP pseudotyped with the NiV glycoprotein, displaying IL7. The NiV-G envelope glycoprotein was truncated by 34 amino acids (Δ34) in its cytoplasmic tail, and four point mutations (E501A, W504A, Q530A, and E533A) were introduced to block its recognition by the natural receptor for ephrin B2 / B3, resulting in the envelope glycoprotein NiV-GΔ34. A targeting domain (CD8-scFv) and / or cytokine (IL7) were then fused to the mutant G protein (NiV-GΔ34) with or without a linker. NiV envelope glycoprotein F is truncated by 22 amino acids in its cytoplasmic tail (NiV-FΔ22). [Figure 9] Figure 1 shows efficient and selective transduction of resting CD4+ T cells by 4H / IL7H-LV. Freshly isolated CD3+ T cells were either left untransduced (ut) or transduced with different types of LV pseudotyped with CD4-DARPin-Hmut (4HΔ18-LV), Hnse (Hnse-LV), or VSVG (VSVG-LV). IL7 was presented on two different forms of Hmut: a C-truncated form with 20 amino acids (HΔ20) or a C-truncated form with 15 amino acids (HΔ15). Thus, two types of 4H / IL7H-LV (4HΔ18 / IL7HΔ15-LV and 4HΔ18 / IL7HΔ20-LV) were generated and transduced into resting T cells. Three days later, cells were stained with CD3, CD8, and CD4 antibodies. GFP expression in CD4- or CD8-gated cells is shown. [Figure 10]Neutralization of LV pseudotyped with NiV glycoproteins. After incubation with serial dilutions of pooled human serum (IVIG) at 37°C for 2 hours, CHO-EpCAM or CHO-EphrinB2 cells were transduced with NiVmutEpCAM-LV (circles), MVEpCAM-LV (squares), NiVwt-LV (triangles), or VSVG-LV (diamonds) at an MOI of 0.4. After 72 hours, GFP+ cells were determined by flow cytometry, and the number of GFP+ cells compared to untreated controls is shown (n=3). [Figure 11] Figure 1 shows selective activation of CD88+ T cells by 8G / IL7G-LV. Freshly isolated CD3+ T cells were either left untransduced (ut) or transduced with different types of LV pseudotyped with CD8-scFv-Gmut (8G-LV) or CD8-scFv-Gmut / IL7-Gmut (8G / IL7G-LV). Three days later, cells were stained with CD8, CD4, and CD71 antibodies. CD71 expression in CD4- or CD8-gated cells is shown. [Figure 12] Figure 1 shows efficient and selective transduction of resting CD8+ T cells by 8G / IL7G-LV. Freshly isolated CD3+ T cells were either left untransduced (ut) or transduced with different types of LV pseudotyped with CD8-scFv-Gmut (8G-LV) or CD8-scFv-Gmut / IL7-Gmut (8G / IL7G-LV). Three days later, cells were stained with CD3, CD8, and CD4 antibodies. GFP expression in CD4- or CD8-gated cells is shown. [Figure 13]Figure 1 shows efficient transduction of the CAR19 gene into resting CD8+ T cells by 8G / IL7G-LV. Freshly isolated CD3+ T cells were either left untransduced (ut) or transduced with different types of LV pseudotyped with CD8-scFv-Gmut (8G-LV), CD8-scFv-Gmut / IL7-Gmut (8G / IL7G-LV), or VSVG (VSV-LV). A therapeutic gene encoding a CD19-specific CAR (CAR19) was delivered by LV. Three days after transduction, CAR19 expression was detected with an anti-cmyc antibody. The percentage of CAR19-expressing cells among CD4+ or CD8+ T cells is shown. [Figure 14] Figure 1 shows that 4H / IL7H-LV enables targeting of CD4+ T cell activation in humanized mice in vivo. NOD / SCID gc- / - mice were injected with cord blood T cells, and two months after engraftment (20% T cell reconstitution), 100 microliters of 4H / IL7H-LV (106 IU) or 4H / IL7H-LV were injected intravenously. Two weeks after vector injection, mice were sacrificed, and spleen cells were assessed for the percentage of CD71+ CD4+ T cells (an activation marker). [Example]

[0315] Materials and Methods Creation of constructs The coding sequence of the EpCAM-specific DARPinAc1 (Stefan et al., 2011), PCR-amplified from pQE30ss_Ac1_corr, was inserted via SfiI / NotI into the backbone of the plasmid pHL3-HRS3opt2#2 (Friedel et al., 2015) to generate pHL3-Ac1, a plasmid encoding the truncated mutant MVHcΔ18mut protein and a (G4S)3 linker (L3) between the H and His-tagged DARPinAc1.

[0316] All plasmids encoding Nipah virus G protein mutants were derived from the pCAGGS-NiV-codonop-Gn plasmid. The Ac1 targeting domain coding sequence was fused to the C-terminus of the G protein reading frame by PCR amplification of each fragment and simultaneously introducing a common AgeI restriction site for ligation, resulting in the pCAGGS-NiV-G-DARPin-Ac1 plasmid. All other targeting domains were also exchanged via AgeI / NotI. Truncated forms of the G protein cytoplasmic tail were introduced by PCR amplification of the G protein reading frame and insertion of the PCR fragment into pCAGGS-NiV-G-DARPin-Ac1, resulting in the pCAGGS-NiV-GcΔ33-DARPin-Ac1 and pCAGGS-NiV-GcΔ34-DARPin-Ac1 plasmids. His-tagged G and GcΔ34 proteins were generated by PCR amplification from pCAGGS-NiV-codonop-Gn. Cloning of the fragments into the pCAGGS-NiV-G-DARPin-Ac1 plasmid backbone via PacI / NotI restriction resulted in pCAGGS-NiV-G-His and pCAGGS-NiV-GcΔ34-His, respectively. Mutations interfering with native receptor recognition were introduced into the protein-coding sequence, NiV-GcΔ34-DARPin-Ac1, by site-directed mutagenesis. Each mutation was generated by amplifying two fragments carrying the designed mutations with regions of homology at the mutation site. These fragments were fused and amplified with flanking primer pairs. The resulting fragment was cloned into pCAGGS-NiV-GcΔ34-DARPin-Ac1 via RsrII / AgeI to generate the plasmid pCAGGS-NiV-GcΔ34EpCAMmut.

[0317] To generate the NiV-F mutants, the coding sequences of FcΔ22 and FcΔ25 were amplified from pCAGGS-NiV-F and cloned via PacI / SacI restriction into the pCAGGS-NiV-codonop-Gn plasmid backbone, resulting in the plasmids pCAGGS-NiV-FcΔ22 and pCAGGS-NiV-FcΔ25. NiV-F mutants were amplified from pCAGGS-NiV-F and simultaneously added with an AU1 tag to the N-terminus to generate AU1-tagged NiV-F mutants used in Western blot analysis of vector particles. The resulting PCR fragments were cloned into the backbone of pCAGGS-NiV-codonop-Gn via restriction digestion with PacI / SacI, resulting in the plasmids pCAGGS-AU1-NiV-F, pCAGGS-AU1-NiV-FcΔ22, and pCAGGS-AU1-NiV-FcΔ25.

[0318] Vector construction Vector particles were generated by transient transfection of HEK-293T cells using polyethyleneimine (PEI). 24 hours before transfection, 2.5 × 10 cells were transfected. 7 The cells were seeded into a T175 flask. On the day of transfection, the cell culture medium was replaced with 10 ml of DMEM supplemented with 15% FCS and 3 mM L-glutamine. A DNA mix was prepared by mixing 35 μg of total DNA with 2.3 ml of DMEM without any additives.

[0319] For example, after optimizing the G to F ratio, 0.9 μg of a plasmid encoding the GcΔ34 DARPin / scFv mutant was mixed with 4.49 μg of a plasmid encoding the F mutant. 14.4 μg of the HIV-1 packaging plasmid pCMVΔR8.9 and 15.1 μg of the transfection LV plasmid were used. A transfection reagent mix was prepared by mixing 140 μl of an 18 mM PEI solution in HO with 2.2 ml of DMEM without additives. This solution was mixed with the DNA mix, vortexed, incubated at room temperature for 20 minutes, and added to HEK-293T cells, resulting in a total of DMEM with 10% FCS and 2 mM L-glutamine. After 24 hours, the medium was replaced with DMEM with 10% FCS and 2 mM L-glutamine to remove any remaining PEI / DNA complexes. Two days after transfection, the cell supernatant containing the lentiviral vector was filtered through a 0.45 μm filter. If necessary, the vector particles were purified by centrifugation on a 20% sucrose cushion at 450 × g for 24 hours. The pellet was resuspended in phosphate-buffered saline (PBS). For transduction, 8 × 10 CHO-EpCAM cells and 8 × 10 SK-OV-3 cells were transfected. 3 or 2 x 10 Molt4.8 cells and 2 x 10 Raji cells 4 The cells were seeded into a single well of a 96-well plate and transduced the following day. When necessary, the cell culture medium was replaced with medium containing different concentrations of Bafilomycin A1 (Santa Cruz Biotechnology, Inc., Dallas, USA), and the cells were preincubated at 37°C for 30 minutes before adding the vector. At least four serial dilutions of vector particles were used for titration. After 72 hours, the percentage of green fluorescent protein (GFP)-positive cells was determined by flow cytometry. Transducing units per ml (tu / ml) were calculated by selecting the dilution that showed a linear correlation between the dilution factor and the number of GFP-positive cells (number of transduced cells / μl of vector volume / 0.001).

[0320] Schematic representations of pseudotyped retrovirus-like particles or retroviral vectors according to the invention are depicted in FIGS.

[0321] Example 1 Selective activation of CD4+ T cells To activate CD4+ lymphocytes but not CD8+ lymphocytes, we created VLPs that display a CD4-specific DARPin as a targeting ligand and IL-7 as an activation domain on the MV H protein together with a fusion protein (F). As a result, 4 H / IL7 H The resulting vector particles were 10-VLPs (containing proteins of the sequences SEQ ID NO:2, SEQ ID NO:14, and SEQ ID NO:15). A transfection protocol was established to generate the particles. Briefly, 0.45 μg of pCG-Hmut-CD4-DARPin (Zhou et al., J Immunol, 2015), 0.45 μg of pCG-HΔ15-IL7 (Els Verhoeyen), 4.7 μg of pCG-FcΔ30 (Funke et al., Molecular therapy, 2008), 14.4 μg of the HIV-1 packaging plasmid pCMVΔR8.9 (Funke et al., Molecular therapy, 2008), and 15.1 μg of pCG-1 were used to transfect HEK293T cells in one T175 flask. After harvesting, the vector particles were further concentrated via ultracentrifugation. For LVs, approximately 10 7 Titers of tu / ml were obtained and for VLPs titers of approximately 1.5ug p24 per ml were obtained.

[0322] On freshly isolated resting T cells, 4 H / IL7 H The functionality of the VLPs was confirmed. Briefly, CD3 T cell was isolated by negative selection using a Pan T cell isolation kit (Miltenyi Biotech). + T cells were isolated from adult peripheral blood. The cells were then cultured for 4 h in the absence of stimulation. H / IL7 HAfter 3 days, CD4+ cells in the cell cultures were incubated with IgG-VLPs, as determined by FACS analysis for the CD71 activation marker. + T cells were activated, but CD4 - T cells were not activated (Figure 3). Non-targeting particles presenting IL-7 stimulated CD4 + T cells and CD4 - Both IL-7 and IL-7-presenting VLPs activated primary T cells. IL-7 is a known T cell survival cytokine. Therefore, we demonstrate that all IL-7-presenting VLPs are as effective as recombinant human IL-7 in preventing primary T cell death, whereas the majority of resting T cells in culture died after 6 days in the presence of VLP particles lacking conventional IL-7 (Figure 4).

[0323] Promotes T cell survival and CD4 + The ability to selectively activate T cells was also demonstrated by IL7-presenting CD4-targeted LV(4 H / IL7 H -LV). Virus particles were generated as described above, except that a transfer plasmid encoding GFP was substituted for pCG-1. In addition, to examine the flexibility of the system and whether particle function could be enhanced by using other variants of Hmut, two different plasmids encoding Hmut-IL7 were used for vector generation. pCG-HΔ15-IL7 contains a 15-amino acid cytoplasmic truncated form of Hmut-IL7 (4 H / IL7 HΔ15 pCG-HΔ20-IL7 contains a 20 amino acid cytoplasmic truncated form of Hmut-IL7 (4 H / IL7 HΔ20 Although not specifically shown, IL7-presenting VLPs and IL7-presenting LVs were produced using pCG-HΔ15-IL7. H / IL7 H -Freshly isolated human CD3 +T cells were incubated with the indicated LVs for 6 days in the absence of stimulation, resulting in 4 H / IL7 H -LV enhances T cell survival and H The rhIL-7-treated group showed a significant increase in viable cells compared with the VSV-LV-transduced group and the non-transduced Hnse-LV (Figure 5). As expected, a high percentage of viable cells was observed in the rhIL-7-treated group, while minimal viable cells were observed in the VSV-LV-transduced group or the non-transduced (ut) group (Figure 5). Furthermore, 4 H / IL7 H -LV has been shown to be effective in selectively stimulating its target cell populations in mixed cell cultures. H / IL7 H As shown in Figure 6, the 4-VLP was as potent as the 4-VLP. H / IL7 H -LV expresses the activation markers CD69 and CD71, and is CD4 + Selectively upregulates CD4 - Cells do not upregulate CD4 + In T cell stimulation, 4 H / IL7 HΔ15 -LV and 4 H / IL7 HΔ20 Thus, we successfully produced targeted LVs that presented functional IL7.

[0324] Example 2 Delivery of tumor-specific chimeric antigen receptors to resting T cells Because IL7-presenting vectors induced activation of resting T cells, these cells are predicted to be permissive to lentiviral transduction. To demonstrate this, we used vectors that deliver a GFP transgene. H / IL7 H Two variants of 4-LV were created. H / IL7 HΔ15 -LV and 4 H / IL7 HΔ20 -None of the LVs contained resting CD4 + T cells can be efficiently and selectively transduced.

[0325] Chimeric antigen receptors (CARs) are powerful tools for cancer therapy. Traditionally, T cell subsets must be purified and activated for gene delivery of CARs. Here, we demonstrate that CARs target resting CD4 T cells. + This demonstrates that ErbB2-specific chimeric antigen receptor (ErbB2-CAR) can be selectively delivered to resting T cells. Particles were produced as described in Example 1, except that pCG-1 was replaced with a transfection plasmid encoding a CAR. H / IL7 H -LV transduction, ErbB2-CAR expression was significantly increased in CD4 + This was observed only within the T cell population. H -LV) compared to 4 H / IL7 H -LV, CD4 + It was more effective in delivering the CAR gene while retaining selectivity for T cells (Figure 1).

[0326] Example 3 Resting human CD8 + The MV glycoproteins can be replaced with those of NiV to selectively activate and transduce T cells. To efficiently pseudotype VLPs or LVs with NiV glycoproteins, NiV-G was truncated by 34 amino acids in its cytoplasmic tail (GcΔ34), and NiV-F was truncated by 22 amino acids in its cytoplasmic tail (FΔ22). Next, four point mutations (E501A, W504A, Q530A, and E533A) were introduced into GcΔ34 to disrupt its recognition of the native receptors for ephrin B2 and B3. LVs pseudotyped with this engineered G protein completely lost binding to the native NiV ephrin B2 and ephrin B3 receptors (Figure 2) and failed to enter cells expressing these receptors.

[0327] Furthermore, NiV-pseudotyped LV has several attractive features, such as high production yield and resistance to intravenous immunoglobulin. Since there is no vaccination against NiV and the incidence is limited to a few cases in Malaysia, Bangladesh, and India (SEARO: WHO South-East Asia Regional Office), neutralizing antibodies should not be present in humans. To support this, we administered intravenous immunoglobulin (IVIG; Intratect®), which covers a wide range of human serum donors, at increasing concentrations to NiVwt-LV, NiVmut-LV, and NiVmut-LV. EpCAM -LV,MV EpCAM The target cells were incubated with VSV-LV and VSV-LV before transduction. GFP expression was then determined by flow cytometry 3 days after transduction. As expected, transduction mediated by VSV-LV and NiVwt-LV was not affected by IVIG treatment. On the other hand, MV EpCAM NiVmut showed a dose-dependent decrease in transduction efficiency, which was completely neutralized by 100 μg / ml of IVIG. EpCAM -LV was resistant to IVIG at all concentrations used and should therefore be at least 10,000-fold less sensitive to human immunoglobulins than the corresponding MV-based vector (Figure 10). These results indicate that receptor-targeting vectors based on NiV glycoproteins are not neutralized when injected into humans.

[0328] Therefore, due to the above-mentioned characteristics of NiV-G, we further pseudotyped it with NiV to generate CD8-targeting particles displaying cytokines. Here, a CD8-specific scFv derived from OKT8 was displayed on NiV-GΔ34mut4 to generate the envelope plasmid pCG-Gmut-CD8scFv, and human IL7 was displayed on NiV-GΔ34mut4 to generate the envelope plasmid pCG-Gmut-IL7. 8 G / IL7 GTo generate LV, 0.45 μg of pCG-Gmut-CD8scFv, 0.45 μg of pCG-Gmut-IL7, 4.7 μg of pCG-FcΔ22, 14.4 μg of the HIV-1 packaging plasmid pCMVΔR8.9, and 15.1 μg of the transduced LV plasmid were used to transfect HEK293T cells in one T175 flask. 7 Titers of tu / ml were obtained and for VLPs titers of approximately 1.5ug p24 per ml were obtained.

[0329] 4 H / IL7 H - As with achievements based on LV, 8 G / IL7 G -LV (containing proteins of the sequences SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 16) also inhibits CD8 in peripheral blood mixtures. + It was possible to selectively stimulate and transduce T cells. As shown in Figure 11, freshly isolated human CD3 + T cells, 8 G / IL7 G Incubation with LV for 3 days resulted in increased CD8 + Not only did a subset of T cells upregulate CD71 expression, but only this cell population efficiently expressed the reporter transgene GFP (Figure 12) or the therapeutic transgene CAR19 (Figure 13). In addition, parental IL7-deficient CD8-targeted LV (8 G -LV) compared to 8 G / IL7 G -LV, CD8 + It was more effective in activating target cells and delivering transgenes without compromising specificity for T cell transduction.

[0330] Example 4 Identification of the optimal ratio of CD8-targeting NiV-G to IL7-presenting NiV-G To hit more target cells, improve titer, and enhance particle specificity for selective activation and transduction of distinct cell populations while maximally reducing off-target effects, the transfection protocol was further optimized for vector production and NiV-G maintained in packaging cells. targeting domain (or MV-H targeting domain ) and NiV-G functional domain (MV-H functional domain Carefully determine the amount of plasmid encoding the .

[0331] G 8 / G IL7 To generate CD8-LV, a total of 0.9 μg of pCG-Gmut plasmid, 14.4 μg of the HIV-1 packaging plasmid pCMVΔR8.9, and 15.1 μg of pSEW, along with 4.7 μg of pCG-FcΔ22, were used to transfect HEK293T cells in one T175 flask, as described in Example 3. During this time, the plasmids pCG-Gmut-CD8scFv and pCG-Gmut-IL7 were mixed at different ratios of 20:1, 10:1, 5:1, 1:1, or 1:5. G / IL7 G Each cell supernatant containing LV was treated with CD8 + Molt cells or CD8 + Used for transduction of A301 cells, 48 ​​hours after transduction, GFP + The percentage of cells is determined by FACS and the titer is calculated. G / IL7 G -LVs, freshly isolated CD3 + used for transduction of CD8 T cells + Intracellular and CD8 - Intracellular expression of CD71 and GFP is determined, and the best ratio is identified as the ratio that gives high titer, high expression of CD71 and GFP in the target population and lowest expression in the non-target population.

[0332] Example 5 Characterization of resting T cells transduced by targeting VLPs and targeting LVs presenting IL7 The T cell compartment is highly heterogeneous, and T cell subsets differ phenotypically and functionally. As far as T cell therapy for cancer is concerned, less differentiated cells usually correlate with better antitumor efficacy in vivo. Therefore, it is important to identify T cell subsets that are activated or modified by the vector particles according to the present invention. G / IL7 G Taking LV-CAR as an example, the phenotype of transduced resting T cells should be characterized in comparison to transduced pre-stimulated T cells (by the most widely used CD3 / CD28 stimulation protocol). Briefly, resting CD3 + T cells or pre-stimulatory CD3 + Three days after transduction of T cells, the expression of multiple T cell markers is monitored, including CD11a, CD11b, CD25, CD27, CD28, CD45RA, CD45RO, CD62L, CD69, CD71, CD95, CD127, and CCR7. These molecules have previously been used to monitor the expression of CD45RA + CD45RO - CD62L high CD95 - CD27 high CCR7 high Naive T cells (T N ), CD45RA + CD45RO - CD62L high CD95 + CD27 high CCR7 high Stem cell memory T cells (T SCM ), CD45RA - CD45RO high CD62L + CD95 + CD27 + Central memory T cells (T CM ), CD45RA - / + CD45RO high CD62L - CD95 + CD27 - / +CCR7 - Effector memory T cells (T EM ), and CD45RA - / + CD45RO + CD62L - CD95 high CD27 - CD28 - CCR7 - Effector T cells (T E CAR was chosen because it has been used to differentiate CAR. + T cell phenotypes are analyzed by flow cytometry.

[0333] In addition, pre-sorted CD4 + T cell subsets (T SCM , T CM , T EM , and T E ) to 4 H / IL7 H -VLPs to demonstrate the advantages of the present invention for T cell activation over conventional TCR stimulation. H / IL7 H After 3 days of incubation with VLPs or stimulation with CD3 / 28 antibodies, the T cell subsets indicated above are analyzed for phenotypic variation and activation levels. IL7-presenting T cell targeting vector particles are expected to activate and transduce less differentiated T cells.

[0334] Example 6 Enhance the function of ex vivo generated CART cells and simplify the CART cell generation procedure. Adoptive T cell immunotherapy has been demonstrated to be an effective clinical regimen for treating a variety of diseases. However, its widespread application is limited by several issues. On the one hand, the generation of CAR-engineered or TCR-engineered T cells is extremely expensive and labor-intensive due to in vitro stimulation, prolonged expansion, and cell isolation and purification. On the other hand, in vitro-expanded effector T cells often fail to persist in vivo and exhibit sustained antitumor effects. The present invention offers a potential solution to these challenges by combining cell stimulation and gene transfer in a single step, enabling transduction of resting T cells. Using the present invention, the T cell generation process can be significantly simplified, thereby reducing the cost of T cell production. Furthermore, compared with conventionally generated cells, CAR / TCR-engineered T cells generated by the present invention may be more potent in vivo due to the potential for efficiently manipulating less differentiated cells, as demonstrated in Example 5.

[0335] To confirm these characteristics, freshly isolated human PBMCs or human CD8 + T cells, e.g., 8 G / IL7 G The cells are transduced with either VSV-LV-CAR or VSV-LV-CAR as a control. Meanwhile, conventional CAR T cells are also prepared in parallel according to the most widely used protocols. Briefly, human PBMCs or CD8 T cells isolated from the same donor are transduced with VSV-LV-CAR or VSV-LV-CAR as a control. + T cells are stimulated with CD3 / CD28 antibodies and IL2. G / IL7 GTransduction of LV-CAR or VSV-LV-CAR was followed by 10-day expansion in the presence of IL2. Two to three days after vector transduction of resting T cells, or 12 days after transduction of stimulated T cells, these cells were co-cultured with target tumor cells in vitro or injected into tumor-bearing humanized mice. In vitro tumor cell lysis and in vivo tumor regression, animal survival, and tumor-reactive T cell persistence and proliferation were then assessed.

[0336] Example 7 Selective activation and transduction by targeting particles displaying IL7 under in vivo-like conditions In the next step, we will explore the feasibility of using viral particles to target different cells and functionally modify them in an in vivo situation. Therefore, we will introduce viral particles, e.g., G 8 / G IL7 -LV transduction is performed in vivo, in the presence of an active human complement system, an obstacle encountered by viral particles, and in the presence of CD8 + It allows assessment of activation of T cell populations and targeting of gene transfer to them.

[0337] Briefly, fresh peripheral blood was collected from 8 G / IL7 G -LV-GFP, or other control vectors (VSV-LV, 8 H / IL7 H -LV, 8 G -LV or VSV / IL7 G The cells are then incubated with CD8 T cells (-LV) for 6-8 hours. Total PBMCs are then isolated from the blood and further cultured in T cell culture medium without the addition of stimulatory reagents. After 3-4 days, the CD8 T cells in the culture + Cells and CD8 -Cells are assessed for CD71 and GFP expression. To confirm stable gene transfer, a small fraction of cells are transferred to culture medium supplemented with CD3 / CD28 antibodies and IL2 for an additional 3 days before expressing GFP. + The percentage of cells is analyzed.

[0338] Example 8 Targeting particles presenting IL7 selectively activate and transduce resting T cells in a humanized mouse model To demonstrate that the particles according to the invention can be applied locally or systemically in vivo to allow cell type-specific activation or modification, these particles were administered to human CD34 cells derived from healthy donors. + This is applied to mice engrafted with hematopoietic stem cells (HSCs), which allows the generation of multilineage human hematopoietic cells that are tolerated by the mouse host.

[0339] Here, G 8 / G IL7 Pseudotyped particles of human CD8 + Take the application to T cell activation and modification as an example. Briefly, human CD3 + After confirming the successful establishment of the human immune system by detecting approximately 10% of T cells, 8 / G IL7 Pseudotyped particles are applied systemically via intravenous injection (iv) or locally via intrasplenic or intrathymic injection. 8 / G IL7 -VLPs were injected to induce in vivo human CD8 + The feasibility of selective activation of T cells is evaluated. In this experimental setting, activation levels / duration and CD8 T cell proliferation were assessed at different time points after VLP injection. + Analyze T cell proliferation. 8 / G IL7In this experimental setting, GFP expression, the phenotype of transduced cells, and their proliferation and survival are analyzed at different time points after LV injection. 8 / G IL7 -LV-CAR will be injected to evaluate therapeutic gene delivery and in vivo generation of CAR TT cells. In this experimental setting, CAR expression, transduced cell phenotype, transduced cell proliferation and persistence in the presence / absence of target tumor cells / antigens, and local / systemic tumor clearance will be analyzed at different time points after LV injection.

[0340] Example 9 Rhesus CD4 + Selective activation of T cells Non-human primate (NHP) T cells are phenotypically similar to human T cells, making them the best animal model for human immunity and immunotherapy. As far as T cell therapy is concerned, the NHP model could be more reliable in predicting efficacy, dosage, route of application, and potential drug safety. Therefore, tools to specifically modify distinct cell types or induce functional changes in these cell types in NHPs are highly desirable.

[0341] Here, 4 H / IL7 H The SIV vector and its application to rhesus monkeys will now be described in detail as an example. First, the rhesus CD4-specific DARPin57.2 (CD4 D57.2 ) and Hmut protein (H-CD4 D57.2 ) as the targeting domain displayed on Hmut (H-rmIL7), and rhesus-reactive IL7 as the functional domain displayed on Hmut (H-rmIL7). The two H proteins, the ratio of which was determined in Example 4, were then used to pseudotype LV derived from simian immunodeficiency virus mac 251 (SIVmac251). Vector yield and titer were determined by P27 ELISA / Nano-sight, and CD4+ After determining whether to transduce monkey cells, freshly isolated monkey CD3 + 4. Delivering GFP or CAR to T Cells H / IL7 H -SIV transduction. Then CD4 - CD4 versus T cells + The expression of activation markers and transgenes in T cells was determined as described above. The potential for in vivo application was then evaluated in an NHP model. To this end, a single dose of vector was slowly injected into two axillary lymph nodes of each enrolled animal (e.g., rhesus macaque). One week after injection, blood samples were collected, and one of the two injected lymph nodes was surgically removed and dissociated into a single cell suspension. Detailed analysis was performed to analyze the phenotype, specific activation, transgene expression level, and / or function (in the case of delivering a therapeutic transgene) of the transduced T cells in vivo. Furthermore, a second dose of the same vector was administered to a second lymph node, and the enrolled animals were monitored for six months to evaluate the proliferation, survival, and / or function (in the case of delivering a therapeutic transgene) of the transduced T cells in vivo.

[0342] Example 10 Selective activation and expansion of antigen-specific T cells in immunocompetent mouse models The potential of targeting cytokines to function at the site of disease while avoiding systemic toxicity can be successfully assessed in an immunocompetent murine melanoma tumor model. Murine CD8-targeting VLPs (m8) presenting IL-12 G / mIL12 G Take m8-VLP as an example. Previous studies have shown that administration of IL12 enhances the tolerance and antitumor efficacy of the introduced tumor-reactive T cells, but results in significant systemic toxicity. To overcome this drawback, the present inventors have investigated the use of m8-VLP. G / mIL12 GThe IL12 function is controlled at the tumor site using IL-12-VLPs. Wild-type C57 / B16 mice are implanted with B16 / OVA melanoma cells. After tumor establishment, the mice are treated with irradiation and transduced with T cells isolated from C57 / B16 OT-I transgenic mice. After implantation of OT-I cells, m8 T cells are injected intravenously. G / mIL12 G Mice are treated with OT-IT cells or mouse IL12 or PBS as a control. Tumor volume, in vivo persistence and function of OT-IT cells, and survival of mice are analyzed.

[0343] Example 11 Targeting human CD4+ T cell activation in vivo Human CD4 + In vivo efficacy of MV-4 for T cell activation H / IL7 H To assess LV, NOD / SCID gc- / - (NSG) mice were transfected with CD3 + Cord blood T cells were engrafted. Human T cell reconstitution in the blood was determined weekly. 20% (hCD3 + Cells / total hCD45 + cells+mCD45 + Once hT cells were detected in 10% of the cells, mice were 6 IU's MV-4 H / IL7 H -LV or MV-4 H Mice were intravenously injected with MV-4. Two weeks after vector injection, the mice were sacrificed and splenocytes and peripheral blood mononuclear cells were isolated. H / IL7 H -MV-4 in the case of LV (25% CD4+ CD71+ cells) HWe found a strong specific activation of IL-7, as revealed by the CD71 late activation marker, for human CD4+ T cells compared to LV (8% CD4+ CD71+ cells) (Figure 14). Furthermore, CD4- T cells, representing CD8+ T cells, show identical expression of CD71 for both targeting vectors. The latter highlights that IL-7 survival signaling activates only targeted CD4+ T cells, but not CD8+ cells.

[0344] In conclusion, in vitro, MV-4 H / IL7 H Following the specific activation of target cells achieved by MV-4, an in vivo human blood system mouse model was also observed. H / IL7 H -LV-induced specific activation of CD4+ T cells was confirmed.

Claims

1. a) at least one cell-targeting fusion protein comprising (i) an envelope glycoprotein G or envelope glycoprotein H, or a protein derived from a transmembrane domain, of a virus of the Paramyxoviridae family, and (ii) at least one cell-targeting domain; b) at least one modulating fusion protein comprising (i) an envelope glycoprotein G or envelope glycoprotein H, or a protein derived from a transmembrane domain, of a virus of the Paramyxoviridae family, and (ii) at least one functional domain; and c) comprises at least one glycoprotein derived from the envelope glycoprotein F of a virus of the Paramyxoviridae family; the cell-targeting fusion protein and / or the modulating fusion protein comprises a protein derived from envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family; The protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family lacks at least a portion of the cytoplasmic region of the envelope glycoprotein G or envelope glycoprotein H; Pseudotyped retrovirus-like particles or retroviral vectors.

2. 2. The pseudotyped retrovirus-like particle or retroviral vector of claim 1, wherein the virus of the Paramyxoviridae family is a virus of the genus Morbillivirus or Henipavirus.

3. 3. The pseudotyped retrovirus-like particle or retroviral vector of claim 1, wherein the protein derived from the envelope glycoprotein G or the envelope glycoprotein H of a virus of the Paramyxoviridae family is at least partially incapable of binding to at least one natural receptor for the envelope glycoprotein G or the envelope glycoprotein H.

4. 4. The pseudotyped retrovirus-like particle or retroviral vector of claim 1, wherein the glycoprotein derived from the envelope glycoprotein F of a virus of the Paramyxoviridae family lacks at least a portion of the cytoplasmic domain of the envelope glycoprotein F.

5. 5. The pseudotyped retrovirus-like particle or retroviral vector of claim 1, wherein the cell targeting domain is selected from the group consisting of a DARPin, an ScFv, a targeting peptide, and combinations thereof.

6. 6. The pseudotyped retrovirus-like particle or retroviral vector of any one of claims 1 to 5, wherein the functional domain is selected from the group consisting of a cytokine, a growth factor, a hormone, a neurotransmitter, an apoptotic ligand, and combinations thereof.

7. 7. Use of a pseudotyped retrovirus-like particle or retroviral vector according to any one of claims 1 to 6 to selectively modulate the activity of, and optionally transduce, a target cell.

8. 10. A method for selectively modulating the activity of and / or transducing a target cell, the method comprising contacting a cell, including said target cell, with a pseudotyped retrovirus-like particle or retroviral vector according to any one of claims 1 to 6.

9. 10. A pseudotyped retrovirus-like particle or retroviral vector according to any one of claims 1 to 6 for use as a medicament.

10. 10. The pseudotyped retrovirus-like particle or retroviral vector for use in immunotherapy, gene therapy, and / or vaccination according to claim 9.

11. 11. The pseudotyped retrovirus-like particle or retroviral vector according to claim 9 or 10 for use in the prevention and / or treatment of immune disorders, cancer, genetic disorders, allergic disorders, inflammatory disorders, infectious disorders, metabolic disorders, neurological disorders, muscular disorders, and combinations thereof.

12. A nucleic acid comprising a sequence encoding a cell-targeting fusion protein and / or a sequence encoding a modulating fusion protein, the cell-targeting fusion protein comprises (i) a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family, or a transmembrane domain, and (ii) at least one cell-targeting domain; the modulating fusion protein comprises (i) a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family, or a transmembrane domain, and (ii) at least one functional domain; Nucleic acid.

13. 13. The nucleic acid of claim 12, wherein the nucleic acid comprises a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 13, and / or comprises a sequence encoding a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO:

14.

14. A vector comprising the nucleic acid of claim 12 or 13.

15. 7. A method for producing a pseudotyped retrovirus-like particle or retroviral vector according to any one of claims 1 to 6, comprising injecting into a packaging cell line: (i) at least one nucleic acid encoding a cell-targeting fusion protein comprising (i) an envelope glycoprotein G or an envelope glycoprotein H, or a protein derived from a transmembrane domain, of a virus of the Paramyxoviridae family, and (ii) at least one cell-targeting domain; (ii) at least one nucleic acid encoding a modulating fusion protein comprising (i) an envelope glycoprotein G or envelope glycoprotein H, or a protein derived from a transmembrane domain, of a virus of the Paramyxoviridae family, and (ii) at least one functional domain; (iii) at least one nucleic acid encoding a glycoprotein derived from the envelope glycoprotein F of a virus of the Paramyxoviridae family; and (iv) at least one vector comprising a nucleic acid encoding a core protein from said retrovirus; co-transfecting The cell-targeting fusion protein and / or the modulating fusion protein comprises a protein derived from the envelope glycoprotein G or envelope glycoprotein H of a virus of the Paramyxoviridae family. method.

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