Cincitin-1 fusion protein and its use for cargo delivery to target cells

Abstract

The inventors have developed a novel system for modifying the tropism of the envelope protein syncytin and used this to functionalize particles such as virus particles or, more specifically, virus-like particles (VLPs), which can be used for gene transfer or other applications. In particular, the inventors have created a fusion protein containing the syncytin-1 (SYN) signal sequence (SS), a targeting moiety (either natural or engineered), the SYN protein, and a flexible linker between SYN and the targeting moiety to enhance the transduction of cell types expressing a receptor or antigen targeted by the targeting moiety, such as hematopoietic stem and progenitor cells (HSPCs). The inventors have demonstrated that the fusion strategy enables modification of the tropism of syncytin towards various receptors, thereby enabling targeting of desired cell types. The system is applicable to other desired antigens, thereby allowing the fusion protein to be retargeted to specific cell types.

Description

Technical Field

[0001] Field of the Invention: The present invention resides in the field of medicine, particularly in the field of cargo delivery to target cells.

[0002] Background of the Invention: Particles are particularly suitable for cargo delivery into cells. In particular, virus particles can be self-assembled spontaneously by viral structural proteins under appropriate conditions in vitro. Furthermore, virus particles have several features, including that they can be produced rapidly in large quantities through existing expression systems and that they closely resemble natural viruses in terms of conformation and appearance. Additionally, virus particles with diameters of 20 - 150 nm also have the characteristics of nanomaterials, such as a large surface area, amino acids (such as lysine residues and glutamic acid residues) that are prone to approaching the surface with reactive moieties, a well-behaved spatial structure, and good biocompatibility. Therefore, constructed virus particles have great potential as delivery systems for specifically transporting a wide variety of cargos. Some results demonstrate the importance of having both viral structural proteins (such as capsids) for forming virus particles and functional fusogenic envelopes on the surface of virus particles for efficient delivery of cargos into cells. In the above context, the G glycoprotein (VSV-G) of the fusogenic envelope of vesicular stomatitis virus has been widely used to enhance the fusogenicity of virus particles. Other fusogenic proteins are also being studied for improving the delivery of virus particles. In particular, interest in the syncytin glycoprotein, which is an envelope protein of the human endogenous retrovirus family W (HERV-W), has been explored. For example, International Publication No. WO 2017 / 182607 describes a method for transducing immune cells using a lentiviral vector pseudotyped with the syncytin glycoprotein of endogenous retrovirus.More recently, virus particles pseudotyped with mouse syncytin and incorporating a mammalian Gag homolog have been engineered to encapsulate, secrete, and deliver specific RNAs (Segel M, Lash B, Song J, Ladha A, Liu CC, Jin X, Mekhedov SL, Macrae RK, Koonin EV, Zhang F. Mammalian retrovirus-like protein PEG10 packages its own mRNA and can be pseudotyped for mRNA delivery. Science. 2021 Aug 20;373(6557):882-889. doi: 10.1126 / science.abg6155. PMID: 34413232; PMCID: PMC8431961). In short, these results demonstrate that syncytin represents a modular platform suitable for development as an efficient therapeutic delivery modality. However, there remains a need to improve the targeting of virus particles to target cells in order to enhance therapeutic efficiency and avoid any unspecific effects.

[0003] Summary of the Invention: The present invention is defined by the claims. In particular, the present invention relates to syncytin-1 fusion proteins and their use for cargo delivery to target cells.

[0004] Detailed Description of the Invention: Main definitions: As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably and refer to amino acid polymers of any length. The term also encompasses polymers of amino acids that are modified; for example, polymers of amino acids that have formed disulfide bonds, been glycosylated, lipidated, phosphorylated, or conjugated to a labeling component. When considered in the context of gene therapy, a polypeptide refers to each intact polypeptide, or any fragment or genetically engineered derivative thereof that retains the desired biochemical function of the intact protein.

[0005] As used herein, the term "fusion protein" means a protein made by connecting two or more polypeptide sequences to each other. The fusion polypeptides encompassed by the present invention include the translation product of a chimeric gene construct in which a nucleic acid sequence encoding a first polypeptide, for example an RNA binding domain, is connected to a nucleic acid sequence encoding a second polypeptide, for example an effector domain, to form one open reading frame. In other words, a "fusion polypeptide" or "fusion protein" is a recombinant protein of two or more proteins connected by peptide bonds or via several peptides. A fusion protein may also contain a peptide linker between the two domains. Within a fusion protein, the term "operably linked" is intended to indicate that the peptides of the present invention and the heterologous polypeptide are fused in-frame with each other.

[0006] As used herein, the term "linker" has its general meaning in the art and refers to an amino acid sequence having a length sufficient for the protein to reliably form an appropriate secondary and tertiary structure. Typically, the linker is a linker that allows the compound to assume an appropriate conformation. The most suitable linker sequences will (1) assume a flexible and extended conformation, (2) not tend to generate an ordered secondary structure that could interact with the functional domains of the fusion protein, and (3) have minimal hydrophobic or charged characteristics that could promote interaction with the functional protein domains.

[0007] As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length, which includes ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-stranded, double-stranded, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-stranded, double-stranded, and single-stranded ribonucleic acid ("RNA"). It also includes polynucleotides in modified forms, for example, modified by alkylation and / or capping, and polynucleotides in unmodified forms. More specifically, the term "polynucleotide" refers to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), such as transfer RNA (tRNA), ribosomal RNA (rRNA), hairpin RNA (hRNA), small interfering RNA (siRNA), and messenger RNA (mRNA) (regardless of whether spliced or unspliced), any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing a non-nucleotide backbone, such as polyamides (e.g., peptide nucleic acid "PNA") and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in a conformation that allows base pairing and stacking of bases as seen in DNA and RNA. In some embodiments, the polynucleotide includes mRNA. In other embodiments, the mRNA is synthetic mRNA. In some embodiments, the synthetic mRNA includes at least one unnatural nucleobase. In some embodiments, all nucleobases of a particular class are replaced with unnatural nucleobases (e.g., all uridines within the polynucleotides disclosed herein may be replaced with an unnatural nucleobase, such as 5-methoxyuridine). In some embodiments, the polynucleotide (e.g., synthetic RNA or synthetic DNA) includes only natural nucleobases, i.e., A, C, T, and G in the case of synthetic DNA, or A, C, T, and U in the case of synthetic RNA.

[0008] As used herein, the term "encoding" refers to the role of a particular nucleotide sequence within a polynucleotide, such as a gene, cDNA, or mRNA, as a template for synthesis in a biological process of other polymers and macromolecules that have either a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the resulting biological properties. Thus, a gene, cDNA, or RNA encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. It can be said that both the nucleotide sequence that is identical to the mRNA sequence and the coding strand typically provided in the sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA, encode the protein or other product of that gene or cDNA. Unless otherwise specified, "polynucleotide sequences encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "polynucleotide sequence or RNA encoding a protein" may include introns to the extent that the nucleotide sequence encoding the protein may include intron(s) in some versions.

[0009] As used herein, the expression "derived from" refers to a process of isolating, deriving, or producing a different second component (e.g., a second polypeptide different from the first polypeptide) using a first component (e.g., a first polypeptide) or information from that first component.

[0010] The "percent identity" between two sequences used in this specification is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for the optimal alignment of the two sequences (i.e., percent identity % = number of identical positions / total number of positions × 100). The comparison of sequences and the determination of the percent identity between two sequences can be accomplished using mathematical algorithms as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-53.). The percent identity between two nucleotide sequences or between amino acid sequences can also be determined using algorithms such as, for example, EMBOSS Needle (pairwise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle can be used with the BLOSUM62 matrix, a "gap open penalty" of 10, a "gap extend penalty" of 0.5, the "end gap penalty" of Felsenstein, an "end gap open penalty" of 10, and an "end gap extend penalty" of 0.5. Generally, "percent identity" is a function of dividing the number of matching positions by the number of positions being compared and multiplying by 100. For example, if 6 out of 10 positions of a sequence are identical between two compared sequences after alignment, the percent identity is 60%. The percent identity is typically determined with respect to the full length of the query sequence for which the analysis is being performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical regardless of any chemical and / or biological modifications.According to the present invention, a first amino acid sequence having at least 70% identity to a second amino acid sequence means that the first sequence has an identity of 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% to the second amino acid sequence.

[0011] As used herein, the term "mutation" has its general meaning in the art and refers to a substitution, deletion or insertion. In particular, the term "substitution" means that a particular amino acid residue at a particular position is removed and another amino acid residue is inserted at the same position. In this specification, a mutation is a reference according to standard mutation nomenclature.

[0012] As used herein, the term "syncytin-1" or "SYN" has its general meaning in the art and refers to a protein found in humans and other primates encoded by the ERVW-1 gene (endogenous retrovirus group W envelope member 1). Syncytin-1 is a cell-cell fusion protein whose function is best characterized in placental development. The term also refers to endogenous retrovirus group W member 1, Env-W, envelope polyprotein gPr73, emerin, HERV-7q envelope protein, HERV-W envelope protein, HERV-W_7q21.2 proviral ancestor Env polyprotein and is also known as syncytin. An exemplary amino acid sequence of syncytin-1 is shown by SEQ ID NO: 1. The signal peptide ranges from the amino acid residue at position 1 to the amino acid residue at position 20 of SEQ ID NO: 1. The extracellular domain of syncytin-1 ranges from the amino acid residue at position 21 to the amino acid residue at position 443 of SEQ ID NO: 1.

[0013]

Chemical formula

[0014] As used herein, the term "ASCT1" refers to human neutral amino acid transporter A, which is encoded by the SLC1A4 gene. Syncytin-1 can bind to ASCT1 (Antony JM, Ellestad KK, Hammond R, Imaizumi K, Mallet F, Warren KG, Power C. The human endogenous retrovirus envelope glycoprotein, syncytin-1, regulates neuroinflammation and its receptor expression in multiple sclerosis: a role for endoplasmic reticulum chaperones in astrocytes. J Immunol. 2007 Jul 15;179(2):1210-24. doi: 10.4049 / jimmunol.179.2.1210. PMID: 17617614).

[0015] As used herein, the term "ASCT2" refers to neutral amino acid transporter B(0), which is encoded by the SLC1A5 gene. ASCT2 has been described as a receptor for syncytin-1 (Blond JL, Lavillette D, Cheynet V, Bouton O, Oriol G, Chapel-Fernandes S, Mandrand B, Mallet F, Cosset FL. An envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells expressing the type D mammalian retrovirus receptor. J Virol. 2000;74:3321-3329. doi: 10.1128 / JVI.74.7.3321-3329.2000.)

[0016] As used herein, the term "SYN480" refers to a polypeptide consisting of an amino acid sequence that is in the range of the amino acid residue at position 21 to the amino acid residue at position 480 of SEQ ID NO: 1.

[0017] As used herein, the term "syncytin-1 polypeptide" or "SYN polypeptide" refers to any polypeptide that is derived from syncytin-1 and contains the conserved motif SDGGGXXDXXR (SEQ ID NO: 2) that is essential for the interaction of syncytin-1 with hASCT2 (see Cheynet V, Oriol G, Mallet F. Identification of the hASCT2-binding domain of the Env ERVWE1 / syncytin-1 fusogenic glycoprotein. Retrovirology. 2006 Jul 4;3:41. doi: 10.1186 / 1742-4690-3-41. PMID: 16820059; PMCID: PMC1524976). According to the present invention, the syncytin-1 polypeptide can bind to the ASCT1 receptor, preferably the ASCT2 receptor, as determined by any assay well known in the art (see, for example, Cheynet V. et al. supra).

[0018] As used herein, the term "particle" refers to a small object that behaves as a complete unit with respect to its transport and properties, i.e., an object of an individual unit, where an atom or molecule (from which it is formed) essentially embodies the particle.

[0019] As used herein, the term "nanoparticle" refers to a particle having a diameter of less than about 1000 nm (e.g., about 500 nm), more specifically less than about 300 nm. In one embodiment, the term "nanoparticle" refers to a particle having a diameter in the nanosize range that does not exceed the micron size range.

[0020] As used herein, the term "functionalized" is used interchangeably with the terms "attached" and "bonded".

[0021] As used herein, the term "viral particle" has its ordinary meaning in the art and refers to the capsid of a virus that is fully or partially constructed. A viral particle may or may not contain a viral genome. Thus, the term encompasses virus-like particles (VLPs). Viral particles with a diameter of about 20 - 150 nm also have characteristics of nanomaterials such as a large surface area, amino acids (e.g., lysine residues and glutamic acid residues) that are prone to approaching a surface with reactive moieties, a well-behaved spatial structure, and good biocompatibility. Therefore, viral particles have great potential as a delivery system for specifically transporting a wide variety of cargos.

[0022] As used herein, the term "virus-like particle" or "VLP" refers to a structure that resembles a viral particle but lacks a viral genome, is non-replicable, and is non-pathogenic. The particle typically contains at least one structural protein derived from a virus. Preferably, there is only one structural protein present. Most preferably, there are no other non-structural components of the virus. Thus, virus-like particles can be spontaneously self-assembled by viral structural proteins under appropriate conditions in vitro while excluding genetic material and the potential for replication. Virus-like particles with a diameter of about 20 - 150 nm also have characteristics of nanomaterials such as a large surface area, amino acids (e.g., lysine residues and glutamic acid residues) that are prone to approaching a surface with reactive moieties, a well-behaved spatial structure, and good biocompatibility. Therefore, constructed virus-like particles have great potential as a delivery system for specifically transporting a wide variety of cargos.

[0023] As used herein, the term "pseudotyped virus particle" refers to a virus particle in which the viral envelope protein has been replaced by a heterologous protein, particularly the syncytin-1 fusion protein of the present invention.

[0024] As used herein, the term "virus particle with an envelope" refers to a virus particle surrounded by a lipid bilayer envelope derived from the plasma membrane. As used herein, the term "lipid bilayer envelope derived from the plasma membrane" refers to the lipid bilayer derived from the plasma membrane of the host cell from which the virus particle was released. This envelope encloses the virus particle, either partially or completely. Preferably, the virus particle is completely (or substantially completely) enclosed within the envelope. The lipid bilayer will have a composition of macromolecules corresponding to that of the plasma membrane of the host cell. The bilayer will have similar ratios of the same lipids, proteins, and sugar chains. Such macromolecules will include transmembrane receptors and channels (kinase receptors and ion channels), cytoskeletal proteins (such as actin), sugar chains linked to lipids or proteins, phospholipids (such as phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine), and cholesterol.

[0025] As used herein, the term "viral envelope protein" refers to a protein encoded by the viral genome and associated with the viral envelope in a virus having a normal envelope, where the protein can specifically interact with a cognate intracellular virus receptor protein, for example, to facilitate the attachment of the virus to a cell. Viral envelope proteins include, but are not limited to, glycoproteins.

[0026] As used herein, the term "viral structural protein" refers to a protein that contributes to the overall structure of the viral capsid protein or core protein. The viral structural proteins of the present invention can be obtained from any virus that can form viral particles. These are typically proteins derived from viruses that naturally have an envelope. Such viruses include, but are not limited to, Retroviridae (e.g., human immunodeficiency virus, Moloney murine leukemia virus, feline leukemia virus, Rous sarcoma virus), Coronaviridae, Herpesviridae, Hepadnaviridae, and Orthomyxoviridae (e.g., influenza virus). However, viruses that do not naturally have an envelope may be capable of forming viral particles with an envelope, and these are also encompassed by the present invention. Examples of viruses that do not naturally have an envelope include Picomaviridae, Reoviridae, Adenoviridae, Papillomaviridae, and Parvoviridae (including adeno-associated virus).

[0027] As used herein, the terms "Gag protein", "GAG protein", or "group-specific antigen" refer to a family of glycoproteins that form the capsid of a particular virus. The Gag protein is processed into the MA (matrix), CA (capsid), and NC (nucleocapsid) portions. Typically, the nucleocapsid protein (NC) contains at least one zinc finger motif flanked by highly basic regions.

[0028] As used herein, the term "target cell" means a cell with which fusion of the viral particles of the present invention is desirable.

[0029] As used herein, the term "cargo" as used herein refers to any molecule, such as a nucleic acid, polypeptide, pharmaceutical, etc., that has a desired biological activity and an appropriate solubility profile and is encapsulated within the viral particles of the present invention.

[0030] As used herein, the term "encapsulated" or "encapsulation" as used herein refers to enclosing the cargo within the viral particles of the present invention.

[0031] The term "targeting moiety" as used herein refers to any molecule that specifically binds to a target.

[0032] As used herein, the term "antibody" refers to an immunoglobulin molecule and immunologically active portions of immunoglobulin molecules, i.e., molecules containing antigen-binding sites that immunospecifically bind to an antigen. In natural antibodies of rodents and primates, the two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, lambda (λ) and kappa (κ). There are mainly five heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. In a typical IgG antibody, the light chain contains two domains, i.e., a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains, i.e., one variable domain (VJ) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity for the antigen. Thus, the term "variable domain" refers to the variable domain of the light chain (VL) or the variable domain of the heavy chain (VH), and thus indicates the domain directly involved in the binding of the antibody to the antigen. The constant region domains of the light chain (CL) and the heavy chain (CH) confer important biological properties, such as antibody chain association, secretion, transplacental transfer, binding to complement, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of an antibody resides in the structural complementarity between the antibody binding site and the antigen determinant. The antibody binding site is mainly made up of residues derived from the hypervariable regions or complementarity determining regions (CDR). Occasionally, residues derived from non-hypervariable regions or framework regions (FR) may be involved in the antibody binding site or may affect the binding site through the overall domain structure. The complementarity determining region, i.e., CDR, refers to the amino acid sequence that together defines the binding affinity and specificity of the natural Fv region of the natural immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, and H-CDR3, respectively.Therefore, the antigen-binding site typically contains six CDRs, including a set of CDRs each derived from the V regions of the heavy and light chains. The framework region (FR) refers to the amino acid sequences inserted between the CDRs. Thus, the variable regions of the light and heavy chains typically contain four framework regions and three CDRs in the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Residues within the antibody variable domain are customarily numbered according to a system devised by Kabat et al. This system is shown in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, National Institutes of Health, U.S.A. (Kabat et al., 1992, hereinafter referred to as "Kabat et al." in this specification). The Kabat residue designations do not always directly correspond to the linear numbering of amino acid residues in the sequence numbering. The actual linear amino acid sequence may have fewer or additional amino acids corresponding to shortening of the structural components (whether framework regions or complementarity-determining regions (CDRs)) of the basic variable domain structure compared to the strict Kabat numbering. For a given antibody, the correct Kabat numbering of residues can be determined by alignment of residues having homology within the antibody's sequence with the sequence of the "standard" Kabat numbering. According to the Kabat numbering system, the CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR1), residues 50-65 (H-CDR2), and residues 95-102 (H-CDR3). According to the Kabat numbering system, the CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2), and residues 89-97 (L-CDR3). For the antibodies described hereinafter in this specification, the CDRs were determined using the CDR discovery algorithm from www.bioinf.org.uk. See the chapter entitled "How to Identify CDRs by Searching the Sequence" within the antibody page.

[0033] As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a conventional four-chain antibody chain or a heavy-chain antibody chain, or a light chain, etc.), or a polypeptide consisting essentially of such a globular region.

[0034] As used herein, the term "antibody fragment" refers to at least one part of an intact antibody that retains the ability to specifically interact (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution) with an epitope of an antigen, preferably the antigen-binding region or variable region of an intact antibody. A "fragment" includes a part of an intact antibody, generally including the antigen-binding site or variable region. Examples of antibody fragments include Fab, Fab’, Fab’-SH, F(ab’)2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of a sequence of one uninterrupted continuous amino acid residue (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide") (which includes, but is not limited to, (1) single-chain Fv molecules, (2) single-chain polypeptides containing only one unassociated light-chain variable domain of the heavy-chain portion or fragments thereof containing three CDRs of the light-chain variable domain, and (3) single-chain polypeptides containing only one unassociated heavy-chain variable domain of the light-chain portion or fragments thereof containing three CDRs of the heavy-chain variable region); and multispecific antibodies formed from antibody fragments. Fragments of the antibodies of the present invention can be obtained using standard methods.

[0035] As used herein, the terms "single-domain antibody", "sdAb" or "VHH" refer to a single heavy-chain variable domain of a type of antibody found in camelid mammals that are naturally lacking a light chain. Such VHHs are also referred to as "nanobodies (registered trademark)". According to the present invention, the sdAb can be, in particular, an sdAb from a llama.

[0036] As used herein, the term "scFv" refers to a fusion protein comprising at least one antibody fragment containing the variable region of the light chain and at least one antibody fragment containing the variable region of the heavy chain, where the variable regions of the light and heavy chains are continuously linked, for example via a synthetic linker, such as a short flexible polypeptide linker, and can be expressed as a single-chain polypeptide, where the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, an scFv used herein can have the VL and VH variable regions in either order with respect to the N- and C-termini of the polypeptide, and the scFv can contain either VL-linker-VH or VH-linker-VL.

[0037] As used herein, the term "chimeric antibody" refers to an antibody that includes the VH and VL domains of a non-human antibody and the CH and CL domains of a human antibody. In some embodiments, a "chimeric antibody" is one in which (a) the constant region (i.e., the heavy and / or light chain) or a portion thereof is modified, substituted, or exchanged such that the antigen-binding site (variable region) is linked to a constant region of a different or altered class, effector function, and / or species, or to a completely different molecule, such as an enzyme, toxin, agonist molecule, such as CD40 ligand, hormone, growth factor, drug, etc., that confers new properties to the chimeric antibody; or (b) the variable region or a portion thereof is modified, substituted, or exchanged with a variable region having a different or altered antigen specificity. Chimeric antibodies also include primatized, particularly humanized, antibodies. Further, a chimeric antibody may contain residues not found in the recipient antibody or donor antibody. These modifications are made to further refine the performance of the antibody. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992) (see U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0038] As used herein, the term "humanized antibody" includes an antibody that has the six CDRs of a mouse antibody but also has a humanized framework and constant region. More specifically, as used herein, the term "humanized antibody" may include an antibody in which the CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto a human framework sequence.

[0039] As used herein, the term "human monoclonal antibody" is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by in vitro random mutagenesis or site-directed mutagenesis, or mutations introduced by somatic mutations in vivo). However, in some embodiments, the term "human monoclonal antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0040] As used herein, the term "specificity" refers to the ability of an antibody to bind to a target molecule (e.g., an epitope presented on an antigen) such that it can be detected, while having relatively little detectable reactivity with other target molecules. Specificity can be determined relatively by a binding assay or a competitive binding assay, such as using a BIAcore instrument, as described elsewhere herein. Specificity can be indicated, for example, by an affinity / avidity ratio of about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1 or more in binding to a specific antigen vs non-specific binding to other irrelevant molecules.

[0041] As used herein, the term "affinity" means the strength of binding of an antibody to a target molecule (e.g., an epitope). The affinity of a binding protein is indicated by the dissociation constant Kd. The Kd for an antibody is defined as [antibody] × [antigen] / [antibody - antigen], where [antibody - antigen] is the molar concentration of the antibody - antigen complex, [antibody] is the molar concentration of unbound antibody, and [antigen] is the molar concentration of unbound antigen. The affinity constant Ka is defined as 1 / Kd. Preferred methods for determining the affinity of a binding protein can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589 - 601(1983), which references are hereby incorporated by reference in their entirety into this specification. One preferred and standard method well - known in the art for determining the affinity of a binding protein is the use of a BIAcore instrument.

[0042] As used herein, the term "bound" refers to a direct association between two molecules due to, for example, covalent interactions, electrostatic interactions, hydrophobic interactions, and ionic interactions, and / or hydrogen - bonding interactions (including interactions such as salt bridges and water bridges). In particular, the term "bound" in the context of the binding of an antibody to a given target molecule (e.g., an antigen or epitope) as used herein typically refers to binding with an affinity corresponding to about 10 -7 M or less, for example about 10 -8 M or less, for example about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 M or even less.

[0043] As used herein, the terms "subject", "host", "individual" or "patient" refer to a mammal, preferably a human male or female of any age in need of therapy.

[0044] As used herein, the terms "treatment" or "treating" refer to both prophylactic or preventive treatment and curative or disease-modifying treatment (including treatment of a patient at risk of having or suspected of having a disease, as well as a patient diagnosed as being ill or suffering from a disease or medical condition), which also includes suppression of clinical recurrence. Treatment can be administered to a patient having a medical disorder or potentially ultimately at risk of having a disorder to prevent, cure, delay the onset of, reduce the severity of, or remit one or more symptoms of the disorder or a recurring disorder, or to extend the survival period of the patient beyond that expected if such treatment were not given. A "treatment regimen" means a pattern of treatment of a disease, such as a dosing pattern used during therapy. A treatment regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a treatment regimen (or a portion of a treatment regimen) used for the initial treatment of a disease. A general goal of an induction regimen is to provide a patient with a high level of drug during an initial period of the treatment regimen. An induction regimen can use a "loading regimen" (in whole or in part), which can include administering more doses of a drug than a physician would use during a maintenance regimen, administering the drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used for the maintenance of a patient during treatment of a disease, for example, to keep a patient in remission over a long period (months or years). A maintenance regimen can use continuous therapy (e.g., administering a drug at regular intervals, such as once a week, once a month, once a year, etc.) or intermittent therapy (e.g., episodic treatment, intermittent treatment, treatment at recurrence, or treatment upon reaching certain predetermined criteria [e.g., pain, signs of disease, etc.]).

[0045] As used herein, the term "pharmaceutical composition" refers to the compositions described herein, or pharmaceutically acceptable salts thereof, including other substances such as carriers and / or excipients. Pharmaceutical compositions as provided herein typically include a pharmaceutically acceptable carrier.

[0046] As used herein, the term "pharmaceutically acceptable carrier" includes any solvent, diluent, or other liquid vehicle, dispersion or suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, solid binder, lubricant, etc. that is suitable for the particular desired dosage form. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in the formulation of pharmaceutical compositions and known techniques for their preparation.

[0047] Syncytin-1 fusion protein: A first object of the present invention relates to a fusion protein in which the syncytin-1 polypeptide is fused to one or more targeting moieties.

[0048] Syncytin-1 polypeptide According to the present invention, the syncytin-1 polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 2 (SDGGGXXDXXR) and is capable of binding to the ASCT1 receptor, preferably the ASCT2 receptor.

[0049] In some embodiments, the syncytin-1 polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 3 (SDGGGVQDQAR).

[0050] In some embodiments, the syncytin-1 polypeptide of the present invention comprises an amino acid sequence as set forth in SEQ ID NO: 3 (SDGGGVQDQAR) and comprises at least 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400 or 450 consecutive amino acids of SEQ ID NO: 1.

[0051] In some embodiments, the syncytin-1 polypeptide of the present invention comprises an amino acid sequence having a 70% identity to the amino acid sequence in the range of the 21st amino acid residue to the 480th amino acid residue of SEQ ID NO: 1 ( "SYN480"). In some embodiments, the syncytin-1 polypeptide of the present invention comprises the amino acid sequence in the range of the 21st amino acid residue to the 480th amino acid residue of SEQ ID NO: 1, wherein the arginine residue (R) at position 393 and the phenylalanine residue (F) at position 399 are mutated. In some embodiments, the syncytin-1 polypeptide of the present invention comprises the amino acid sequence in the range of the 21st amino acid residue to the 480th amino acid residue of SEQ ID NO: 1, wherein the arginine residue (R) at position 393 is substituted by a glutamine residue (Q), and the phenylalanine residue (F) at position 399 is substituted by an alanine residue (A).

[0052] In some embodiments, the syncytin-1 polypeptide of the present invention comprises an amino acid sequence having a 70% identity to the amino acid sequence in the range of the 21st amino acid residue to the 538th amino acid residue of SEQ ID NO: 1 ( "SYN"). In some embodiments, the syncytin-1 polypeptide of the present invention comprises the amino acid sequence in the range of the 21st amino acid residue to the 538th amino acid residue of SEQ ID NO: 1, wherein the arginine residue (R) at position 393 and the phenylalanine residue (F) at position 399 are mutated. In some embodiments, the syncytin-1 polypeptide of the present invention comprises the amino acid sequence in the range of the 21st amino acid residue to the 538th amino acid residue of SEQ ID NO: 1, wherein the arginine residue (R) at position 393 is substituted by a glutamine residue (Q), and the phenylalanine residue (F) at position 399 is substituted by an alanine residue (A).

[0053] Targeting moiety: According to the present invention, the targeting moiety is a polypeptide having a binding domain. As used herein, the term "binding domain" refers to one or more regions of a polypeptide that mediate specific binding to a target molecule (e.g., an antigen, ligand, receptor, substrate, or inhibitor). Exemplary binding domains include antibody variable domains, ligand receptor-binding domains, receptor ligand-binding domains, or enzyme domains. As used herein, the term "ligand-binding domain" refers to any native receptor (e.g., a cell surface receptor), or any region or derivative thereof that retains at least homologous ligand-binding ability to the corresponding native receptor. As used herein, the term "receptor-binding domain" refers to any native ligand, or any region or derivative thereof that retains at least homologous receptor-binding ability to the corresponding native ligand. In some embodiments, the polypeptide comprises at least 1, 2, 3, 4, or 5 binding sites. The polypeptide can be either a monomer or a multimer. For example, in some embodiments, the polypeptide is a dimer. In some embodiments, the dimer is a homodimer, which comprises two identical monomer subunits. In some embodiments, the dimer is a heterodimer, which comprises two non-identical monomer subunits. The subunits of the dimer can comprise one or more polypeptide chains. For example, in some embodiments, the dimer comprises at least 2 polypeptide chains. In some embodiments, the dimer comprises 2 polypeptide chains. In some embodiments, the dimer comprises 4 polypeptide chains (as in the case of an antibody molecule).

[0054] In some embodiments, the targeting moiety is a ligand. As used herein, the term "ligand" refers to a polypeptide that binds to a polypeptide receptor and typically causes a change in receptor activity and / or a change in receptor conformation and / or affects the binding of another receptor to the targeted receptor. Thus, a ligand includes one or more receptor binding domain(s) as defined above. Receptor ligands are selected from the group consisting of, for example, cytokines, growth factors, hormones, neurotransmitters, apoptotic ligands, chemokines, glucose transporters, and combinations thereof.

[0055] In some embodiments, the targeting moiety is an antibody or antibody fragment that includes one or more variable domain(s). Typically, the antibody fragment is an scFv or VHH, or other functional fragment, such as an immunoglobulin lacking a light chain, Fab, Fab’, F(ab * )2, Fv, antibody fragment, diabody, single-chain antibody, single-domain heavy-chain antibody, single-domain light-chain antibody, Fd, CDR region, or any portion or peptide sequence of an antibody that can bind to an antigen or epitope. Thus, in some embodiments, the polypeptide having a binding domain is an immunoglobulin light chain. In some embodiments, the polypeptide having a binding domain is an immunoglobulin heavy chain. In some embodiments, the polypeptide having a binding domain is a single heavy-chain variable domain of a type of antibody found in camelid mammals that are naturally lacking a light chain. Such single-domain antibodies are also referred to as VHH or "nanobodies (registered trademark)". For a general description of (single)-domain antibodies, reference is also made to the prior art cited above, as well as European Patent No. 0368684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and International Publication Nos. 06 / 030220, 06 / 003388.

[0056] Techniques for preparing and using constructs and fragments based on various antibodies are well known in the art (see, for example, Kohler and Milstein, Nature, 256:495, 1975).

[0057] In some embodiments, the antibody is a monoclonal antibody.

[0058] In some embodiments, the antibody does not internalize. As used herein, the term "non-internalizing antibody" refers to an antibody that has the property of binding to a target antigen present on the cell surface and that, upon binding to its target antigen, does not enter the cell and will be degraded within the lysosome.

[0059] In some embodiments, the targeting moiety is an antibody-independent recognition scaffold. Examples of antibody-independent recognition scaffolds include, for example, affibodies; engineered knotted domains; monobodies (adnectins); anticalins; designed ankyrin repeat domains (DARPins); binding sites of cysteine-rich polypeptides (e.g., cysteine-rich knottin peptides); avimers; affilins, etc. See, for example, Gebauer and Skerra (2009) Curr. Opin. Chem. Biol. 13:245.

[0060] Antibody-independent scaffolds (also referred to herein as "antibody mimetic molecules") can be identified by the selection or isolation of target-binding variants from a library of binding molecules having artificially diversified binding sites. Diversified libraries can be created using a completely random approach (e.g., error-prone polymerase chain reaction (PCR), exon shuffling, or directed evolution) or may be assisted by design strategies recognized in the art. For example, positions of amino acids that are normally involved when the binding site interacts with its cognate target molecule can be randomized by insertion of degenerate codons, trinucleotides, random peptides, or entire loops into the corresponding positions within the nucleic acid encoding the binding site (see, e.g., U.S. Patent Publication No. 20040132028). The location of amino acid positions can be identified by investigation of the crystal structure of the binding site within the protein entity using the target molecule. Candidates for positions for randomization include loops, flat surfaces, helices, and binding cavities of the binding site. After randomization, the diversified library can then be subjected to selection or screening procedures to obtain binding molecules having the desired binding characteristics. For example, selection can be accomplished by methods recognized in the art, such as phage display, yeast display, or ribosome display.

[0061] In some embodiments, the antibody - based backbone contains a binding site derived from an affibody. Affibodies are derived from the immunoglobulin - binding domain of staphylococcal protein A (SPA) (see, for example, Nord et al., Nat. Biotechnol., 15: 772 - 777 (1997)). An affibody is an antibody - mimetic with a unique binding site that binds to a specific target. Affibodies can be small (for example, consisting of three α - helices with 58 amino acids and having a molar mass of about 6 kDa), have an inert format (having no Fc function), and have been successfully tested in humans as targeting moieties. The binding site of an affibody can be synthesized by mutagenizing a staphylococcal protein A - related protein (such as protein Z) derived from the domain of staphylococcal protein A (such as domain B) and selecting a mutant staphylococcal protein A - related polypeptide that has a binding affinity for the target antigen or epitope. Other methods for creating the binding site of an affibody are described in U.S. Patent Nos. 6,740,734 and 6,602,977, and International Publication No. 00 / 63243.

[0062] In some embodiments, the antibody - based backbone contains a binding site derived from an anticalin. Anticalins are functional antibody - mimetics derived from human lipocalins. Lipocalins are a family of natural binding proteins that bind and transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. The main structure of an anticalin is similar to that of wild - type lipocalin. The central element of this protein structure is an eight - stranded, antiparallel β - barrel structure that supports four loops at its open ends. These loops form the natural binding site of the lipocalin and can be reshaped in vitro by extensive amino - acid substitutions to create new binding specificities. Anticalins have functional properties similar to those of antibodies in that they have high affinity and specificity for their ligands and rapid binding kinetics. Anticalins are described, for example, in U.S. Patent No. 7,723,476.

[0063] In some embodiments, the antibody - based backbone comprises a binding site derived from a cysteine - rich polypeptide. The cysteine - rich domain does not form an α - helix structure, a β - sheet structure, or a β - barrel structure in some embodiments. In some embodiments, disulfide bonds facilitate the folding of the domain into a three - dimensional structure. In some embodiments, the cysteine - rich domain has at least two disulfide bonds, such as at least three disulfide bonds. An exemplary cysteine - rich polypeptide is the A - domain protein. The A - domain (sometimes called a "complement - like repeat") has about 30 - 50 or 30 - 65 amino acids. In some embodiments, the domain comprises about 35 - 45 amino acids, and in some embodiments about 40 amino acids. Within 30 - 50 amino acids, there are about six cysteine residues. Among the six cysteines, disulfide bonds are typically found between the following cysteines: C1 and C3, C2 and C5, C4 and C6. The A - domain constitutes a ligand - binding moiety. The cysteine residues of the domain form disulfide bonds to form a compact, stable, and functionally independent moiety. Clusters of these repeats create a ligand - binding domain, and discriminative clustering can confer specificity with respect to binding to a ligand. Exemplary proteins containing the A - domain include, for example, complement components (such as C6, C7, C8, C9, and factor I), serine proteases (such as enteropeptidase, matriptase, and kallikrein), transmembrane proteins (such as ST7, LRP3, LRP5, and LRP6), and endocytosis receptors (such as sortilin - related receptor, LDL receptor, VLDL receptor, LRP (LDL - receptor - related protein) 1, LRP2, and ApoER2). Methods for generating A - domain proteins with desired binding specificities are disclosed, for example, in WO 02 / 088171 and WO 04 / 044011.

[0064] In some embodiments, the antibody - independent scaffold comprises a binding site derived from a repeat protein. A repeat protein is a protein that contains contiguous copies, i.e., repeats, of a small (e.g., from about 20 to about 40 amino acid residues) structural unit that stack to form a continuous domain. The repeat protein may be modified to fit a particular target binding site by adjusting the number of repeats within the protein. Exemplary repeat proteins include designed ankyrin repeat proteins (i.e., DARPins) (see, e.g., Binz et al., Nat. Biotechnol., 22: 575 - 582 (2004)) or leucine - rich repeat proteins (i.e., LRRPs) (see, e.g., Pancer et al., Nature, 430: 174 - 180 (2004)).

[0065] In some embodiments, DARPins are used as an antibody - independent scaffold. As used herein, the term "DARPin" (an acronym for designed ankyrin repeat proteins) refers to antibody - mimetic proteins that typically exhibit binding to highly specific and high - affinity target proteins. DARPins were first derived from natural ankyrin proteins. In some embodiments, DARPins contain three, four, or five repeat motifs of ankyrin proteins. In some embodiments, one unit of ankyrin repeat consists of 30 - 34 amino acid residues and functions to mediate protein - protein interactions. In some embodiments, each ankyrin repeat exhibits a helix - turn - helix conformation, and a series of such tandem repeats are aligned and packed in a nearly linear fashion to form a helix - turn - helix bundle connected by relatively mobile loops. In some embodiments, the overall structure of the ankyrin repeat protein is stabilized by hydrophobic and hydrogen - bonding interactions within and between the repeats. The repetitive and extensible nature of the ankyrin repeats provides the molecular basis for the unique characteristics of ankyrin repeat proteins in terms of protein stability, folding and unfolding, and binding specificity. For DARPins with four or five repeats, the molecular weight of the DARPin domain can be approximately 14 or 18 kDa, respectively. DARPins are described, for example, in U.S. Patent No. 7,417,130. In some embodiments, the tertiary structure of the ankyrin repeat unit is composed of two antiparallel α - helices followed by a β - hairpin and ends with a loop that connects the repeat unit to the next repeat unit. Domains constructed from ankyrin repeat units can be formed by stacking the repeat units into an extended and curved structure.LRRP binding sites form part of the adaptive immune system of sea lampreys and other jawless fishes, and they are similar to antibodies in that they are formed by recombination of a series of leucine-rich repeat genes during lymphocyte maturation. Methods for making Darpins or LRRP binding sites are described in WO 02 / 20565 and WO 06 / 083275.

[0066] In some embodiments, the antibody - independent scaffold comprises a binding site derived from a small disulfide - bonded protein scaffold such as a Src homology domain (e.g., SH2 domain or SH3 domain), PDZ domain, β - lactamase, high - affinity protease inhibitor, or scorpion venom. Methods for generating binding sites from these molecules are disclosed in the art. See, for example, Panni et al., J. Biol. Chem., 277: 21666 - 21674 (2002), Schneider et at, Nat. Biotechnol., 17: 170 - 175 (1999); Legendre et al., Protein Sci., 11:1506 - 1518 (2002); Stoop et al., Nat. Biotechnol., 21: 1063 - 1068 (2003); and Vita et al., PNAS, 92: 6404 - 6408(1995). Further binding sites can be derived from binding domains selected from the group consisting of EGF (epidermal growth factor) - like domains, kringle domains, PAN domains, Gla domains, SPCR domains, kunitz / bovine pancreatic trypsin inhibitor domains, Kazal - type serine protease inhibitor domains, trefoil (P - type) domains, von Willebrand factor type C domains, anaphylatoxin - like domains, CUB domains, thyroglobulin type I repeats, LDL receptor class A domains, sushi domains, link domains, thrombospondin type I domains, immunoglobulin - like domains, C - type lectin domains, MAM domains, von Willebrand factor type A domains, somatomedin B domains, WAP - type four - disulfide - core domains, F5 / 8C - type domains, hemopexin domains, laminin - type EGF - like domains, C2 domains, binding domains derived from monomeric or trimeric tenectins, and other such domains known to those of skill in the art, as well as derivatives and / or variants thereof.Exemplary antibody-independent scaffolds and methods of making them can also be found in Stemmer et al., “Protein scaffolds and uses thereof”, U.S. Patent Publication No. 20060234299 (October 19, 2006) and Hey, et al., Artificial, Non-Antibody Binding Proteins for Pharmaceutical and Industrial Applications, TRENDS in Biotechnology, Vol. 23, No. 10, Table 2 and pages 514-522 (October 2005).

[0067] In some embodiments, the antibody - free backbone comprises a knotted domain. As used herein, the term "knotted domain" refers to a conserved protein domain that inhibits a specific protease, e.g., a serine protease. The knotted domain is relatively small, typically about 50 to 60 amino acids in length and has a molecular weight of about 6 kDa. The knotted domain typically has a basic charge and is characterized by the substitution of two, four, six, eight or more folded peptides that form disulfide bonds contributing to its compact and stable nature. For example, many knotted domains have six conserved cysteine residues that form three disulfide bonds. The disulfide - rich α / β - folded knotted domain may contain two, three (typical), or four or more disulfide bonds. The knotted domain has a pear - shaped structure, which is stabilized, for example, by three disulfide bonds and which contains a reactive - site region that features the major determinant P1 residue within a rigid conformation. These inhibitors competitively prevent the access of the target protein (e.g., a serine protease) to its physiologically relevant macromolecular substrate through the insertion of the P1 residue into the active - site cleft. The P1 residue within the protease - inhibitory loop provides the major specificity determinant and dictates much of the inhibitory activity that a particular knottin protein has against a target protease. Generally, the N - terminal side of the reactive site (P) is more energetically important than the C - terminal side of P. In most cases, lysine or arginine occupies the P1 position and inhibits proteases that cleave adjacent to such residues within the protein substrate. Other residues, particularly those within the inhibitory - loop region, contribute to the binding strength. Generally, about 10 - 12 amino - acid residues within the target protein and 20 - 25 residues within the protease are directly involved in the formation of a stable protease - inhibitory protein entity and provide a buried surface area of about 600 - 900 Å. By modifying the residues within the P - site and the surrounding residues, the knotted domain can be designed to target a selected protein. The knotted domain is described, for example, in U.S. Patent No. 6,057,287.

[0068] In some embodiments, the antibody - based scaffold is an affilin. Affilins are small antibody - mimicking proteins that are designed for specific affinity to proteins and small compounds. Novel affilins can be selected very rapidly from two libraries, each of which is based on a different human - derived scaffold protein. Affilins show no structural homology to immunoglobulin proteins. There are two commonly used affilin scaffolds, one being γ - crystallin, a human eye lens structural protein, and the other being a "ubiquitin" superfamily protein. Both human scaffolds are very small, show stability to high temperatures, and are largely resistant to pH changes and denaturing agents. This high stability is mainly due to the extended β - sheet structure of the proteins. Examples of γ - crystallin - derived proteins are described in WO 2001 / 04144, and examples of "ubiquitin - like" proteins are described in WO 2004 / 106368.

[0069] In some embodiments, the antibody - based scaffold is an affimer. Affimers are generated from large human extracellular receptor domain families by in vitro exon shuffling and phage display to create multi - domain proteins with binding and inhibitory properties. The linkage of multiple independent binding domains has been shown to create binding force, resulting in improved affinity and specificity compared to conventional single - epitope binding proteins. In some embodiments, an affimer consists of two or more peptide sequences, each 30 - 35 amino acids, connected by a spacer region peptide. The individual sequences are derived from the A domains of various membrane receptors and have a robust structure stabilized by disulfide bonds and calcium. Each A domain can bind to a specific epitope of a target protein. Combinations of domains that bind to different epitopes of the same protein enhance the affinity for this protein, an effect known as avidity (hence the name). Affimers with sub - nanomolar affinities for a wide variety of targets have been obtained. Alternatively, the domains can be directed against epitopes on different target proteins. Additional information regarding affimers can be found in U.S. Patent Application Publication Nos. 2006 / 0286603, 2006 / 0234299, 2006 / 0223114, 2006 / 0177831, 2006 / 0008844, 2005 / 0221384, 2005 / 0164301, 2005 / 0089932, 2005 / 0053973, 2005 / 0048512, 2004 / 0175756.

[0070] According to the present invention, the targeting moiety is not a protein tag. As used herein, the term "tag" refers to a chemical moiety, i.e., a nucleotide, oligonucleotide, polynucleotide, or an amino acid, peptide or protein or other chemical substance, which when added to another sequence confers additional utility or imparts properties useful particularly in detection or isolation to that sequence. According to the present invention, the targeting moiety does not contain histidine residues (e.g., 4 to 8 contiguous histidine residues) that are normally added to either the amino or carboxy terminus of a polypeptide to facilitate isolation of the protein by chelate metal chromatography. Alternatively, an amino acid sequence, peptide, protein or fusion pair that is reactive with a specific antibody molecule or other molecule that can be added to a protein to facilitate isolation of the protein by procedures such as such affinity chromatography or immunoaffinity chromatography (e.g., flag epitope, c-myc epitope, transmembrane epitope of influenza A virus hemagglutinin protein, protein A, cellulose binding domain, calmodulin binding protein, maltose binding protein, chitin binding domain, glutathione S-transferase, etc.) is not considered a targeting moiety according to the present invention.

[0071] According to the present invention, the targeting moiety is not a fluorescent protein. As used herein, the term "fluorescent protein" refers to fluorescent proteins produced by various organisms such as Renilla and Aequorea that can fluoresce in various visible colors, as well as modified forms of these natural fluorescent proteins. Generally, the terms "fluorescent protein" and "GFP (green fluorescent protein)" are sometimes used interchangeably; however, other specific colors may sometimes be mentioned. Since the system is strictly for memory aid, for example, RFP refers to red fluorescent protein, YFP refers to yellow fluorescent protein, BFP refers to blue fluorescent protein, and so on. Visible light of a wide range of wavelengths is emitted by these proteins depending on the specific modifications made.

[0072] In some embodiments, the targeting moiety is not selected from the group consisting of biotin carboxyl carrier protein (BCCP), glutathione-S-transferase (GST), green fluorescent protein (GFP), maltose binding protein (MBP), Nus-tag (NusA protein), thioredoxin (Trx), Fc-tag (immunoglobulin Fc domain), such as rabbit IgG, mouse IgG, goat IgG, rat IgG, bovine IgG, or canine IgG, carbohydrate binding module (CBM), yellow fluorescent protein, mCherry, β-galactosidase, digoxigenin, biotin, small ubiquitin-like modifier (SUMO), Avi-tag, calmodulin-tag, polyglutamate tag, E (epitope)-tag, Flag-tag, HA (hemagglutinin)-tag, His-tag, Myc-tag, S-tag, streptavidin binding peptide tag, streptavidin-tag, tetracysteine-tag, V5 tag, vesicular stomatitis virus-tag, Xpress tag, Isopeptag, and Spy tag.

[0073] In some embodiments, the targeting moiety has binding affinity for a cell surface molecule of the target cell. In some embodiments, the cell surface molecule is a receptor. In some embodiments, the cell surface molecule is a transmembrane protein. In some embodiments, the targeting moiety is specific for a target protein antigen, carbohydrate antigen, or glycosylated protein. For example, an antibody can target the glycosylation moiety of an antigen that is preferentially produced by transformed (neoplastic or cancerous) cells, infected cells, and the like (cells associated with disorders related to other immune system).

[0074] A partial list of suitable mammalian cells that can be targeted by the targeting moiety of the present invention includes, but is not limited to, blood cells, myoblasts, bone marrow cells, peripheral blood cells, umbilical cord blood cells, cardiomyocytes (and their precursors), chondrocytes (cells of cartilage), dendritic cells, fetal neural tissue, fibroblasts, hepatocytes (liver cells), pancreatic islet cells, keratinocytes (skin cells), and stem cells.

[0075] In some embodiments, the targeting moiety is particularly suitable for targeting immune cell populations. Recognized immune cell populations include lymphocytes such as B lymphocytes (Fc receptor, MHC class II, CD19 positive, CD21 positive), helper T lymphocytes (CD3 positive, CD4 positive, CD8 negative), cytotoxic T lymphocytes (CD3 positive, CD4 negative, CD8 positive), natural killer cells (CD16 positive), mononuclear phagocytes (including monocytes), neutrophils and macrophages, and dendritic cells. Other cell types of potential interest include eosinophils and basophils.

[0076] In some embodiments, the targeting moiety is particularly suitable for targeting hematopoietic cell populations.

[0077] As used herein, the term "hematopoietic cell" generally refers to blood cells derived from both the myeloid and lymphoid lineages. In particular, the term "hematopoietic cell" includes undifferentiated or poorly differentiated cells such as hematopoietic stem cells and progenitor cells, and differentiated cells such as T lymphocytes, B lymphocytes or dendritic cells. Preferably, the hematopoietic cells are selected from the group consisting of hematopoietic stem cells, CD34 positive progenitor cells, particularly peripheral blood CD34 positive cells, CD34 positive early progenitor cells, CD19 positive B progenitor cells, CD13 positive myeloid progenitor cells, T lymphocytes, B lymphocytes, monocytes, dendritic cells, cancerous B cells, particularly B cell chronic lymphocytic leukemia (BOLL) cells, and marginal zone lymphoma (MZL) B cells, and thymocytes.

[0078] Accordingly, in some embodiments, the targeting moiety is specific for an immunocyte regulatory molecule such as CD3, CD4, CD8, CD25, CD28, CD26, CTLA-4 (cytotoxic T lymphocyte antigen 4), ICOS (inducible T cell co-stimulator) or CD11a. Other suitable antigens include antigens associated with immune cells such as T cell-associated molecules such as TCR (T cell receptor) / CD3 or CD2; natural killer cell-associated targets such as NKG2D, FcγRIIIa (CD16), CD38, CD44, CD56 or CD69; granulocyte-associated targets such as FcγRI (CD64), FcαRI (CD89), and CR3 (CD11b / CD18); monocyte / macrophage-associated targets (e.g., FcγRI (CD64), FcαRI (CD89), CD3 (CD11b / CD18), or mannose receptor; dendritic cell-associated targets such as FcγRI (CD64) or mannose receptor; and erythrocyte-associated targets such as CRI (CD35), but are not limited thereto.

[0079] In some embodiments, the targeting moiety is particularly suitable for targeting malignant cell populations. Thus, in some embodiments, the targeting moiety is specific for a cancer antigen. Known cancer antigens include, but are not limited to, breast, ovarian, and colorectal tumor cells, and c-erbB-2 (erbB-2 is also known as c-neu or HER-2), which is particularly associated with neuroblastoma, lung cancer, thyroid cancer, pancreatic cancer, prostate cancer, kidney cancer, and cancers of the gastrointestinal tract. Another class of cancer antigens are the fetal proteins with non-enzymatic functions. These antigens are found in a wide variety of neoplasms and are often referred to as "tumor-associated antigens". Carcinoembryonic antigen (CEA), and alpha-fetoprotein (AFP) are two examples of such cancer antigens. AFP levels are elevated in patients with hepatocellular carcinoma: 69% of liver cancer patients express high levels of AFP in their sera. CEA is a 200 kDa serum glycoprotein found in adenocarcinoma of the colon, as well as cancers of the lung and urogenital tract. Yet another class of cancer antigens are antigens specific to a particular tumor, sometimes referred to as "tumor-specific antigens", such as heat shock proteins (e.g., hsp70 or hsp90 proteins) derived from a particular type of tumor. Other targets include the MICA / B ligands of NKG2D. These molecules are expressed on many types of tumors but are not normally expressed on healthy cells.Additional specific examples of cancer antigens include epithelial cell adhesion molecule (Ep-CAM / TACSTD1), mesothelin, tumor-associated glycoprotein 72 (TAG-72), gp100, Melan-A, MART-1, KDR, RCAS1, MDA7, cancer-associated viral vaccines (such as human papillomavirus antigens), prostate-specific antigen (PSA, PSMA), RAGE (kidney antigen), CAMEL (antigen recognized by cytotoxic T lymphocytes on malignant melanoma), CT antigens (such as MAGE-B5, -B6, -C2, -C3, and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin antigens (such as MUC1, mucin-CA125, etc.), cancer-associated ganglioside antigens, tyrosinase, gp75, C-myc, Mart1, Melan A, MUM-1, MUM-2, MUM-3, HLA-B7, Ep-CAM, tumor-derived heat shock proteins, etc. (see also, for example, Acres et al., Curr Opin Mol Ther 2004 February, 6:40-7; Taylor-Papadimitriou et al., Biochim Biophys Acta. 1999 Oct. 8; 1455(2-3):301-13; Emens et al., Cancer Biol Ther. 2003 July-August; 2(4 Suppl 1):S161-8; and Ohshima et al., Int J Cancer. 2001 Jul. 1; 93(1):91-6). Other exemplary cancer antigen targets include CA195 tumor-associated antigen-like antigens (see, for example, U.S. Patent No. 5,324,822) and squamous cell carcinoma-like antigens contained in female urine (see, for example, U.S. Patent No. 5,306,811), and the breast cell carcinoma antigen described in U.S. Patent No. 4,960,716.

[0080] In some embodiments, the targeting moiety has a binding affinity for a pancreatic antigen. In some embodiments, the targeting moiety is specific for the LP1R (glucagon-like peptide 1 receptor) receptor or for the IA-2 receptor found on type 1 diabetic pancreatic cells.

[0081] In some embodiments, the targeted moiety is CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD5, CD6, CD7, CD8 alpha, CD8 beta, CD9, CD10, CD11a, CD11b, CD11c, CDw12, CD13, CD14, CD15u, CD16a, CD16b, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD44R, CD45, CD46, CD47R, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CDw93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CDw113, CD114, CD115, CD116, CD117, CD118, CDw119, CD120a, CD120b, CD121a, CDw121b, CD122, CD123, CD124, CDw125, CD126, CD127, CDw128a, CDw128b, CD129, CD130, CD131, CD132, CD133, CD134, CD135, CDw136, CDw137, CD138, CD139, CD140a, CD140b, CD141CD142, CD143, CD144, CDw145, CD146, CD147, CD148, CDw149, CD150, CD151, CD152, CD153, CD154, CD155, CD156a, CD156b, CDw156C, CD157, CD158, CD159a, CD159c, CD160, CD161, CD162, CD162R, CD163, CD164, CD165, CD166, CD167a, CD168, CD169, CD170, CD171, CD172a, CD172b, CD172g, CD173, CD174, CD175, CD175s, CD176, CD177, CD178, CD179a, CD179b, CD180, CD181, CD182, CD183, CD184, CD185, CDw186, CD191, CD192, CD193, CD195, CD196, CD197, CDw198, CDw199, CDw197, CD200, CD201, CD202b, CD203c, CD204, CD205, CD206, CD207, CD208, CD209, CDw210, CD212, CD213a1, CD213a2, CDw217, CDw218a, CDw218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235b, CD235ab, CD236, CD236R, CD238, CD239, CD240CE, CD240D, CD240DCE, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD252, CD253, CD254, CD256, CD257, CD258, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD271, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD289, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a, CD300c, CD300e, CD301, CD302, CD303, CD304, CD305It has binding affinity for CD (cluster of differentiation) molecules selected from the group consisting of CD306, CD307, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CDw325, CD326, CDw327, CDw328, CDw329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CDw338, and CD339.

[0082] In some embodiments, the targeting moiety has binding affinity for cell surface molecules of the hematopoietic system. In some embodiments, the targeting moiety is selected from the group consisting of 2B4 / CD244 / SLAMF4, ABCG2, aldehyde dehydrogenase 1-A1 / ALDH1A1, BMI-1, C1qR1 / CD93, CD34, CD38, CD44, CD45, CD48 / SLAMF2, CD90 / Thy1, CD117 / c-kit, CD133, CDCP1, CXCR4, endoglin / CD105, EPCR, erythropoietin R, ESAM, EVI-1, integrin α6 / CD49f, SLAM / CD150, VCAM-1 / CD106, and VEGFR2 / KDR / Flk-1 and has binding affinity for cell surface molecules.

[0083] In some embodiments, the targeting moiety is stem cell factor (also known as SCF, kit ligand (KITL)) that binds to the CD117 (c-kit) receptor. In some embodiments, thus, the targeting moiety comprises an amino acid sequence having at least 70% identity to the amino acid sequence as set forth in SEQ ID NO: 4.

[0084]

Chemical formula

[0085] In some embodiments, the targeting moiety is a single-chain fragment variant (scFv) against the CD133 receptor ("scFvCD133"). In some embodiments, thus, the targeting moiety comprises an amino acid sequence having at least 70% identity to the amino acid sequence as set forth in SEQ ID NO: 5.

[0086]

Chemical formula

[0087] In some embodiments, the targeting moiety is a DARPin against CD4 ("DARPinCD4"). In some embodiments, thus, the targeting moiety comprises an amino acid sequence having at least 70% identity to the amino acid sequence as set forth in SEQ ID NO: 6.

[0088]

Chemical formula

[0089] In some embodiments, the targeting moiety is a single-chain fragment variant (scFv) against CD8 ("scFvCD8"). In some embodiments, thus, the targeting moiety comprises an amino acid sequence having at least 70% identity to the amino acid sequence as set forth in SEQ ID NO: 7.

[0090]

Chemical formula

[0091] In some embodiments, the targeting moiety is a single-chain fragment variant (scFv) directed against the IA-2 receptor ("scFvIA-2"). In some embodiments, thus, the targeting moiety comprises an amino acid sequence having at least 70% identity to the amino acid sequence as set forth in SEQ ID NO: 8.

[0092]

Chemical formula

[0093] In some embodiments, the targeting moiety is GLP1 ("GLP1"). In some embodiments, therefore, the targeting moiety comprises an amino acid sequence having at least 70% identity to the amino acid sequence as set forth in SEQ ID NO: 9.

[0094]

Chemical Formula

[0095] Fusion: In some embodiments, the C-terminus of the targeting moiety is fused to the N-terminus of the synctin-1 polypeptide.

[0096] In some embodiments, the synthitin-1 polypeptide and the targeting moiety of the invention are fused directly to each other (i.e., without using a linker) or via a linker. The linker is typically a linker peptide and would be selected according to the invention to enable the attachment of the polypeptide to the targeting moiety. Suitable linkers will be apparent to those skilled in the art based on the disclosure herein and, optionally, after some limited routine experimentation. Suitable linkers are described herein and include, for example but not limited to, amino acid sequences which preferably have a length of two or more amino acids. Typically the linker has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. However, the upper limit is not critical and is selected for reasons of convenience, for example, in the production of biopharmaceuticals of such fusion proteins. The linker sequence may be a natural sequence or a non-natural sequence. When used for therapeutic purposes, the linker is preferably non-immunogenic in the subject to whom the fusion protein of the invention is administered. One group of useful linker sequences are linkers derived from the hinge region of heavy chain antibodies as described in WO 96 / 34103 and WO 94 / 04678. Another example is a poly-alanine linker sequence such as alanine-alanine-alanine. Further preferred examples of linker sequences are glycine / serine linkers of various lengths such as (gly4ser)3, (gly4ser)4, (gly4ser), (gly3ser), gly3 and (gly3ser2)3.

[0097] In some embodiments, the synthitin-1 polypeptide is fused to 2, 3, 4, 5, 6, 7 or 8 targeting moieties, which may be fused directly to each other or indirectly via a linker.

[0098] In some embodiments, the fusion protein comprises a signal peptide sequence. As used herein, the term "signal peptide" has its general meaning in the art and refers to a pre-peptide that exists as an N-terminal peptide on a precursor form of a protein. The function of the signal peptide is to facilitate the translocation of the expressed polypeptide attached thereto into the endoplasmic reticulum. The signal peptide is usually cleaved and removed during the course of this process. The signal peptide may be heterologous or homologous to the organism used for the production of the polypeptide.

[0099] In some embodiments, the signal peptide is the synaptopodin-1 (SYN) signal sequence (SS). In some embodiments, the signal peptide consists of an amino acid sequence in the range of the amino acid residue at position 1 to the amino acid residue at position 20 of SEQ ID NO: 1.

[0100] In some embodiments, the C-terminus of the signal peptide is fused to the N-terminus of the targeting moiety.

[0101] In some embodiments, therefore, the synaptopodin-1 fusion protein of the present invention comprises, in the following order, a synaptopodin-1 (SYN) signal sequence (SS), a targeting moiety, and a synaptopodin-1 polypeptide.

[0102] In some embodiments, therefore, the synaptopodin-1 fusion protein of the present invention comprises a tag. In some embodiments, the tag is an HA epitope and consists of an amino acid sequence as set forth in SEQ ID NO: 40.

[0103]

Chemical formula

[0104] Specific embodiments of the syncytin-1 fusion protein: In some embodiments, the syncytin-1 fusion protein of the present invention consists of an amino acid sequence as set forth in SEQ ID NO: 10 ("SCF-SYN"), SEQ ID NO: 11 ("scFvCD133-SYN"), SEQ ID NO: 12 ("DARPinCD4-SYN"), SEQ ID NO: 13 ("scFVCD8-SYN"), or SEQ ID NO: 14 ("scFVIA-2-SYN"), SEQ ID NO: 15 ("GLP1-SYN").

[0105]

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

[0106] In some embodiments, the syncytin-1 fusion protein of the present invention consists of an amino acid sequence as set forth in SEQ ID NO: 16 ("SCF-SYN480"), SEQ ID NO: 17 ("scFvCD133-SYN480"), SEQ ID NO: 18 ("DARPinCD4-SYN480"), SEQ ID NO: 19 ("scFVCD8-SYN480"), or SEQ ID NO: 20 ("scFVIA-2-SYN480"), or SEQ ID NO: 21 ("GLP1-SYN480").

[0107]

Chemical Structure

Chemical Structure

[0108] The nucleic acids, vectors and host cells of the present invention: A further object of the present invention relates to a polynucleotide encoding the syncytin-1 fusion protein of the present invention.

[0109] Typically, the polynucleotide is a DNA molecule or an RNA molecule, which may be contained in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector.

[0110] Thus, a further object of the present invention relates to a vector containing the polynucleotide of the present invention.

[0111] As used herein, the terms "vector", "cloning vector" and "expression vector" mean a vehicle by which a DNA sequence or an RNA sequence (e.g., a foreign gene) can be introduced into a host cell, thereby transforming the host and promoting the expression (e.g., transcription and translation) of the introduced sequence.

[0112] Such a vector may contain regulatory sequences, such as a promoter, enhancer, terminator, etc., that cause or direct the expression of the antibody upon administration to a subject.

[0113] As used herein, the term "regulatory sequence" refers to a nucleic acid sequence (e.g., a DNA sequence) that is recognized by the synthetic machinery of a cell or an introduced synthetic machinery and is required to initiate specific transcription of a polynucleotide sequence, thereby enabling the expression of a gene product operably linked to the promoter sequence / regulatory sequence. In some cases, this sequence can be a core promoter sequence, and in other cases, this sequence can also include enhancer sequences and other regulatory elements required for the expression of the gene product. The promoter sequence / regulatory sequence can, for example, be one that expresses a gene product in a tissue-specific manner.

[0114] As used herein, the term "operably linked" or "transcriptional control" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, such that the latter is expressed. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences may or may not be contiguous with each other, and for example, when it is necessary to connect two protein-coding sequences, they are within the same reading frame.

[0115] Examples of promoters and enhancers used in expression vectors for animal cells include the early promoter and enhancer of SV40, the LTR promoter and enhancer of Moloney murine leukemia virus, the promoter and enhancer of immunoglobulin H chain, and the like. Any expression vector for animal cells may be used as long as the gene encoding the human antibody constant region can be inserted and expressed. Examples of suitable vectors include pAGE107, pAGE103, pHSG274, pKCR, pSG1βd2-4, and the like. Other examples of plasmids include replicating plasmids containing an origin of replication, or integrating plasmids, such as pUC, pcDNA, pBR, and the like. Other examples of viral vectors include adenoviral vectors, retroviral vectors, herpesviral vectors, and adeno-associated viral vectors. Such recombinant viruses can be produced by techniques known in the art, such as transfection of packaging cells, or transient transfection using helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GP envelope-positive cells, 293 cells, and the like. Detailed protocols for producing such replication-deficient recombinant viruses can be found, for example, in International Publication No. 95 / 14785, International Publication No. 96 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6,013,516, U.S. Patent No. 4,861,719, U.S. Patent No. 5,278,056, and International Publication No. 94 / 19478.

[0116] A further object of the present invention relates to host cells transfected, infected, or transformed with the polynucleotides and / or vectors described in the present invention.

[0117] As used herein, the term "transformation" means the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA sequence or RNA sequence into a host cell, whereby the host cell will express the introduced gene or sequence to produce the desired sequence, typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA is "transformed."

[0118] Using the polynucleotides of the present invention, the syncytin-1 fusion protein of the present invention can be produced in a suitable expression system. Common expression systems include E. coli host cells and plasmid vectors, insect host cells, and baculovirus vectors, as well as mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces, or Saccharomyces yeast. Mammalian host cells include Chinese hamster ovary (CHO) cells, e.g., dhfr-negative CHO cells used with a dihydrofolate reductase (DFHR) selection marker ( 1 described therein), CHOK1 dhfr-positive cell lines, NS0 myeloma cells, COS cells, and SP2 cells, e.g., GS CHO cell lines that together include a GS (glutamine synthetase) Xceed™ gene expression system (Lonza), or HEK cells.

[0119] The present invention also relates to a method for producing a recombinant host cell expressing the syncytin-1 fusion protein of the present invention, the method comprising: (i) introducing, in vitro or ex vivo, a recombinant polynucleotide or vector as described above into a competent host cell; (ii) culturing the resulting recombinant host cell in vitro or ex vivo; and (iii) optionally, selecting cells that express and / or secrete the antibody. Such recombinant host cells can be used for the production of the antibody of the present invention.

[0120] Thus, host cells as disclosed herein are particularly suitable for producing the syncytin-1 fusion protein of the invention. Indeed, when recombinant expression is introduced into mammalian host cells, the polypeptide is produced by culturing the host cells for a sufficient time for expression of the antibody within the host cell and, optionally, secretion of the antibody into the culture medium in which the host cells are growing. The antibody can be recovered and purified, for example, from the culture medium after their secretion, using standard protein purification methods.

[0121] Particles functionalized with syncytin-1 fusion protein: The present invention relates to particles functionalized with the syncytin-1 fusion protein of the invention.

[0122] The syncytin-1 fusion protein of the invention is particularly suitable for enabling i) fusion of the particles to the target cell membrane by the syncytin-1 polypeptide and ii) specific targeting to the target cell by the targeting moiety(ies).

[0123] Any particles described in the art for intracellular cargo delivery may be used. Such nanoparticles include, for example, liposomes and micelles, nanospheres or nanoparticles, nanotubes, nanocrystals, hydrogels, carbon-based nanoparticles, etc. In addition to the above, biological particles can also be used as particles according to the present invention. Examples of these include virus particles (which usually have a size of 20 nm to 300 nm), virus-like particles (e.g., particles composed only of the shell of virus particles), HDL and LDL nanoparticles (which usually have a size of 5 to 30 nm), self-assembling nanoparticles, bacterial particles, and cells. Any such particles that can be functionalized with the syncytin-1 fusion protein can be used according to the present invention. The particles can act as carriers for carrying one or more cargo(es) and can bind to the target cell and release the cargo(es) into the cell.

[0124] In some embodiments, the particles are nanoparticles having an average diameter of 1 to 2000 nm, such as 10 to 500 nm, or 10 to 200 nm. For most nanoparticles, the size of the nanoparticle is the distance between the two most distant points within the nanoparticle. The size of the nanoparticle can be determined by various methods such as dynamic light scattering (DLS), small angle X-ray scattering (SAXS), scanning mobility particle sizer (SMPS), scanning electron microscope (SEM), transmission electron microscope (TEM), etc. (Orts-Gil, G., K. Natte, et al. (2011), Journal of Nanoparticle Research 13(4): 1593-1604; Alexandridis, P. and B. Lindman (2000), Amphiphilic Block Copolymers: Self-Assembly and Applications, Elsevier Science; Hunter, R. J. and L. R. White (1987). Foundations of colloid science, Clarendon Press.).

[0125] In other embodiments, the nanoparticles comprise at least one core with one or more polymers, or copolymers thereof, such as one or more dextrans, carboxymethyl dextran, chitosan, trimethyl chitosan, polyvinyl alcohol (PVA), polyanhydrides, polyacrylates, polymethacrylates, polyacrylamides, cellulose, hydromellose, starch, dendrimers, polyamino acids, polyethylene glycol, copolymers of polyethylene glycol-propylene glycol, aliphatic polyesters such as polylactic acid (PLA), poly(glycolic acid), and copolymers thereof such as polylactic acid-glycolic acid copolymer (PLGA), or poly(ε-caprolactone). Other suitable polymers may include polyamino acids selected from the group consisting of poly(g-glutamic acid), poly(a-aspartic acid), poly(e-lysine), poly(a-glutamic acid), poly(a-lysine), poly-asparagine, or derivatives thereof, and mixtures thereof. Generally, the surface of the nanoparticles may also be functionalized or coated to provide desired physical characteristics such as solubility, biocompatibility, etc., and to facilitate chemical bonding with other biomolecules such as the syncytin-1 fusion protein of the present invention. In some embodiments, the surface of the nanoparticles may be functionalized by incorporating one or more chemical linkers such as, but not limited to, carboxyl groups, amine groups, carboxyl / amine groups, hydroxyl groups, polymers such as silanes, dextrans, or polyethylene glycol or derivatives thereof.

[0126] In some embodiments, the particles are virus particles, more specifically virus-like particles pseudotyped with the syncytin-1 fusion protein of the present invention.

[0127] In some embodiments, the virus particles of the present invention are virus particles having an envelope.

[0128] In some embodiments, the virus particles of the present invention comprise one or more viral structural proteins.

[0129] Preferred structural proteins are the Gag proteins of the Retroviridae family. Particularly preferred as a structural protein is the protein corresponding to the HIV-1 gag gene. This is because the production and construction of Gag virus particles are very efficient and these virus particles have low cytotoxicity. The gag gene of the lentivirus HIV-1 encodes the polyprotein Pr55Gag, which is a precursor of the structural proteins p17 matrix (MA), p24 capsid (CA), p7 nucleocapsid (NC) and p6. Gag is cleaved into individual proteins within the mature infectious virions of HIV-1, however, within Gag virus particles, Gag remains as a single protein because the required viral protease is absent. The mechanisms underlying Gag virus particle formation and the proteins involved therein have been fully considered in the prior art (see Carriere et al., 1995 J. Virol. 69:2366-2377; Wilk et al., 2001 J. Virol. 75:759-77130; US2002 / 0052040; Chazal and Gerlier, 2003 Microbiol. Molec. Biol. Rev. 67:226-237; Hong and Boulanger, 1993 J, Virol. 67:2787-2798; Royer et al., 1992 J. Virol. 66:3230-3235; Spearman et al, 1994 J. Virol. 68:3232-3242 and the references cited therein).

[0130] Thus, in some cases, the virus particles of the present invention contain a Gag protein, most preferably a Gag protein derived from a virus selected from the group comprising Rous sarcoma virus (RSV), feline immunodeficiency virus (FIV), simian immunodeficiency virus (SIV), Moloney leukemia virus (MLV) and human immunodeficiency virus (HIV-1 and HIV-2), particularly human immunodeficiency virus type 1 (HIV-1).

[0131] As will be readily understood by those skilled in the art, the virus particles used in accordance with the present invention can be selected from the group comprising Moloney murine leukemia virus-derived vector particles, bovine immunodeficiency virus-derived particles, simian immunodeficiency virus-derived vector particles, feline immunodeficiency virus-derived vector particles, human immunodeficiency virus-derived vector particles, equine infectious anemia virus-derived vector particles, caprine arthritis encephalitis virus-derived vector particles, baboon endogenous virus-derived vector particles, rabies virus-derived vector particles, influenza virus-derived vector particles, norovirus-derived vector particles, respiratory syncytial virus-derived vector particles, hepatitis A virus-derived vector particles, hepatitis B virus-derived vector particles, hepatitis E virus-derived vector particles, Newcastle disease virus-derived vector particles, Norwalk virus-derived vector particles, parvovirus-derived vector particles, papillomavirus-derived vector particles, yeast retrotransposon-derived vector particles, measles virus-derived vector particles, and bacteriophage-derived vector particles.

[0132] In some embodiments, the viral particles of the present invention are retrovirus-derived particles. In some embodiments, the viral particles of the present invention are lentivirus-derived particles. Lentiviruses belong to the retrovirus family and have the unique ability to infect non-dividing cells. Such lentiviruses can be selected from bovine immunodeficiency virus, simian immunodeficiency virus, feline immunodeficiency virus, human immunodeficiency virus, equine infectious anemia virus, and caprine arthritis encephalitis virus. For the preparation of Moloney murine leukemia virus-derived vector particles, those skilled in the art can refer particularly to the methods disclosed by Sharma et al. (1997, Proc Natl Acad Sci USA, Vol. 94: 10803-10808), Guibingua et al. (2002, Molecular Therapy, Vol. 5(n°5): 538-546). Moloney murine leukemia virus-derived (MLV-derived) vector particles can be selected from the group comprising MLV-A-derived vector particles and MLV-E-derived vector particles. For the preparation of bovine immunodeficiency virus-derived vector particles, those skilled in the art can refer particularly to the methods disclosed by Rasmussen et al. (1990, Virology, Vol. 178(n°2): 435-451). For the preparation of simian immunodeficiency virus-derived vector particles, those skilled in the art can refer particularly to the methods disclosed by Mangeot et al. (2000, Journal of Virology, Vol. 71(n°18): 8307-8315), Negre et al. (2000, Gene Therapy, Vol. 7: 1613-1623), Mangeot et al. (2004, Nucleic Acids Research, Vol. 32 (n° 12), e102).For the preparation of feline immunodeficiency virus-derived vector particles, one of ordinary skill in the art may refer, in particular, to the methods disclosed by Saenz et al. (2012, Cold Spring Harb Protoc, (1): 71-76; 2012, Cold Spring Harb Protoc, (1): 124-125; 2012, Cold Spring Harb Protoc, (1): 118-123). For the preparation of human immunodeficiency virus-derived vector particles, one of ordinary skill in the art may refer, in particular, to the methods disclosed by Jalaguier et al. (2011, PlosOne, Vol. 6(n°11), e28314), Cervera et al. (J Biotechnol, Vol. 166(n°4): 152-165), Tang et al. (2012, Journal of Virology, Vol. 86(n°14): 7662-7676). For the preparation of equine infectious anemia virus-derived vector particles, one of ordinary skill in the art may refer, in particular, to the method disclosed by Olsen (1998, Gene Ther, Vol. 5(n°11): 1481-1487). For the preparation of caprine arthritis encephalitis virus-derived vector particles, one of ordinary skill in the art may refer, in particular, to the method disclosed by Mselli-Lakhal ety al. (2006, J Virol Methods, Vol. 136(n°1-2): 177-184).

[0133] In some embodiments, capsids derived from mammalian endogenous retroviruses are used. Among these are homologs of LTR retrotransposons and the capsid proteins (known as Gag) of retroviruses. In recent years, several mammalian Gag homologs that form virus particles have been identified (Campillo et al. 2006 PMID: 16979784 (computation analysis); Pastuzyn et al. 2018 PMID: 29328916 (ARC); Ashley et al. 2018 PMID: 29328915 (ARC) and Abed et al. 2019 PMID: 30951545 (10)). For example, Arc, MOAP1, ZCCHC12, RTL1, PNMA3, PNMA5, PNMA6a, and PEG10 self-associate to form capsid-like particles and can thus be used for the formation of the virus particles of the present invention.

[0134] Accordingly, in some embodiments, the viral structural protein is PEG10. As used herein, the term "PEG10" refers to a protein derived from a retrotransposon, which is encoded by the PEG10 gene. In particular, PEG10 has a CCHC-type zinc finger domain containing sequences characteristic of the gag proteins of most retroviruses. Exemplary amino acid sequences of PEG10 are shown by SEQ ID NO: 22 (isoform 1) or SEQ ID NO: 23 (isoform 2).

[0135]

Chemical formula

Chemical formula

[0136] Thus, in some embodiments, the viral structural protein has an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23.

[0137] Methods for generating viral particles are well known in the art. Typically, vectors for expressing the required viral structural proteins and the syncytin-1 fusion protein of the present invention are used to express nucleic acid sequences within the desired packaging cells. In particular, vectors for expressing the required proteins or nucleic acids include open reading frames that are placed under the control of regulatory elements and are functional within the packaging cells in which their expression is sought. In particular, these vectors include open reading frames and polyadenylation sequences that are placed under the control of appropriate promoter sequences for each protein or nucleic acid to be expressed. As is well known in the art, nucleic acid vectors are introduced into packaging cells by any of a variety of techniques (e.g., calcium phosphate co-precipitation, lipofection, electroporation). The viral proteins produced by the packaging cells mediate the insertion of the viral protein(s), the syncytin-1 fusion protein of the present invention, and optionally cargo (e.g., polypeptide or polynucleotide) into virus-derived particles, which are then released into the culture supernatant. The nucleic acid vectors used may be derived from retroviruses (e.g., lentiviruses). For example, a retroviral vector suitable for the production of virus-derived particles described herein enables (1) the formation of a packaging cell line that produces virus particles pseudotyped with the syncytin-1 protein of the present invention by transfection of a packaging vector and an envelope vector into a host cell, and (2) the packaging of cargo (e.g., Cas protein and optionally CRISPR guide RNA(s)) into virus-derived particles.Exemplarily, vectors for expressing viral structural proteins, such as Gag protein or Gag-Pro-Pol fusion protein, and optionally also viral envelope proteins, such as VSV-G protein or BAEV (endogenous primate retrovirus)-G protein, can be prepared by those skilled in the art according to the techniques of Negre et al. (2000, Gene Ther, Vol. 7: 1613-1623) and of Yee et al. (1994, Methods Cell Biol, Vol. 43 PtA: 99-112).

[0138] Any suitable acceptable packaging cell known in the art can be used in the production of the virus-derived particles described herein. Mammalian cells or insect cells are preferred. Examples of cells useful in the production of virus-derived particles in the practice of the present invention include, for example, human cell lines such as VERO cells, WI38 cells, MRC5 cells, A549 cells, HEK293 cells, HEK293T cells, B-50 cells or any other HeLa cells, HepG2 cells, Saos-2 cells, HuH7 cells, and the HT1080 cell line. Exemplary cell lines for use as packaging cells also include insect cell lines. A number of cell types can be used, which include the following: a) NIH-3T3 mouse cells (Takahara et al., Journal of Virology, (June 1992), 66 (6) 3725-32), which are currently widely used as packaging cells for producing recombinant retroviruses for clinical use, and b) TK -Cell lines have already been described, which include NIH-3T3 TK cells (F. Wagner et al., EMBO Journal (1985), Vol. 4 (n°3): 663-666); these cells can die when cultured in a selective culture medium such as HAT. When they are complemented with a thymidine kinase function, for example, a thymidine kinase function derived from the HSV1-TK (thymidine kinase) virus, they can grow in the selective medium; thus, such strains offer the possibility of using the HSV1-TK gene as a selectable gene. The gene encoding one of the thymidine kinases of HSV1 or a functional derivative thereof is also widely used as a transgene that converts ganciclovir or acyclovir into a prodrug that is cytotoxic to cells, and thus it can be applied to the selective destruction of cells, such as cancer cells (see, for example, International Publication No. WO 95 / 22617).

[0139] Therefore, a further object of the present invention relates to a cell line for generating virus particles as described herein, which comprises i) one or more polynucleotides encoding the structural viral proteins required for the formation of said virus particles, ii) a polynucleotide encoding the syncytin-1 fusion protein of the present invention, and iii) one or more polynucleotides encoding the cargo(es).

[0140] Cargo: According to the present invention, the particles of the present invention encapsulate one or more cargos. Typically, the cargo can be of any nature suitable for encapsulation into the particles, such as virus particles.

[0141] In some embodiments, the cargo is selected from the group consisting of organic molecules, polymers, polypeptides, polynucleotides, and small organic compounds having a molecular weight greater than 50 and less than about 2,500 daltons. The cargo can also be found among biomolecules, including peptides, polysaccharides, fatty acids, lipids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

[0142] In some embodiments, cargos include chemotherapeutic agents, anti-inflammatory agents, hormones, or hormone antagonists, ion channel modifiers, and neuroactive agents. Examples of suitable pharmaceuticals for the present invention are those described in “The Pharmacological Basis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth edition, under the sections: Drugs Acting at Synaptic and Neuroeffector Junctional Sites; Drugs Acting on the Central Nervous System; Autacoids: Drug Therapy of Inflammation; Water, Salts and Ions; Drugs Affecting Renal Function and Electrolyte Metabolism; Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function; Drugs Affecting Uterine Motility; Chemotherapy of Parasitic Infections; Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Used for Immunosuppression; Drugs Acting on Blood-Forming organs; Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology (all of which are incorporated herein by reference). Toxins, and biological and chemical warfare agents are also included. See, for example, Somani, S. M. (Ed.), “Chemical Warfare Agents,” Academic Press, New York, 1992).

[0143] In some embodiments, the cargo is a polynucleotide. In some embodiments, the polynucleotide is an RNA molecule or a DNA molecule.

[0144] In some embodiments, the polynucleotide can be introduced into the target cells of a tissue or organ and expressed under appropriate conditions or, alternatively, can confer beneficial properties to the cells. Thus, the polynucleotide is selected based on the desired therapeutic outcome. For example, the polynucleotide encodes a polypeptide that confers beneficial properties or a desired therapeutic outcome to the cell. Examples of polynucleotides of interest include protective polypeptides (e.g., neuroprotective polypeptides such as GDNF (glial cell line-derived neurotrophic factor), CNTF (ciliary neurotrophic factor), NT4 (neurotrophin), NGF (nerve growth factor), and NTN (netrin)); anti-angiogenic polypeptides (e.g., soluble vascular endothelial growth factor (VEGF) receptor; VEGF-binding antibody; VEGF-binding antibody fragment (e.g., single-chain anti-VEGF antibody)); and anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-X); and those encoding polypeptides selected from the group consisting of the like, but are not limited thereto.

[0145] In some embodiments, the polynucleotide encodes an antigen. As used herein, the term "antigen" has its general meaning in the art and generally refers to a substance or a fragment thereof that is recognized and selectively bound by an antibody or by a T cell antigen receptor, resulting in the induction of an immune response. The antigens described in the present invention are typically, but not exclusively, peptides and proteins. An antigen in the context of the present invention can include any subunit, fragment, or epitope of any proteinaceous molecule (including proteins or peptides of viral origin, bacterial origin, parasitic origin, fungal origin, protozoal origin, prion origin, cellular origin, or extracellular origin) that ideally elicits an immune response in a mammal and preferably results in protective immunity. In some embodiments, the antigen is a tumor antigen. In particular, the antigen can be a peptide isolated from any virus, including but not limited to viruses derived from any of the following virus families: Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae (e.g., norovirus (also known as "Norwalk-like virus"), Capillovirus, Carlavirus, Cauliflower mosaic virus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae (e.g., coronavirus, e.g., severe acute respiratory syndrome (SARS) virus, or SARS-CoV-2), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus,Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g., Zaire, Reston, Cote d'Ivoire, or Sudan strains)), Flaviviridae (e.g., hepatitis C virus, dengue virus type 1, dengue virus type 2, dengue virus type 3, and dengue virus type 4), Hepadnaviridae (e.g., hepatitis B virus or hepatitis C virus), Herpesviridae (e.g., human herpesvirus (HSV) types 1, 2, 3, 4, 5, and 6, cytomegalovirus, and Epstein-Barr virus (EBV)), Hypoviridae, Iridoviridae, Leviviridae, Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g., influenza virus types A and B), Papovaviridae, Papillomaviridae (e.g., human papillomavirus (HPV)), Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus (RSV)), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus, hepatovirus, and aphthovirus) (e.g., hand, foot, and mouth disease virus)), Poxviridae (e.g., vaccinia virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentivirus, e.g., human immunodeficiency virus (HIV) types 1 and 2), Rhabdoviridae, and Totiviridae.

[0146] In some embodiments, the polynucleotides of the invention are RNA molecules, particularly messenger RNA (mRNA). In some embodiments, the particles of the invention are capable of inducing: i) the introduction of one or more endogenous or exogenous coding sequences of interest into target cells, ii) the introduction of one or more non-coding RNAs, such as RNAs capable of inducing an effect on gene expression, for example, small hairpin RNAs, microRNAs, single-guide RNAs, long non-coding RNAs or circular RNAs, iii) the introduction of intracellular RNAs, messenger RNAs of a particular type or others (such as microRNAs), sub-genomic replicons of RNA viruses (such as hepatitis C virus) or complete genomes of RNA viruses, iv) the co-expression of endogenous or exogenous coding or non-coding sequences of target cells, or vi) the involvement in the modification of the genome of target cells by a genome engineering system, such as a CRISPR system, and encapsulate one or more RNA molecules.

[0147] In some embodiments, the RNA molecules encapsulated in the viral particles of the invention comprise at least one capsid-forming sequence. By "capsid-forming sequence" is meant an RNA motif (sequence and three-dimensional structure) that is specifically recognized by an RNA-binding domain as described above.

[0148] In some embodiments, the polynucleotide is an antisense sequence or a short interfering RNA sequence that acts to reduce the expression of the target sequence. Antisense or short interfering RNA nucleic acids are designed to specifically bind to RNA such that an RNA-DNA or RNA-RNA hybrid is formed, resulting in the cessation of DNA replication, reverse transcription, or messenger RNA translation. Gene expression is reduced through a variety of mechanisms. Antisense nucleic acids based on a selected nucleic acid sequence can interfere with the expression of the corresponding gene. Antisense oligodeoxynucleotides (ODNs) include synthetic ODNs having chemical modifications derived from natural nucleic acids, or nucleic acid constructs that express antisense molecules such as RNA. One antisense molecule or a combination thereof can be administered, where the combination may include multiple different sequences. Antisense oligonucleotides generally have a length of at least about 7, usually at least about 12, more usually at least about 20 nucleotides, not exceeding about 500, usually not exceeding about 50, and more usually not exceeding about 35 nucleotides, where the length is determined by factors such as inhibitory efficiency, specificity (including the absence of cross-reactivity), etc.

[0149] There is also interest in RNA interference agents. RNA interference agents are small ribonucleic acid molecules (also referred to herein as interfering ribonucleic acids), i.e., oligoribonucleotides that are in a double-stranded structure, for example, two distinct oligoribonucleotides that hybridize to each other, or a single ribooligonucleotide (which is assumed to form a small hairpin to generate a double-stranded structure). The oligoribonucleotide means a ribonucleic acid that does not exceed about 100 nucleotides in length, typically does not exceed about 75 nucleotides in length, and where the length is less than about 70 nucleotides in some embodiments. When the RNA agent is a double-stranded structure of two distinct ribonucleic acids that hybridize to each other, such as a small interfering RNA, the length of the double-stranded structure is typically in the range of about 15 - 30 bp, usually about 15 - 29 bp, where lengths of about 20 - 20 bps, such as 21 bp, 22 bp, are of particular interest in some embodiments. When the RNA substance is a double-stranded structure of a single ribonucleic acid present in hairpin formation, i.e., a small hairpin-type RNA, the length of the hybridized portion of the hairpin is typically the same as the length provided above for small interfering RNA-type substances or only 4 - 8 nucleotides longer.

[0150] In some embodiments, the cargo is a polynucleotide encoding an endonuclease, a base editing enzyme, an epigenome editor, or a prime editor as described later herein.

[0151] In some embodiments, the cargo is a polypeptide. Polypeptides of interest include biologically active proteins such as transcription factors, proteins involved in signal transduction pathways, cytokines, chemokines, toxins, and the like. Such polypeptides can include proteins not found in the target cells, proteins from different species, or cloned versions of proteins found within the target cells. Preferred target proteins of the present invention will be proteins that have the same state as found in the expressing target cells such that post-translational modifications are the same as those found within the target cells. Such modifications include glycosylation or lipid modification, addition of cofactors, or formation of quaternary structure. Most preferred will be wild-type proteins corresponding to proteins that are found mutated or absent within the target cells. In some embodiments, the polypeptide is a membrane protein or a non-membrane protein. Non-limiting examples of membrane proteins include ion channels, tyrosine kinase receptors such as the PDGF receptor and the SCF-R receptor (stem cell factor receptor, or c-kit, or CD117), G protein-coupled receptors such as the adrenergic receptor. Non-limiting examples of non-membrane proteins include cytosolic proteins such as actin, Ras, ERK1 / 2, and nuclear proteins such as steroid receptors, histone proteins, or transcription factors.

[0152] In some embodiments, the cargo is an endonuclease that provides site-specific knockdown of gene function. For example, here the endonuclease knocks out alleles associated with genetic diseases. For example, if a defective copy of a gene is encoded by a dominant allele that is a retinal structural protein and / or provides normal retinal function in the wild-type case, the site-specific endonuclease can be targeted to the defective allele to knock out the defective allele. In addition to knocking out the defective allele, site-specific nucleases can be used to stimulate homologous recombination with donor DNA encoding a functional copy of the protein encoded by the defective allele. Thus, for example, the methods of the invention can be used to deliver both site-specific endonucleases that knock out the defective allele and to deliver a functional copy of the defective allele, resulting in the defective allele being repaired, thereby resulting in the production of a functional protein.

[0153] In some embodiments, the DNA-targeting endonuclease is a transcription activator-like effector nuclease (TALEN). TALENs are artificially created by fusing a TAL effector ("TALE") DNA-binding domain, such as one or more TALEs, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 TALEs, to a DNA-modifying domain, such as a FokI nuclease domain. The transcription activator-like effector (TALE) may be engineered to bind any desired DNA sequence (Zhang (2011), Nature Biotech. 29: 149-153). By combining the engineered TALE with a DNA cleavage domain, a restriction enzyme that is specific for any desired DNA sequence can be generated. These can then be introduced into cells, where they can be used for genome editing (Boch (2011) Nature Biotech. 29: 135-6; and Boch et al. (2009) Science 326: 1509-12; Moscou et al. (2009) Science 326: 3501). TALEs are proteins secreted by Xanthomonas bacteria. The DNA-binding domain contains a repeated and highly conserved 33-34 amino acid sequence, except for the 12th and 13th amino acids. These two positions are highly variable and show a strong correlation with the recognition of specific nucleotides. Thus, they can be engineered to bind to a desired DNA sequence (Zhang (2011), Nature Biotech. 29: 149-153). To generate TALENs, the TALE protein is fused to a nuclease (N), such as wild-type or mutant FokI endonuclease.Several mutations to FokI have been made for its use in TALENs; these improve cleavage specificity or activity, for example (Cermak et al. (2011) Nucl. Acids Res. 39: e82; Miller et al. (2011) Nature Biotech. 29: 143-8; Hockemeyer et al. (2011) Nature Biotech. 29: 731-734; Wood et al. (2011) Science 333: 307; Doyon et al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) Nature Biotech. 25: 786-793; and Guo et al. (2010) J. Mol. Biol. 200: 96). The FokI domain functions as a dimer and requires two constructs with unique DNA-binding domains for sites within the target genome in the appropriate orientation and spacing. Both the number of amino acid residues between the TALE DNA-binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving a high level of activity (Miller et al. (2011) Nature Biotech. 29: 143-8). TALENs can be used intracellularly to cause double-strand cleavage within a target nucleic acid, for example at a site within a gene. If the repair mechanism inappropriately repairs the cleavage via non-homologous end joining, mutations may be introduced at the cleavage site (Huertas, P., Nat. Struct. Mol. Biol. (2010) 17: 11-16). For example, inappropriate repair can introduce frameshift mutations.Alternatively, foreign DNA may be introduced into the cell together with TALENs according to the foreign DNA sequence and chromosomal sequence, and this process can be used to modify a target gene via the homologous direct repair pathway, for example, by correcting a defect in the target gene, it is possible to cause the expression of the repaired target gene, or by introducing such a defect into, for example, a wild-type gene, it is possible to reduce the expression of the target gene.

[0154] In some embodiments, the DNA-targeting endonuclease is a zinc finger nuclease (ZFN). Similar to TALENs, ZFNs include a DNA-modifying domain, such as a nuclease domain, such as the FokI nuclease domain (or a derivative thereof), fused to a DNA-binding domain. In the case of ZFNs, the DNA-binding domain includes one or more zinc fingers, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 zinc fingers (Carroll et al. (2011) Genetics Society of America 188: 773-782; and Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160). Zinc fingers are small protein structural motifs stabilized by one or more zinc ions. Zinc fingers can include, for example, Cys2His2 and can recognize a sequence of about 3 bp. By combining various zinc fingers with known specificities, a multi-finger polypeptide that recognizes a sequence of about 6, 9, 12, 15, or 18 bp can be generated. To create zinc fingers (and combinations thereof) that recognize specific sequences, various selection techniques and modular construction techniques (including phage display, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells) are available. Zinc fingers can be engineered to bind to a predetermined nucleic acid sequence. The criteria for engineering zinc fingers to bind to a predetermined nucleic acid sequence are known in the art (Sera (2002), Biochemistry, 41:7074-7081; Liu (2008) Bioinformatics, 24:1850-1857). Zinc finger nucleases using the FokI nuclease domain or other dimeric nuclease domains function as dimers. Thus, a pair of zinc finger nucleases is required for targeting to a DNA site that is not a palindrome.Two individual zinc finger nucleases must bind to opposite strands of DNA while being appropriately spaced with respect to their nucleases (Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10570-5). Similarly to TALENs, zinc finger nucleases can create double-strand breaks within DNA, which, if repaired inappropriately, can create frameshift mutations, for example via non-homologous end joining, which can result in a decrease in the expression of the target gene within the cell.

[0155] In some embodiments, the DNA-targeting endonuclease is a CRISPR-associated endonuclease. In bacteria, the CRISPR / Cas locus encodes an RNA-guided adaptive immune system against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). Three types (I-VI) of CRISPR systems have been identified. The CRISPR cluster contains spacers, which are sequences complementary to previous mobile elements. The CRISPR cluster is transcribed and processed into mature CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) RNA (crRNA). The CRISPR-associated endonucleases Cas9 and Cpf1 belong to type II and type V CRISPR / Cas systems and have potent endonuclease activity to cleave target DNA. Cas9 is guided by a mature crRNA containing a unique target sequence of about 20 nucleotides (referred to as a spacer) and a trans-activating small RNA (tracrRNA) that serves as a guide for the processing of the pre-crRNA assisted by ribonuclease III. The crRNA:tracrRNA duplex directs Cas9 to target DNA through complementary base pairing between the spacer on the crRNA and the complementary sequence (referred to as a protospacer) on the target DNA. Cas9 recognizes a trinucleotide (NGG) protospacer adjacent motif (PAM) to specify the cleavage site (the third or fourth nucleotide from the PAM). The crRNA and tracrRNA may be expressed separately or engineered into an artificial chimeric single-guide RNA (sgRNA) through a synthetic stem-loop to mimic the native crRNA / tracrRNA duplex. Such single-guide RNAs may be synthesized, transcribed in vitro for direct RNA transfection, or expressed from an RNA expression vector promoted by U6 or H1, similar to small hairpin RNAs.

[0156] In some embodiments, the CRISPR-associated endonuclease is Cas9 nuclease. The Cas9 nuclease may have a nucleotide sequence identical to the wild-type Streptococcus pyrogenes sequence. In some embodiments, the CRISPR-associated endonuclease may be a sequence derived from other species, such as other Streptococcus species, such as thermophilus; Pseudomonas aeruginosa, Escherichia coli, or other sequenced bacterial genomes and archaea, or other prokaryotic microorganisms. Alternatively, the Cas9 sequence of wild-type Streptococcus pyogenes may be modified. The nucleic acid sequence may be codon-optimized for efficient expression in mammalian cells, i.e., "humanized". The humanized Cas9 nuclease sequence may be, for example, the Cas9 nuclease sequence encoded by any of the expression vectors listed in Genbank accession numbers KM099231.1, GL669193757; KM099232.1, GL669193761; or KM099233.1, GL669193765. Alternatively, the Cas9 nuclease sequence may be a sequence contained within a commercially available vector such as pX330, pX260 or pMJ920 (Addgene, Cambridge, Massachusetts). In some embodiments, the Cas9 endonuclease may have an amino acid sequence that is a variant or fragment of any of the Cas9 endonuclease sequences of Genbank accession numbers KM099231.1, GL669193757; KM099232.1; GL669193761; or KM099233.1, GL669193765, or the Cas9 amino acid sequence of pX330, pX260 or pMJ920 (Addgene, Cambridge, Massachusetts).

[0157] In some embodiments, the CRISPR-associated endonuclease is a Cpf1 nuclease. As used herein, the term "Cpf1 protein" refers to a Cpf1 wild-type protein derived from a type V CRISPR-Cpf1 system, a modified form of a Cpf1 protein, a variant of a Cpf1 protein, an ortholog of Cpf1, and combinations thereof. The cpf1 gene encodes a Cpf1 protein that has a RuvC-like nuclease domain that is homologous to each domain of Cas9, but lacks the HNH nuclease domain present in the Cas9 protein. The type V system has been identified in several bacteria, including Parcubacteria bacterium GWC2011_GWC2_44_17 (PbCpf1), Lachnospiraceae bacterium MC2017 (Lb3 Cpf1), Butyrivibrio proteoclasticus (BpCpf1), Peregrinibacteria bacterium GW2011_GWA 33_10 (PeCpf1), Acidaminococcus BV3L6 (AsCpf1), Porphyromonas macacae (PmCpf1), Lachnospiraceae bacterium ND2006 (LbCpf1), Porphyromonas crevioricanis (PcCpf1), Prevotella disiens (PdCpf1), Moraxella bovoculi 237 (MbCpf1), Smicella sp. SC_K08D17 (SsCpf1), Leptospira inadai (LiCpf1), Lachnospiraceae bacterium MA2020 (Lb2Cpf1), Franciscella novicida U112 (FnCpf1), Candidatus methanoplasma termitum (CMtCpf1), and Eubacterium eligens (EeCpf1).In recent years, Cpf1 has also been demonstrated to have ribonuclease activity and be involved in the processing of pre-crRNA (Fonfara, I., et al., “The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA,” Nature 28; 532(7600):517-21 (2016)).

[0158] In some embodiments, the cargo is a base editing enzyme. As used herein, the term “base editing enzyme” refers to a fusion protein that includes a defective CRISPR / Cas nuclease linked to a deaminase polypeptide. The term is also known as a “base editor”. As used herein, the term “deaminase” refers to an enzyme that catalyzes a deamination reaction. As used herein, the term “deamination” refers to the removal of an amino group from a molecule. In some embodiments, the deaminase is a cytidine deaminase, which catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine to inosine, which is processed by the cell like guanosine to create a change from A to G (or T to C). Two classes of base editing enzymes, namely cytosine base editors (CBEs) and adenine base editors (ABEs), can be used to perform single-base pair editing without causing double-strand breaks. Typically, cytosine base editors are created by fusing a defective CRISPR / Cas nuclease to a deaminase.

[0159] In some embodiments, the base editing enzyme comprises a defective CRISPR / Cas nuclease. The sequence recognition mechanism is the same as that of a non-defective CRISPR / Cas nuclease. Typically, the defective CRISPR / Cas nuclease of the present invention comprises at least one RNA-binding domain. The RNA-binding domain interacts with a guide RNA molecule as defined hereinafter in this specification. However, the defective CRISPR / Cas nuclease of the present invention is a modified version that has no nuclease activity at all. Thus, the defective CRISPR / Cas nuclease specifically recognizes the guide RNA molecule, thereby guiding the base editing enzyme to its target DNA sequence. In some embodiments, the CRISPR / Cas nuclease consists of a mutant CRISPR / Cas nuclease, i.e., a protein, fusion protein, or combination thereof having one or more point mutations, insertions, deletions, truncations. In some embodiments, the mutant has RNA-guided DNA-binding activity but lacks one or both of its nuclease active sites. In some embodiments, the CRISPR / Cas nuclease of the present invention is a nickase, more specifically Cas9 nickase, i.e., Cas9 derived from Streptococcus pyogenes having one mutation selected from the group consisting of D10A and H840A.

[0160] The second component of the base editing enzyme disclosed herein comprises a DNA-modifying enzyme that is a deaminase and not a nuclease.

[0161] In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is a deaminase of the apolipoprotein B mRNA editing complex (APOBEC) family. In some embodiments, the deaminase is a deaminase of the APOBEC1 family. In some embodiments, the deaminase is an activation-induced cytidine deaminase (AID). In some embodiments, the deaminase is an ACF1 / ASE deaminase.

[0162] In some embodiments, the deaminase is AID: activation-induced cytidine deaminase, APOBEC1: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 1, APOBEC3A: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A, APOBEC3B: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3B, APOBEC3C: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3C, APOBEC3D: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3D, APOBEC3F: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3F, APOBEC3G: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G, APOBEC3H: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3H, ADA: adenosine deaminase, ADAR1: adenosine deaminase acting on RNA 1, Dnmt1: DNA (cytosine-5-)-methyltransferase 1, Dnmt3a: DNA (cytosine-5-)-methyltransferase 3α, Dnmt3b: DNA (cytosine-5-)-methyltransferase 3β, and Tet1: methylcytosine dioxygenase.

[0163] In some embodiments, the deaminase is derived from activation-induced cytidine deaminase (AID). AID is a cytidine deaminase that can catalyze the deamination reaction of cytosine in the context of DNA or RNA. When AID comes to the targeting site, it changes the C base to a U base. In dividing cells, this can result in a point mutation from C to T. Alternatively, the change from C to U can trigger the intracellular DNA repair pathway, mainly the excision repair pathway, which will remove the U-G base pair of the inappropriate base pair and replace it with a T-A, A-T, C-G, or G-C pair. As a result, a point mutation will occur at the target C-G site. In some embodiments, the DNA modification enzyme is AID*Δ, which is an AID mutant with increased somatic hypermutation activity from which its nuclear export signal (NES) has been removed (Hess GT, Fresard L, Han K, Lee CH, Li A, Cimprich KA, Montgomery SB, Bassik MC: Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat Methods 2016, 13(12):1036-1042).

[0164] In some embodiments, the deaminase is adenosine deaminase. In some embodiments, the deaminase is a deaminase of the ADAT family. In some embodiments, the adenosine deaminase variant is TadA deaminase (adenosine deaminase of tRNA). In some embodiments, the adenosine deaminase variant is TadA of Staphylococcus aureus, TadA of Bacillus subtilis, TadA of Salmonella typhimurium, TadA of Shewanella putrefaciens, TadA of Haemophilus influenzae F3031, TadA of Caulobacter crescentus, or TadA of Geobacter sulfurreducens, or a fragment thereof. In some embodiments, the TadA deaminase is the deaminase of TadA of E. coli (ecTadA). In some embodiments, the TadA deaminase is a truncated and shortened TadA deaminase of E. coli. In some embodiments, the TadA deaminase is TadA*7.10. In some embodiments, the TadA deaminase is a TadA*8 variant. For example, the deaminase is described in International PCT applications WO 2018 / 027078, WO 2017 / 070632, WO / 2020 / 168132, WO / 2021 / 050571, each of which is incorporated herein by reference in its entirety.Komor, A.C., et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, N.M., et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, A.C., et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017) ), and Rees, H.A., et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 Dec;19(12):770-788. doi: 10.1038 / s41576-018-0059-1 (the entire content of which is incorporated herein by reference) should also be referred to.

[0165] In some embodiments, the cargo is an effector of epigenome editing (an "epi - editor") that can activate and suppress the expression of endogenous genes and can perform step - by - step control over gene regulation (Nakamura, M., Gao, Y., Dominguez, A.A. et al. CRISPR technologies for precise epigenome editing. Nat Cell Biol 23, 11 - 22 (2021)). The recruitment of epigenome - editing effector domains typically involves the CRISPR / Cas system, which enables site - specific control with respect to modifications to DNA, histones, and chromatin structure.

[0166] In some embodiments, the cargo is a prime editor consisting of a fusion protein, where the catalytically impaired Cas9 endonuclease is fused to an engineered reverse transcriptase. Through complex formation with a prime - editing guide RNA (pegRNA), the prime editor can identify the target site and replace target DNA nucleotides by providing new genetic information. It mediates targeted insertions, deletions, and base - to - base conversions without the need for double - strand breaks (DSB) or donor DNA templates (Anzalone, Andrew V.; Randolph, Peyton B.; Davis, Jessie R.; Sousa, Alexander A.; Koblan, Luke W.; Levy, Jonathan M.; Chen, Peter J.; Wilson, Christopher; Newby, Gregory A.; Raguram, Aditya; Liu, David R. (21 October 2019). "Search - and - replace genome editing without double - strand breaks or donor DNA". Nature. 576 (7785): 149 - 157.).

[0167] In some embodiments, the particles of the invention encapsulate i) a polypeptide (or polynucleotide encoding the same) selected from the group consisting of a CRISPR-associated endonuclease, a base editing enzyme, an epigenome editing factor, and a prime editor, and ii) one or more guide RNA molecules.

[0168] As used herein, the term "guide RNA molecule" generally refers to an RNA molecule (or a group of RNA molecules collectively) that can bind to a Cas9 protein and target the Cas9 protein to a specific location within a target DNA. The guide RNA can include the following two segments: a DNA-targeting guide segment and a protein-binding segment. The DNA-targeting segment contains a nucleotide sequence that is complementary to the target sequence (or can hybridize under at least stringent conditions). The protein-binding segment interacts with a CRISPR protein, such as Cas9 or a Cas9-related polypeptide. These two segments may be located within the same RNA molecule or within two or more separate RNA molecules. When the two segments are in separate RNA molecules, the molecule containing the DNA-targeting guide segment is sometimes referred to as a CRISPR RNA (crRNA), and the molecule containing the protein-binding segment is referred to as a trans-activating RNA (tracrRNA).

[0169] In some embodiments, the cargo polypeptide is fused either directly or via a linker to a viral structural protein (such as the GAG or PEG10 protein). For example, in some embodiments, a nuclease (such as Cas9) is fused either directly or via a linker to a viral structural protein.

[0170] In some embodiments, the cargo polypeptide (such as a nuclease like Cas9) and the viral structural protein (such as GAG or PEG10 protein) form a dimer. The means by which the viral structural protein and the cargo polypeptide form a dimer is not particularly limited. In some embodiments, the viral structural protein (such as GAG or PEG10 protein) and the cargo polypeptide (such as a nuclease like Cas9) are each fused to a respective domain capable of forming a dimer in the presence of a compound, either directly or via a linker. For example, it is possible to use a system in which FK506 binding protein (“FKBP12 domain”) and FKBP12-rapamycin associated protein 1, FRAP1 fragment (“FRB domain”) form a heterodimer in the presence of rapamycin. Thus, in some embodiments, the viral structural protein (such as GAG of PEG10) is fused to the FRB domain and the cargo polypeptide (such as a nuclease like Cas9) is fused to the FKBP12 domain (or vice versa), and it is possible to dimerize the FKBP12 domain and the FRB domain in the presence of rapamycin during the production of viral particles.Alternatively, a system in which GAI (gibberellin-insensitive) and GIDI (gibberellin-insensitive dwarf1) form a heterodimer in the presence of gibberellin or GA3-AM (see, for example, Miyamoto T., et al., Rapid and Orthogonal Logic Gating with a Gibberellin-induced Dimerization System, Nat Chem Biol., 8 (5), 465-470, 2012), that is, a system in which PyL (PYR1-like, consisting of amino acids from position 33 to 209) and ABI1 (consisting of amino acids from position 126 to 423) form a heterodimer in the presence of S-(+)-abscisic acid (ABA) (see, for example, Liang F. S., et al., Engineering the ABA plant stress pathway for regulation of induced proximity, Sci Signal., 4 (164), rs2, 2011), etc. can be used.

[0171] Other cargos of interest include detectable markers such as luciferase, luciferin, green fluorescent protein, fluorescent dyes such as FITC (fluorescein isothiocyanate), etc. Detectable markers can also include imaging diagnostic entities such as metal nanoparticles such as gold, platinum, silver, etc., which can be provided as nanoparticles, usually nanoparticles less than 10 nm, less than about 5 nm, etc.

[0172] Therapeutic use: The present invention provides particle compositions and kits suitable for use (in vivo or ex vivo) in a treatment method. According to the present invention, the therapeutic effect is brought about by one or more cargoes (groups) encapsulated within the particles of the present invention. For example, the particles and the compositions containing them can be used for gene therapy or vaccine purposes.

[0173] Accordingly, a further object of the present invention is to provide a method of treatment in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the particles of the present invention.

[0174] Examples of types of diseases and disorders that can be treated by the method of the present invention include, but are not limited to, retinal diseases such as age-related macular degeneration; diabetic retinopathy; infectious diseases such as human immunodeficiency virus, influenza pandemic, Category 1 and 2 biological warfare agents, or any newly emerging viral infection; autoimmune diseases; cancer; multiple myeloma; diabetes; systemic lupus erythematosus (SLE); hepatitis C; multiple sclerosis; Alzheimer's disease; Parkinson's disease; amyotrophic lateral sclerosis (ALS), Huntington's disease; epilepsy; chronic obstructive pulmonary disease (COPD); joint inflammation, arthritis; myocardial infarction (MI); congestive heart failure (CHF); hemophilia A; or hemophilia B.

[0175] The infectious diseases that can be treated or prevented by the method of the present invention are caused by infectious pathogens including, but not limited to, viruses, bacteria, fungi, protozoa, helminths, and parasites. The present invention is not limited to the treatment or prevention of infectious diseases caused by intracellular pathogens. Many medically relevant microorganisms are well described in the literature, see, for example, C. G. A Thomas, Medical Microbiology, Bailliere Tindall, Great Britain 198 (the entire content of which is hereby incorporated by reference herein).

[0176] The types of cancer that can be treated or prevented by the method of the present invention include human sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary cell carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic lung cancer, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, malignant melanoma, neuroblastoma, retinoblastoma; leukemias, such as acute lymphocytic leukemia, and acute myelogenous leukemia (myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, and erythroleukemia); chronic leukemias (chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphomas (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenström's macroglobulinemia, and heavy chain disease, but are not limited thereto.

[0177] In some embodiments, when the particles of the present invention are designed for the targeting of hematopoietic cells, the treatment methods disclosed herein are particularly suitable for the treatment of β - hemoglobinopathies.

[0178] As used herein, the term "β - hemoglobinopathy" has its general meaning in the art and refers to any abnormality in the structure or function of any hemoglobin in an individual, and this includes any mutation, such as a deletion mutation or substitution mutation in the coding region of the HBB gene, or a mutation or deletion in the promoter or enhancer of such a gene that causes a decrease in the amount of hemoglobin produced as compared to normal or standard conditions, and includes abnormalities in the primary, secondary, tertiary, or quaternary structure of hemoglobin.

[0179] In some embodiments, the particles of the present invention are particularly suitable for the treatment of sickle cell disease.

[0180] As used herein, the term "sickle cell disease" has its ordinary meaning in the art and refers to a group of autosomal recessive hereditary blood disorders that result from mutations in the globin genes and are characterized by abnormal, rigid, sickle-shaped red blood cells. They are defined by the presence of the βS-globin gene, which encodes a β-globin chain variant in which the glutamic acid at the 6th amino acid of the peptide is replaced by valine: the incorporation of βS-globin into the hemoglobin tetramer (HbS, sickle hemoglobin) results in hemoglobin polymerization and the clinical phenotype. The term includes sickle cell anemia (HbSS), sickle cell hemoglobin C disease (HbSC), sickle cell disease with β-thalassemia (HbS / β+), or sickle cell disease without β-thalassemia (HbS / β0).

[0181] In some embodiments, the particles of the present invention are particularly suitable for the treatment of β-thalassemia.

[0182] As used herein, the term "β-thalassemia" refers to a hemoglobinopathy that results from a change in the ratio of α-globin polypeptide chains to β-like globin polypeptide chains, resulting in insufficient production of normal hemoglobin tetramer proteins and precipitation of free unpaired α-globin chains.

[0183] Compositions such as those described herein are included in pharmaceutical compositions for use in practicing methods of treatment in a subject in need thereof, including mammalian and human individuals other than humans in need thereof. The compositions of the invention can be formulated for delivery to animals (e.g., livestock, e.g., cows, pigs, etc.) for veterinary purposes, and to other mammalian subjects other than humans, as well as to human subjects. For example, the particles may be formulated with a physiologically acceptable carrier for use in applications to gene transfer and gene therapy. In some embodiments, the composition further comprises one or more transduction helper compounds. The transduction helper compound is preferably selected from the group comprising cationic polymers, as specifically described by Zuris et al. (2015, Nat Biotechnol, Vol. 33(n°1): 73-80).The transfection helper compound can be selected from the group comprising polybrene (which can also be referred to as hexadimethrine bromide), protamine sulfate, 12-myristate 13-acetate (also known as phorbol myristate acetate or PMA as described in Johnston et al., 2014, Gene Ther, Vol. 21(12): 1008-1020), Vectofusin (as described in Fenard et al., 2013, Molecular Therapy Nucleic Acids, Vol. 2: e90), poloxamer P338 (as described in Anastasov et al., 2016, Lentiviral vectors and exosomes as gene and protein delivery tools, in Methods in Molecular Biology, Vol. 1448: 49-61), RetroNectin® reagent (commercially available from Clontech Laboratories), Viral PLUS® transfection enhancer (commercially available from Applied Biological Materials), Transplus® viral transfection enhancer (commercially available from Clinisciences), LentiBOOST® (commercially available from Sirion Biotech), or ExpressMag® transfection system (commercially available from Sigma-Aldrich). As shown in the examples herein, the cationic transfection helper compound may consist of polybrene. The particles can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. The particles can be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Injectable formulations can be presented in unit dosage forms, for example in ampoules, or in multi-dose containers with the addition of preservatives. The particle composition can take the form of a suspension, a solution, or an emulsion in an oily or aqueous vehicle, and can contain formulating agents such as suspending agents, stabilizers and / or dispersing agents.The liquid preparation of the particle composition can be prepared by conventional means together with pharmaceutically acceptable additives such as a suspending agent (e.g., sorbitol syrup, cellulose derivative or hardened edible fat); an emulsifier (e.g., lecithin or acacia); a non-aqueous vehicle (e.g., almond oil, oily ester, ethyl alcohol or fractionated vegetable oil); and a preservative (e.g., methyl or propyl-p-hydroxybenzoate or sorbic acid). The preparation may also contain buffer salts. Alternatively, the composition may be in powder form for constitution with a suitable vehicle, e.g., pyrogen-free water, before use.

[0184] The particle composition of the present invention can be administered to a subject in a therapeutically effective dose that provides a therapeutic effect. In some embodiments, the amount of the particle composition of the present invention is administered in a dosage unit that ranges from about 0.1 to 5 micrograms (μg) per kilogram (kg) of body weight. To achieve this purpose, the particle composition of the present invention can be formulated in a dosage ranging from about 7 mg to about 350 mg for treating a subject with an average body weight of 70 kg. The amount of the particle composition of the present invention that can be administered can be selected from the group comprising 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg or 5.0 mg / kg. In particular, for treating a subject with an average body weight of 70 kg, the dosage of the particles in a unit dose of the composition can be selected from the group comprising 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg or 750 mg. These dosages can be administered once or repeatedly, for example, once a day, every other day, once a week, once every two weeks, or once a month. In some embodiments, the virus-like particle composition may be administered to the subject in 1 dose, or 2 doses, or 3 doses, or 4 doses, or 5 doses, or 6 doses, or more. The required dosing interval can be determined based on the decision of the practitioner.

[0185] The particulate composition, if desired, may be presented in a packaging or a dosing device that contains one or more unit dosage forms containing the active ingredient. Examples of the packaging may include a metal foil or a plastic foil, such as a blister pack. The packaging or the dosing device may be accompanied by instructions for administration. In some embodiments, the particulate composition may be in a liquid dosage form or a solid (e.g., lyophilized) dosage form.

[0186] Administration of the particles to a human or animal subject in need thereof can be by any means known in the art for the administration of viral vectors. Exemplary administration forms include rectal, transmucosal, topical, transdermal, inhalation, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, and intra-articular) administration, etc., as well as direct injection into a tissue or organ, or intrathecal injection, direct intramuscular injection, intracardiac injection, intravenous injection, intraperitoneal injection, intranasal injection, or intraocular injection. The injection solution can be prepared in a conventional dosage form, either as a liquid solution or suspension, as a solid dosage form suitable for making a solution or suspension in a liquid before injection, or as an emulsion. Alternatively, the virus may be administered locally rather than systemically, for example, in a depot formulation or a sustained release formulation.

[0187] The present invention will be further illustrated by the following drawings and examples. However, these examples and drawings should not be construed as limiting the scope of the present invention in any way.

Brief Description of the Drawings

[0188] Figure 1(A) Design of pseudotyped virus particles using syncytin-1 (SYN) fused to a ligand. To acquire specificity for CD117-positive cells or CD133-positive cells, a scFv antibody fragment against CD133 or the natural ligand of CD117 (stem cell factor (SCF)) was inserted between the signal sequence (SS) and the protein sequence of SYN. The inventors inserted a GGGS flexible linker between the ligand and SYN. (B) Hematopoietic stem and progenitor cells from umbilical cord blood were transfected with lentiviral particles pseudotyped with SYN fused to various ratios of ligand: wild-type SYN. Flow cytometry analysis of GFP expression 48 hours after transduction in hematopoietic stem and progenitor cells from umbilical cord blood. Various ratios were obtained by transfecting plasmids encoding SYN fused to various stoichiometric ratios of ligand (stem cell factor or scFvCD133): plasmid expressing wild-type SYN. A virus pseudotyped with VSV-G (G protein of vesicular stomatitis virus) was used as a control. The inventors plotted the fold change compared to the SYN wild-type lentivirus. All lentiviruses expressed green fluorescent protein under the control of the phosphoglycerate kinase (PGK) promoter. Figure 2(A) Design of pseudotyped virus particles using a short mutant of SYN (SYN480) fused to a ligand. (B) The physical titer of lentivirus was measured by p24 ELISA and expressed as the mean ± standard deviation (n = 3 for SYN480, scFv-SYN480, and SCF-SYN480 lentiviruses; n = 2 for VSV-G). (C) Sorting strategy for hematopoietic stem and progenitor cells of umbilical cord blood based on either CD117 or CD133 expression. Hematopoietic stem and progenitor cells of umbilical cord blood with low or high CD133 or CD117 cells were sorted by FACS (fluorescence-activated cell sorting). (D) Various populations of hematopoietic stem and progenitor cells of umbilical cord blood (CD133low, CD133high, CD117low, or CD117high) were transduced with equal amounts of lentivirus pseudotyped with VSV-G, SYN480, or SYN480 fused to a ligand. Flow cytometry analysis of green fluorescent protein expression in hematopoietic stem and progenitor cells of umbilical cord blood was performed 48 hours after transduction. Quantification was a comparison with the results observed in the population transduced with lentivirus pseudotyped with SYN480. Figure 3 (A) FACS analysis of CD133 and CD117 expression in HEK293T cells. (B) HEK293T cells were transduced with various volumes (10, 5, and 1 μl) of lentivirus with various pseudotypes of either VSVG, SYN480, scFvCD133-SYN480, or SCF-SYN480. Flow cytometry analysis of green fluorescent protein expression in HEK293T cells 48 hours after transduction. (C) Quantification of GFP-positive HEK293T cells after transduction with lentivirus pseudotyped with various envelopes. Figure 4(A) CD177low cells and CD117high cells were transduced with VSVG, SYN480, or lentiviruses pseudotyped with various ratios of SYN480 and SCF-SYN480 (33%, 67%, and 100%) generated in HEK293T cells transfected with various amounts of envelope plasmid (6 μg, 12 μg, and 18 μg). Flow cytometry analysis of GFP expression in hematopoietic stem and progenitor cells of umbilical cord blood was performed 48 hours after transduction. (B) Quantification was a comparison with the results observed in the population transduced with lentivirus pseudotyped with SYN480. Data are presented as mean ± standard deviation (n = 3 biologically independent experiments). (C) Vector copy number (VCN) was analyzed in cells transduced 13 days after transduction. Data are presented as mean ± standard deviation (n = 3 biologically independent experiments). Figure 5(A) Design of pseudotyped virus particles using a short mutant of SYN480 fused to a ligand targeting T cells. To acquire specificity for CD4-positive T cells or CD8-positive T cells, a DARPin against CD4, or an scFv antibody fragment against CD8, was inserted between the signal sequence (SS) and the protein sequence of SYN. (B) Flow cytometry analysis after selection to analyze the purity of CD4-positive T cells and CD8-positive T cells, respectively. (C) CD4-positive T cells and CD8-positive T cells were transduced with lentiviruses pseudotyped with VSVG, SYN480, or SYN480 fused to an appropriate ligand at various ratios (33%, 67%, and 100%). Lentiviruses were produced in HEK293T cells transfected with various amounts (6 μg, 12 μg, and 18 μg) of envelope plasmid. Flow cytometry analysis of GFP expression in T cells was performed 7 days after transduction. (D) Frequency of appearance of GFP-positive cells observed 1 week after transduction. Data are presented as mean ± standard deviation (n = 4 biologically independent experiments). (E) Quantification is a comparison with the results observed in the population transduced with lentiviruses pseudotyped with SYN480. Data are presented as mean ± standard deviation (n = 4 biologically independent experiments). Figure 6(A) Design of pseudotyped virus particles using a short mutant of SYN480 fused to a ligand for targeting IA2-positive cells. To acquire specificity for IA2-positive (also known as PTPRN) cells, an scFv antibody fragment against IA2 was inserted between the signal sequence (SS) and the protein sequence of SYN. (B) Flow cytometry analysis to evaluate IA2 expression in HEK293T cells and HCT116 cells. (C) HEK293T cells and HCT116 cells were transduced with lentivirus pseudotyped with SYN or 33% scFv-IA2-SYN480. Lentivirus was generated in HEK293T cells transfected with various amounts of envelope plasmid (6 μg, 12 μg, and 18 μg). Flow cytometry analysis of GFP expression in HEK293T cells and HCT116 cells was performed 48 hours after transduction. (D) Quantification is a comparison with the results observed in the population transduced with lentivirus pseudotyped with SYN480. Data are presented as mean ± standard deviation (n = 3 biologically independent experiments). (E) Vector copy number (VCN) was analyzed 7 days after transduction. Data are presented as mean ± standard deviation (n = 3 biologically independent experiments). Figure 7(A) Design of virus particles pseudotyped with a short mutant of SYN480 fused to a ligand for targeting Glp1R-positive cells. To acquire specificity for Glp1R-positive cells, the native ligand of Glp1R (GLP1) was inserted between the signal sequence (SS) and the protein sequence of SYN. To generate Glp1R-positive cells, cells were transduced with a lentiviral construct containing Glp1R and the hygromycin resistance (Hygro) gene to overexpress Glp1R. Stably transduced cells were selected using hygromycin. (B) Flow cytometry analysis to evaluate Glp1R expression in HEK293T cells and HCT116 cells. (C) HEK293T cells and HEK293T Glp1R-positive cells were transduced with lentiviruses pseudotyped with SYN480 or 33% GLP1-SYN480. Lentiviruses were produced in HEK293T cells transfected with various amounts of envelope plasmid (6 μg, 12 μg, and 18 μg). Flow cytometry analysis of GFP expression in HEK293T cells and HEK293T Glp1R-positive cells was performed 48 hours after transduction. Data are presented as mean ± standard deviation (n = 3 independent experiments). (D) Quantification is a comparison with the results observed in populations transduced with lentiviruses pseudotyped with SYN480 in HEK293T Glp1R-positive cells and HEK293T Glp1R-positive cells and HCT116Glp1R-positive cells and HCT116Glp1R-positive cells. Data are presented as mean ± standard deviation (n = 3 biologically independent experiments).

[0189]

Table 1

[0190] Example: Method: Plasmid Cloning:

Chemical Structure

Chem.

Chem.

Chem.

Chem.

Chem.

[0191] The synstatin-1 (SYN) coding sequence was obtained from the Ensembl database (ENST00000493463), and two point mutations were inserted to determine the substitutions of R393Q and F399A amino acids, enhancing the immunosuppressive activity of synstatin-1. 2 The scFvCD133 coding sequence has been previously published 3 and the stem cell factor coding sequence was obtained from the gene database (Ensemble ENSG00000049130). Fragments containing scFvCD133, SYN wild type, and stem cell factor were purchased from Twist Bioscience.

[0192] The scFvCD133 insert was digested with EcoRI. The scFvCD133-SYN insert was ligated into the EcoRI-digested PMD2.G plasmid (Addgene, No. 12259) to generate the scFvCD133-SYN plasmid. The integrity of the plasmid was confirmed by Sanger sequencing. In this plasmid, scFvCD133-SYN is under the control of the cytomegalovirus (CMV) promoter and enhancer.

[0193] The stem cell factor (SCF) insert was digested with XbaI and BspEI. The SCF insert was ligated into the scFvCD133-SYN plasmid digested with XbaI and BspEI to generate the SCF-SYN plasmid. The integrity of the plasmid was confirmed by Sanger sequencing. In this plasmid, SCF-SYN is under the control of the CMV promoter.

[0194] SYN480 or scFvCD133 fused to the SCF ligand was generated by amplification of the scFvCD133-SYN plasmid by PCR using the following primers:

Chemical formula

[0195] The PCR product was digested using BspE1 and XhoI and inserted into the scFvCD133-SYN or SFC-SYN plasmid digested with BspEI and XhoI to generate the scFvCD133-SYN480 and SCF-SYN480 plasmids, respectively. The integrity of the plasmid was confirmed by Sanger sequencing.

[0196] SYN480 alone was generated by amplification of the scFvCD133-SYN480 plasmid by PCR using the following primers.

Chemical formula

[0197] The PCR product was digested with AflII and XhoI and inserted into the scFvCD133-SYN480 plasmid digested with AflII and XhoI.

[0198] Both the DARPin targeting the CD4 array and the scFv targeting the CD8 array were obtained from the patent of International Publication No. WO 2018 / 033544. The GLP1 coding sequence was obtained from the Ensemble database (ENSG00000115263). The scFv targeting sequence of IA2 was extracted from the previously published monoclonal antibody sequence. 4 .

[0199] The DARPin CD4, scFv CD8, scFv IA2, and GLP1 inserts were digested with AflII or XbaI and BspEI. The scFvCD133-SYN480 plasmid was also digested using the same enzymes. Each insert was ligated into the scFvCD133-SYN480 plasmid. The integrity of the plasmid was confirmed by Sanger sequencing. In all these plasmids, the ligand-SYN480 is under the control of the CMV promoter.

[0200] The PCR products were obtained using Phusion High-Fidelity Polymerase (New England Biolabs, NEB). The restriction enzymes were purchased from NEB.

[0201] [Chemical formula]

[0202] GlP1R transgene: The GlP1R coding sequence was obtained from the Ensemble database (ENSG00000112164). The GlP1R-P2A-Hygromycin™ sequence and the Hygromycin insert were purchased from Twist Bioscience. The PGK (phosphoglycerate kinase)-GFP plasmid and the GlP1R-P2A-Hygromycin® sequence (Addgene, 19070) were digested with AgeI and SalI. The GlP1R-P2A insert was ligated into the PGK-GFP plasmid. The integrity of the plasmid was confirmed by Sanger sequencing.

[0203] The PCR products were obtained using Phusion High-Fidelity Polymerase (NEB). The restriction enzymes were purchased from NEB.

[0204] Generation of Lentivirus HEK293T cells were cultured in DMEM + Glutamax supplemented with Glutamax (Gibco), non-essential amino acids (Gibco), and penicillin / streptomycin (Gibco). The medium was changed 2 hours before transfection. 293T cells were transfected with the following plasmids when they reached 80 - 90% confluence: (i) plasmid expressing the envelope (0.7 μg for P60 plate (21 cm 2 ), 6 μg or 12 μg or 18 μg for P150 plate (152 cm 2 ), (ii) pRSV-Rev plasmid (Addgene, 12253) (1.1 μg for P60 plate (21 cm 2 ), 7.25 μg for P150 plate (152 cm 2 ); (iii) pMDlg / pRRE plasmid (Addgene, 12251) (2.2 μg for P60 plate (21 cm 2 ), 14.5 μg for P150 plate (152 cm 2 ); and (iv) PGK-GFP transduction plasmid (Addgene, 19070) (3 μg for P60 plate (21 cm 2 ), 18 μg for P150 plate (152 cm 2 ). The inventors used PEI as a transfection reagent at a ratio of 1:3 DNA:PEI (polyethyleneimine). The transfection mixture was prepared in DMEM and dropped onto HEK293T cells. The medium was changed 12 to 16 hours after transfection. The virus supernatant was collected 24 hours later, centrifuged at 500 g for 5 minutes, filtered using a 0.45 μm filter, and concentrated by ultracentrifugation at 100,000 g at 4°C for 2 hours. The virus pellet was resuspended in the medium (StemSpan or X-VIVO20 or phosphate-buffered saline) used for virus transduction and used directly or stored at -80°C.

[0205] Titration of lentivirus: The titer of physical particles of lentivirus pseudotyped with VSV-G or SYN was determined by ELISA for p24 (Alliance (copyright), HIV-1 Elisa kit, PerkinElmer, Villebon / Yvette, France).

[0206] Cell culture and transduction: The inventors obtained CD34-positive hematopoietic stem and progenitor cells from human umbilical cord blood from healthy donors. Umbilical cord blood samples eligible for the research purpose were obtained from the umbilical cord blood bank of Saint-Louis Hospital (Paris, France) for simplicity.

[0207] Hematopoietic stem and progenitor cells were purified by Ficoll gradient centrifugation (Eurobio, Les Ulis, France) and selection with CD34-positive magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were stored in liquid nitrogen. Hematopoietic stem and progenitor cells were thawed 48 to 96 hours before transduction and cultured in X-VIVO or StemSpan (StemCell Technologies) medium supplemented with the following cytokines (PeproTech): stem cell factor (SCF) (300 ng / ml), Flt-3L (300 ng / ml), thrombopoietin (TPO) (100 ng / ml), interleukin-3 (IL-3) (60 ng / ml), and stemregenin (250 nM) (StemCell Technologies).

[0208] If necessary, hematopoietic stem and progenitor cells were stained with anti-CD117 antibody or anti-CD133 antibody (Miltenyi Biotec) and sorted by FACS using an SH800 cell sorter (Sony Biotechnology). Cells (10 6Cells / mL) were transduced overnight using lentivirus in the presence of VFI (vectofusin) (12 μg / mL) (Miltenyi Biotec), then washed with PBS and resuspended again in fresh X-VIVO20 supplemented with the above cytokines. The next day, hematopoietic stem and progenitor cells were analyzed for GFP expression by flow cytometry using a Fortessa X20 (BD Biosciences) analyzer and Diva and FlowJo version 10 (BD Biosciences) software.

[0209] 200,000 - 250,000 HEK293T cells were transduced overnight using lentivirus in the presence of VFI (12 μg / mL). The next day, the medium was removed and replaced with fresh medium. On day 3, 293T HEK cells were analyzed for GFP expression by flow cytometry using a Fortessa X20 (BD Biosciences) analyzer and Diva and FlowJo version 10 (BD Biosciences) software.

[0210] CD4-positive T cells and CD8-positive T cells were purified by Ficoll gradient centrifugation (Eurobio, Les Ulis, France) and selection with CD4 or CD8 magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were activated for 3 days using PHA (phytohemagglutinin) (2.5 μg / mL, Millipore Sigma) in Panserin 401 (Pan Biotech) supplemented with 5% human antibody serum (BioWest), penicillin (100 U / mL), and streptomycin (100 μg / mL). After 3 days, dead cells were removed by Ficoll gradient centrifugation and the cells were cultured in RPMI1640 + 10% fetal bovine serum + IL-2 (100 U / mL). 100,000 cells (10 6Cells / mL) were transduced overnight with lentivirus in the presence of VF1 (12 μg / mL) (Miltenyi Biotec), then washed with PBS and resuspended again in fresh medium supplemented with IL-2 as described above. On day 7, CD4-positive T cells and CD8-positive T cells were analyzed for GFP expression by flow cytometry using a Fortessa X20 (BD Biosciences) analyzer and Diva and FlowJo version 10 (BD Biosciences) software.

[0211] Flow cytometry staining 300,000 HEK293T cells or HCT116 were stained with either anti-IA2 antibody (Thermo Fisher) or anti-Glp1R antibody (Thermo Fisher). The stained cells were incubated with a secondary antibody (anti-rabbit IgG antibody) conjugated to Alexa Fluor 647. 200,000 CD4-positive T cells and CD8-positive T cells were stained with either anti-CD4 antibody (BioLegend) or anti-CD8 antibody (BioLegend). The cells were analyzed for the expression of their respective markers by flow cytometry using a Fortessa X20 (BD Biosciences) analyzer and Diva and FlowJo version 10 (BD Biosciences) software.

[0212] Overexpression of cell lines Lentivirus was generated as described above, however, the transduction plasmid PGK-Glp1R-P2A-BleoR was used. The virus supernatant was collected, centrifuged, and filtered as described above. 5×10 5 HEK293T cells or HCT116 cells were transduced with fresh virus supernatant and collected 24 hours later. Forty-eight hours after transduction, hygromycin was added for selection over 14 days (300 μg / mL). The expression of GlP1R was confirmed by flow cytometry analysis.

[0213] Vector copy number Genomic DNA (gDNA) was extracted using the PureLink Genomic DNA Kit according to the manufacturer's instructions (Invitrogen). For hematopoietic stem and progenitor cells in umbilical cord blood, digital droplet dPCR (ddPCR) was performed 13 days after transduction. Vector copy numbers were analyzed according to the protocol described by Corre et al. 8 .

[0214] For HEK293T and HCT116 cells, ddPCR was performed 7 days after transduction. The number of diploid genomes was determined using amplification of the human ALB gene with Alb For, Alb Rev, and Alb Pro. Vector copies were determined using GFP For, GFP Rev, and GFP PRO.

[0215] The sequences of the primers and probes are reported below:

Chemical Formula

[0216] Reactions were performed using 10 units of DraI restriction enzyme in the mix (ddPCR Supermix for probes from Bio-Rad (without dUTP)) according to the manufacturer's recommendations on a Bio-Rad system (Bio-Rad QX200 AutoDG), and 30 ng of gDNA was used for each reaction.

[0217] Results: The inventors have developed a novel system for modifying the tropism of the envelope protein syncytin, which can be used to pseudotype viruses or virus-like particles (VLPs) for gene transfer or other applications. Syncytin is encoded by a gene derived from endogenous retroviruses that have entered the germ line of mammalian hosts. 5。The structure of syncytin resembles that of typical retroviral envelope glycoproteins. Furthermore, syncytins have immunosuppressive proteins, which makes them suitable for gene delivery that shows good tolerance in prospective in vivo.

[0218] Human hematopoietic stem and progenitor cells specific for lentivirus In order to enhance the transduction of cell types expressing a receptor or antigen targeted by a ligand, such as hematopoietic stem and progenitor cells (HSPC), the inventors created a fusion protein containing a syncytin-1 (SYN) signal sequence (SS), a ligand (either natural or engineered), the SYN protein, and a flexible linker between SYN and the ligand (Figure 1A). The inventors used either stem cell factor (SCF) that binds to the CD117 (c-kit) receptor or a single-chain fragment variant (scFv) against the CD133 receptor (scFvCD133) as the ligand. Both ligands are known markers of hematopoietic stem and progenitor cells 6、3 。To evaluate the efficiency of the inventors' strategy and to determine the optimal envelope conformation, the inventors transduced hematopoietic stem and progenitor cells from umbilical cord blood (CB) with lentiviral particles containing mRNA encoding GFP and pseudotyped with various ratios of the inventors' fusion protein: wild-type SYN. For this purpose, the inventors generated lentiviral vectors (LV) by transfecting HEK293T cells with either a plasmid encoding wild-type (WT) SYN or a plasmid expressing SCF-SYN or a plasmid expressing scFvCD133-SYN at various stoichiometric ratios. Lentiviral particles generated using ratios of 33%, 67% or 100% were selected based on the native conformation of SYN, a trimeric protein. Thus, the inventors used these ratios and assumed that each envelope protein was composed of one (33% ratio), two (67% ratio) or three (100%) ligand-SYN monomers, and the remaining monomers were wild-type SYN.

[0219] The inventors observed low transduction efficiency in hematopoietic stem and progenitor cells using a lentivirus pseudotyped with wild-type SYN (17%) compared to a lentivirus pseudotyped with VSV-G. However, a substantial increase was observed when SYN was fused to a ligand. Independently of the ratio, the fusion of stem cell factor or scFvCD133 with SYN almost doubled the ratio of GFP-positive hematopoietic stem and progenitor cells in cord blood (Figure 1B), indicating that the addition of a ligand can increase lentiviral transduction when cells have a targeted receptor or antigen on their cell surface. Interestingly, lentiviral particles having only the ligand-SYN envelope (100%) were able to efficiently transduce hematopoietic stem and progenitor cells. Therefore, to potentially enhance the specificity of these lentiviruses, the inventors used lentiviruses generated using a 100% ratio for the following experiments.

[0220] Deletion of the last amino acid at the cytoplasmic side terminus of SYN has previously been reported to increase its fusion ability. 7、8 Therefore, the inventors designed a novel fusion protein by replacing SYN with its C-terminal truncated short form (SYN480; Figure 2A), which has 480 amino acids instead of 538 amino acids. Lentiviruses pseudotyped with these different engineered envelope proteins (scFvCD133- and SCF-SYN480) showed titers similar to those of lentiviruses pseudotyped with VSV-G as measured by p24 ELISA and almost twice as high as those of lentiviruses pseudotyped with SYN480 (Figure 2B). Notably, batches of lentiviruses pseudotyped with SYN480, as previously reported for wild-type SYN 9However, due to fusion of HEK293T cells transfected with a plasmid expressing SYN480 (data not shown), lentivirus could not be generated by pooling two successive harvests from the same producer cells harvested at 48 and 72 hours after transfection. Conversely, HEK293T cells transfected with an SCF-SYN480 expression plasmid and an scFvCD133-SYN480 expression plasmid did not fuse or only fused poorly (data not shown), allowing successive lentivirus harvests from the same HEK293T producer cells. Thus, addition of a ligand to SYN facilitates lentivirus generation by increasing virus titers and by allowing multiple harvests from the same producer cells.

[0221] To evaluate the selective targeting of cells expressing CD117 or CD133 by SCF-SYN480 and scFvCD133-SYN480, respectively, we compared the transduction levels of subpopulations of cord blood hematopoietic stem and progenitor cells expressing different levels of CD117 or CD133. Cord blood hematopoietic stem and progenitor cells were sorted according to their expression levels of either CD133 or CD117 and transduced with various lentiviruses (Figure 2C). high Transduction of the CD117 population increased GFP-positive cells by 69% (Figure 2D). low and CD117 high The SCF-SYN480 lentivirus was observed in a population of CD117 high Increased transduction efficiency was observed in cord blood hematopoietic stem and progenitor cells (55% increase). Thus, addition of ligand to SYN480 increases specificity for the desired cell type expressing the targeted antigen.

[0222] Finally, the inventors asked whether the fusion of SYN480 to the ligand affects the binding of SYN480 to cell types that do not express the receptor targeted by the ligand. Therefore, the inventors evaluated the transduction efficiency of lentiviruses of scFvCD133-SYN480 or SCF-SYN480 in HEK293T cells that do not express CD133 and express low levels of CD117 (39% positive cells) (Figure 3A). The lentivirus of SYN480 showed a transduction efficiency of 35%, which is higher compared to that previously reported for wild-type SYN (about 25%) 9 The fusion of scFvCD133 to SYN480 almost abolished the binding of SYN480 to HEK293T cells, and the transduction efficiency decreased from 35% to less than 1% (Figures 3B and 3C), indicating a complete retargeting of the tropism of SYN480. Furthermore, the lentiviral particles of SCF-SYN480 showed a decreased transduction efficiency compared to the SYN480 lentivirus (from 35% to 15%), indicating that SCF-SYN480 can bind only to HEK293T cells expressing CD117 on their surface (Figures 3B and 3C), suggesting a partial retargeting of the tropism of SYN480.

[0223] Since the inventors observed hematopoietic stem and progenitor cells that were GFP-positive but transduced with low fluorescence intensity (Figures 1 and 2), the inventors asked whether they could improve the intensity signal of GFP in the transduced cells. The amount of plasmid used for the production of viral particles has been shown to affect the titer and infectivity using several viral envelope proteins 10 Therefore, the inventors decided to transfect HEK293T cells with various amounts of envelope plasmid and produced their lentiviruses (6 μg, 12 μg, and 18 μg). The lentiviral particles produced using ratios of 33%, 67%, or 100% were selected based on the native conformation of the trimeric protein SYN as previously described.

[0224] Following the same strategy as before, the inventors tested their novel lentivirus in subpopulations of hematopoietic stem and progenitor cells of cord blood expressing various CD117 levels. The inventors also focused their analysis on GFP-positive cells with higher fluorescence intensity. Using SYN480, the inventors observed low transduction efficiency in hematopoietic stem and progenitor cells, which increased dose-dependently in the range of 3 ± 0.8% to 8 ± 2%, where 6 μg and 18 μg are the amounts of plasmid used for the production of virus particles, respectively (Figures 4A and 4B). The lentivirus produced using the largest amount of plasmid had the ability to better transduce hematopoietic stem and progenitor cells, despite having a lower titer compared to the lentivirus produced using the lowest amount of plasmid (Table 1). By fusing SYN480 to the ligand, the inventors observed a substantial increase in transduction efficiency. Using 33% SCF-SYN480 envelope, the inventors were able to increase the ratio of GFP-positive CD117 hematopoietic stem and progenitor cells by at least two-fold, regardless of the amount of plasmid used for pseudotyping the lentivirus (Figures 4A and 4B). The transduction efficiency increased with the increase in the amount of SCF-SYN480 envelope plasmid used, as observed with SYN480 lentivirus. 33% SCF-SYN480 envelope was able to increase the transduction efficiency, but the inventors observed very low levels of GFP-positive cells using 67% SCF-SYN480 and no GFP-positive cells using 100% SCF-SYN480, suggesting that having two or three SYN480 chains fused to the ligand affects the infectivity of the lentivirus. high high

[0225] The inventors observed a similar trend in CD117 hematopoietic stem and progenitor cells transduced with SYN480 alone, with the transduction level increasing with the largest amount of plasmid used for the production of the lentivirus. Conversely, the GFP-positive CD117 low ​​​low The proportion of cells strongly decreased using 33% of the envelope of SCF-SYN480, regardless of the amount of envelope plasmid used, and partially blocked SYN480 from binding to its native receptor. For the 67% and 100% ratios between SCF-SYN480 and SYN480, the inventors obtained extremely low or no transduction efficiency, respectively (Figures 4A and 4B).

[0226] Finally, 13 days after transduction with lentiviruses pseudotyped with either SYN480 or 33% SYN-SYN480, the inventors high and CD117 low evaluated transduction efficiency and proviral integration into the genome by analyzing the vector copy number (VCN) in both hematopoietic stem and progenitor cells. The inventors used a protocol to specifically quantify proviral integration events and episomal non-integrated proviruses (i.e., pseudotransduction). 11 The inventors observed a consistent increase in the vector copy number in CD117 high using 33% SCF-SYN480 compared to SYN480, regardless of the amount of envelope plasmid used, which is consistent with their flow cytometry analysis (Figures 4A, 4B, and 4C). The increased fold change in vector copy number levels was consistent with what was observed in GFP-positive cells, confirming an increase in transduction efficiency and re-targeting of the tropism of native SYN480 by the addition of a ligand targeting the receptor present on the target cell surface. Finally, the inventors observed higher vector copy numbers using the highest amount of envelope plasmid (Figure 4C). The vector copy number observed in CD117 low hematopoietic stem and progenitor cells decreased using 6 and 12 μg of the SCF-SYN480 envelope compared to the SYN480 envelope, from which the flow cytometry data and CD117 highRedirection of SYN targeting to hematopoietic stem cells and progenitor cells was confirmed. Notably, the decrease in vector copy number was not as strong as the decrease in the ratio of GFP-positive cells.

[0227] Human T cells specific for lentivirus To demonstrate that our approach can be applied to target other cell types using ligands that target those specific cell surface receptors, we decided to focus on immune T cells, particularly CD4-positive and CD8-positive cells.

[0228] For this purpose, the inventors used previously described strategies to develop fusion proteins containing either a DARPin against the CD4 receptor or a scFv against the CD8 receptor to target CD4-positive T cells and CD8-positive T cells, respectively (Figure 5A). The inventors collected peripheral blood mononuclear cells (PBMCs) from healthy individuals and purified CD4-positive T cells and CD8-positive T cells (Figure 5B). To confirm previous results observed using hematopoietic stem and progenitor cells from umbilical cord blood, the inventors generated batches of lentiviruses with various types of envelopes by using various amounts of envelope plasmid and SYN480 fused to SYN480 at various stoichiometric ratios of SYN480:ligand. SYN480 envelopes showed a very low ability to transduce both CD4-positive T cells and CD8-positive T cells, typically ranging from less than 1% to 8%, using lentiviruses generated with 18 μg and 6 μg of envelope plasmid, respectively (Figures 5C and 5D). Surprisingly, the addition of the ligand strongly increased the transduction efficiency of transducing CD4-positive T cells or CD8-positive T cells, respectively, using either 33% of the DARPinCD4-SYN480 or 33% of the scFvCD8-SYN480 envelope (Figures 5C and 5D). The inventors observed GFP-positive cells ranging from 80 ± 10% to a maximum of 95 ± 7% using 33% of the DARPinCD4-SYN480 lentivirus, depending on the amount of plasmid used for lentivirus production (Figures 5C and 5D). In summary, the addition of the DARPin targeting CD4-positive T cells and the use of 33% of the DARPinCD4-SYN480 envelope plasmid enabled a significant increase in the average transduction efficiency of CD4-positive T cells (at least a 25-fold change) compared to the SYN480 lentivirus (Figure 5E). Notably, the inventors observed very low or no transduction levels using ratios of 67% or 100% of DARPinCD4-SYN480, thus confirming the results obtained with hematopoietic stem and progenitor cells from umbilical cord blood.

[0229] Similarly, the fusion of scFv targeting CD8+ T cells enhanced the transduction efficiency of CD8+ T cells (Figure 5C). Using 33% of the lentivirus of scFvCD8-SYN480, we were able to transduce CD8+ T cells at efficiencies ranging from 62% using 12 μg of envelope plasmid to 95% using 6 μg of envelope plasmid (Figure 5D). Thus, regardless of the amount of envelope used to pseudotype the lentivirus, 33% of the scFvCD8-SYN480 envelope enabled efficient transduction of CD8+ T cells. We observed at least a 22-fold increase in the ratio of GFP-positive cells compared to the SYN480 lentivirus (Figure 5E). The use of 67% and 100% of scFvCD8-SYN480 did not increase the level of transduction (Figures 5C and 5D). Importantly, the use of 33% of the DARPinCD4-SYN480 lentivirus and 33% of the scFvCD8-SYN480 lentivirus did not result in the transduction of CD8+ T cells and CD4+ T cells, thus confirming the specificity of the engineered envelope for cells with the targeted cell surface receptor. In particular, neither 67% nor 100% ratios of DARPinCD4-SYN480 and scFvCD8-SYN480 were able to transduce CD8+ T cells and CD4+ T cells, respectively (data not shown).

[0230] Lentivirus targeting human GLP1-positive cells or IA2-positive cells Finally, we designed a fusion protein containing either a glucagon ligand (GLP1) or an scFv targeting IA2, a well-known receptor overexpressed on pancreatic cells of type 1 diabetic patients; autoantibodies against IA2 are found in the majority of these patients 12、13(Figs. 6A and 7A). HEK293T cells and HCT116 cells were shown to naturally express IA2 on their surfaces as demonstrated by flow cytometry analysis (Fig. 6B). Based on our previous results, we determined to test only the ratio of 33% scFvIA2-SYN480 using various amounts of envelope plasmid. The fusion of scFvIA2 consistently increased transduction by at least 50% and up to 95% (compared to SYN480 alone) in both HEK293T cells and HCT116 cells depending on the amount of envelope plasmid used for lentivirus production (Figs. 6C and 6D). As observed in hematopoietic stem and progenitor cells of umbilical cord blood, the ratio of GFP-positive cells became higher as more amount of envelope plasmid was used, despite the decrease in virus titer (Table 1). We also evaluated the transduction efficiency by analyzing proviral integration one week after transduction. We observed increased vector copy numbers using 33% scFvIA2-SYN480 lentivirus compared to SYN480 lentivirus in both HEK293T cells and HCT116 cells, thereby confirming the results observed by flow cytometry (Fig. 6E). Therefore, we were able to modify the tropism of SYN again using alternative ligand / receptor combinations in a novel cell type.

[0231] To target pancreatic cells using alternative receptors, the inventors developed a fusion protein containing GLP1 between a signal sequence and the SYN480 sequence (Figure 7A). HEK293T cells and HCT116 cells do not naturally express the Glp1R receptor on their surface, so the inventors stably transduced them using constructs to overexpress both Glp1R and hygromycin as a selectable marker (Figure 7B). Following the same strategy used for the IA2 receptor, the inventors transduced both Glp1 receptor-positive and Glp1 receptor-negative cells with either SYN480 or 33% GLP1-SYN480 lentivirus. The inventors observed a decreased transduction efficiency of HEK293T cells and HCT116 cells (Glp1 receptor-negative) using 33% GLP1-SYN480 lentivirus compared to SYN480 lentivirus, regardless of the different amounts used for lentivirus production (Figures 7C and 7D). The inventors did not observe an increase in the transduction efficiency of Glp1 receptor-positive cells using 33% GLP1-SYN480 lentivirus compared to SYN480 lentivirus, but rather observed a rescue of the decreased transduction efficiency observed in Glp1 receptor-negative cells (Figures 7C and 7D).

[0232] In summary, the inventors developed a fusion strategy that allows for modification of the tropism of SYN for various receptors to target desired cell types. The inventors demonstrated that they were able to transduce several different cell types using appropriate ligands to target them. As shown, the inventors' system is adaptable to multiple desired antigens to retarget syncytin to cell types of interest.

[0233] References: Throughout this application, various references describe the state of the art in the technical fields to which the present invention pertains. The disclosures of these references are hereby incorporated herein by reference.

Table 2

Claims

1. A fusion protein in which a syncytin-1 polypeptide is fused to one or more targeting moieties, characterized in that the syncytin-1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2 (SDGGGXXDXXR) and is capable of binding to the ASCT1 receptor and / or the ASCT2 receptor, preferably the ASCT2 receptor.

2. The fusion protein of claim 1, wherein the syncytin-1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3 (SDGGGVQDQAR).

3. The fusion protein of claim 2, wherein the syncytin-1 polypeptide of the present invention comprises the amino acid sequence shown in SEQ ID NO: 3 (SDGGGVQDQAR) and further comprises at least 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400 or 450 consecutive amino acids of SEQ ID NO:

1.

4. The fusion protein of claim 2, wherein the syncytin-1 polypeptide comprises an amino acid sequence having 70% identity to the amino acid sequence ranging from amino acid residue 21 to amino acid residue 480 of SEQ ID NO:1 ("SYN480").

5. The fusion protein of claim 2, wherein the syncytin-1 polypeptide comprises an amino acid sequence ranging from amino acid residue 21 to amino acid residue 480 of SEQ ID NO:1, wherein the arginine residue (R) at position 393 is substituted with a glutamine residue (Q) and the phenylalanine residue (F) at position 399 is substituted with an alanine residue (A).

6. The fusion protein of claim 1, wherein the targeting moiety is selected from the group consisting of a ligand, an antibody, an antibody fragment (e.g., a single-chain Fv or VHH or other functional fragment such as a light-chain deleted immunoglobulin), and a non-antibody-based recognition scaffold (e.g., an affibody; an engineered Kunitz domain; a monobody (adnectin); anticalin; a designed ankyrin repeat domain (DARPin); a binding site for a cysteine-rich polypeptide (e.g., a cysteine-rich knottin peptide); an avimer; or an affilin).

7. 2. The fusion protein of claim 1, wherein the targeting moiety is suitable for targeting a cell population selected from the group consisting of an immune cell population, a hematopoietic cell population, and a malignant cell population.

8. The targeting moiety may be selected from the group consisting of CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD5, CD6, CD7, CD8 alpha, CD8 beta, CD9, CD10, CD11a, CD11b, CD11c, CDw12, CD13, CD14, CD15u, CD16a, CD16b, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD 36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD44R, CD45, CD46, CD47R, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66a, CD66b, C D66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75 , CD75s, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD8 7, CD88, CD89, CD90, CD91, CD92, CDw93, CD94, CD95, CD96, CD97, CD98, CD99 , CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CDw113, CD114, CD115, CD116, CD117, CD118, C Dw119, CD120a, CD120b, CD121a, CDw121b, CD122, CD123, CD124, CDw125, CD1 26, CD127, CDw128a, CDw128b, CD129, CD130, CD131, CD132, CD133, CD134, C D135, CDw136, CDw137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143,CD144、CDw145、CD146、CD147、CD148、CDw149、CD150、CD151、CD152、CD153、CD154、CD155、CD156a、CD156b、CDw156C、CD157、CD158、CD159a、CD159c、CD160、CD161、CD162、CD162R、CD163、CD164、CD165、CD166、CD167a、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD181、CD182、CD183、CD184、CD185、CDw186、CD191、CD192、CD193、CD195、CD196、CD197、CDw198、CDw199、CDw197、CD200、CD201、CD202b、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CDw210、CD212、CD213a1、CD213a2、CDw217、CDw218a、CDw218b、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235b、CD235ab、CD236、CD236R、CD238、CD239、CD240CE、CD240D、CD240DCE、CD241、CD242、CD243、CD244、CD245、CD246、CD247、CD248、CD249、CD252、CD253、CD254、CD256、CD257、CD258、CD261、CD262、CD263、CD264、CD265、CD266、CD267、CD268、CD269、CD271、CD272、CD273、CD274、CD275、CD276、CD277、CD278、CD279、CD280、CD281、CD282、CD283、CD284、CD289、CD292、CDw293、CD294、CD295、CD296、CD297、CD298、CD299、CD300a、CD300c、CD300e、CD301、CD302、CD303、CD304、CD305、CD306、CD307、The fusion protein according to claim 1, which has binding affinity for a CD (cluster of differentiation) molecule selected from the group consisting of CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CDw325, CD326, CDw327, CDw328, CDw329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CDw338 and CD339.

9. 2. The fusion protein of claim 1, wherein the targeting moiety has binding affinity for a cell surface molecule selected from the group consisting of 2B4 / CD244 / SLAMF4, ABCG2, aldehyde dehydrogenase 1-A1 / ALDH1A1, BMI-1, C1qR1 / CD93, CD34, CD38, CD44, CD45, CD48 / SLAMF2, CD90 / Thy1, CD117 / c-kit, CD133, CDCP1, CXCR4, endoglin / CD105, EPCR, erythropoietin R, ESAM, EVI-1, integrin α6 / CD49f, SLAM / CD150, VCAM-1 / CD106, and VEGFR2 / KDR / Flk-1.

10. 2. The fusion protein of claim 1, wherein the targeting moiety is stem cell factor (SCF), which binds to the CD117 (c-kit) receptor.

11. The fusion protein of claim 10, wherein the targeting moiety comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:

4.

12. The fusion protein of claim 1 , wherein the targeting moiety is a single chain fragment variant (scFv) against the CD133 receptor ("scFvCD133").

13. The fusion protein of claim 12, wherein the targeting moiety comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:

5.

14. The fusion protein of claim 1 , wherein the targeting moiety is a DARPin against CD4 ("DARPinCD4").

15. The fusion protein of claim 14, wherein the targeting moiety comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:

6.

16. 2. The fusion protein of claim 1, wherein the targeting moiety is a single chain fragment variant (scFv) against CD8 ("scFvCD8").

17. 17. The fusion protein of claim 16, wherein the targeting moiety comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:

7.

18. 2. The fusion protein of claim 1, wherein the targeting moiety is a single chain fragment variant (scFv) against the IA-2 receptor ("scFvIA-2").

19. 19. The fusion protein of claim 18, wherein the targeting moiety comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:

8.

20. The fusion protein of claim 1 , wherein the targeting moiety is GLP1.

21. 21. The fusion protein of claim 20, wherein the targeting moiety comprises an amino acid sequence having at least 70% identity to the amino acid sequence set forth in SEQ ID NO:

9.

22. The fusion protein of claim 1, wherein the C-terminus of the targeting moiety is fused to the N-terminus of the syncytin-1 polypeptide.

23. 2. The fusion protein of claim 1, wherein the syncytin-1 polypeptide and the targeting moiety are fused to each other directly or via a linker.

24. The fusion protein according to claim 1, further comprising a signal peptide sequence, in particular the signal peptide being the signal sequence (SS) of syncytin-1 (SYN) consisting of an amino acid sequence ranging from the amino acid residue at position 1 to the amino acid residue at position 20 of SEQ ID NO:

1.

25. The fusion protein of claim 1, consisting of the amino acid sequence set forth in SEQ ID NO: 10 ("SCF-SYN"), SEQ ID NO: 11 ("scFvCD133-SYN"), SEQ ID NO: 12 ("DARPinCD4-SYN"), SEQ ID NO: 13 ("scFVCD8-SYN"), SEQ ID NO: 14 ("scFVIA-2-SYN"), or SEQ ID NO: 15 ("GLP1-SYN").

26. 2. The fusion protein of claim 1, comprising the amino acid sequence set forth in SEQ ID NO: 16 ("SCF-SYN480"), SEQ ID NO: 17 ("scFvCD133-SYN480"), SEQ ID NO: 18 ("DARPinCD4-SYN480"), SEQ ID NO: 19 ("scFVCD8-SYN480"), SEQ ID NO: 20 ("scFVIA-2-SYN480"), or SEQ ID NO: 21 ("GLP1-SYN480").

27. A polynucleotide encoding the fusion protein of any one of claims 1 to 26.

28. A vector comprising the polynucleotide of claim 27.

29. 28. A host cell transfected, infected or transformed with the polynucleotide of claim 27 and / or a vector comprising said polynucleotide.

30. A particle functionalized with the fusion protein of claim 1 and comprising one or more viral protein(s) and one or more cargo(s).

31. 31. The particle of claim 30, which is a virus particle, more particularly a virus-like particle pseudotyped with the fusion protein of claim 1.

32. 32. The virus or virus-like particle of claim 31, comprising Gag protein, most preferably Gag protein originating from a virus selected from the group comprising Rous Sarcoma Virus (RSV), Feline Immunodeficiency Virus (FIV), Simian Immunodeficiency Virus (SIV), Moloney Leukemia Virus (MLV), and Human Immunodeficiency Virus (HIV-1 and HIV-2), in particular Human Immunodeficiency Virus Type 1 (HIV-1).

33. 33. The virus or virus-like particle of claim 32, wherein the viral structural protein has an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO:22 or SEQ ID NO:

23.

34. 31. The particle of claim 30, wherein the cargo is selected from the group consisting of organic molecules, polymers, polypeptides, polynucleotides, and small organic compounds having a molecular weight greater than 50 and less than about 2,500 daltons.

35. 35. The particle of claim 34, wherein the cargo is a polynucleotide, more particularly an RNA molecule or a DNA molecule.

36. i) a polypeptide (or a polynucleotide encoding the same) selected from the group consisting of a CRISPR-associated endonuclease, a base editing enzyme, an epigenetic editing factor, and a primer editor, and ii) one or more guide RNA molecules 31. The particle of claim 30, wherein the particle encapsulates

37. 31. The particle of claim 30, wherein the cargo polypeptide is fused to a viral structural protein (e.g., a GAG or PEG10 protein) either directly or via a linker.

38. 38. The particle of claim 37, wherein a viral structural protein (e.g., a GAG or PEG10 protein) and a cargo polypeptide (e.g., a nuclease such as Cas9) are fused, either directly or via a linker, to their respective domains that can dimerize in the presence of a compound.

39. 39. A cell line for producing a virus or virus-like particle according to any one of claims 30 to 38, comprising i) one or more polynucleotides encoding viral structural proteins required for the formation of said virus particles, ii) the polynucleotide of claim 17, and iii) one or more polynucleotides encoding cargo(s).

40. A pharmaceutical composition comprising an amount of particles according to any one of claims 30 to 38.

41. A pharmaceutical composition for treatment in a subject in need thereof, comprising a therapeutic amount of particles according to any one of claims 30 to 38.

42. 42. The pharmaceutical composition of claim 41, wherein the subject is suffering from a β-hemoglobinopathy.

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