Rotavirus-derived proteins and uses thereof

Abstract

Provided herein are nucleic acids, vectors, cells, and systems comprising engineered fusion-associated small transmembrane (FAST) proteins from fusogenic Rotaviruses that can mediate cell-cell membrane fusion and syncytium formation. Also provided herein are liposome nanoparticles and lipid nanoparticles comprising fusogenic Rotavirus-derived FAST proteins and their use for the delivery of genetic cargo to a cell.

Description

ROTAVIRUS-DERIVED PROTEINS AND USES THEREOFRELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application, U.S.S.N. 63 / 672,612, filed July 17, 2024, which is incorporated herein by reference.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (F093870000WO00-SEQ-CHB.xml; Size: 369,093 bytes; and Date of Creation: July 8, 2025) is herein incorporated by reference in its entirety.BACKGROUND

[0003] Fusion-associated small transmembrane (FAST) proteins are nonstructural proteins that induce cell-cell fusion and syncytium formation. FAST proteins are composed of three small functional domains: an ectodomain, transmembrane domain (TMD), and endodomain. Despite limited sequence similarity, comparable domains between different FAST proteins are in many cases functionally interchangeable, although not all combinations are tolerated, suggesting that a functional FAST protein requires specific combinations of membrane remodeling motifs.

[0004] Utilizing the structural and functional features of FAST proteins to design new FAST proteins that promote enhanced cell-cell fusion would be beneficial in the fields of membrane protein bioengineering, synthetic biology, genetic therapies, and drug delivery.SUMMARY

[0005] The present disclosure relates to the modification and engineering of fusion-associated small transmembrane (FAST) proteins and chimeras thereof. Specifically, the present disclosure utilizes structure-function relationships between different Rotavirus FAST proteins (e.g., different NSP1-1 and NSP1-2 proteins) to guide the generation of modified FAST proteins (e.g., a wild type FAST protein comprising an amino acid sequence with one or more mutations) and chimeras (e.g., FAST proteins comprising domains from two or more different FAST proteins) that display enhanced fusion activity.

[0006] FAST proteins are small (typically -100-200 amino acids) proteins that induce cell-cell fusion and are encoded by Reoviridae viruses (e.g., Rotaviruses). Infection with a virus or expression of a recombinant FAST protein induces progressive syncytium formation in cultured cells. Unlike fusogenic peptides of enveloped viruses, FAST proteins are nonstructural proteins (NSPs) involved only in inducing cell-cell fusion. Notably, FAST proteins lack receptor-binding capacity and therefore can induce cell-cell fusion of a wide range of cells at neutral pH and without a specific trigger (e.g., receptor binding or acidic pH).

[0007] The genera Aquareovirus and Orthoreovirus of the Reoviridae family are known to encode the following FAST proteins: plO, pl3, pl4, pl5, pl6, and p22. Rotaviruses, on the other hand, are known to encode only one FAST protein, NSP1-1. Specifically, NSP1-1 is encoded by Rotavirus serogroup B, G, and I (RVB, RVG, and RVI, respectively). Although FAST proteins display divergent amino acid sequences, they share a common structure consisting of an N-terminal exoplasmic domain (e.g., referred to herein as the “ectodomain”), a transmembrane domain (TMD), and a C-terminal cytoplasmic domain (e.g., referred to herein as the “endodomain”) (Figure 1). Commonly, acylation (e.g., myristoylation or palmitoylation) is essential for fusion activity. The transmembrane domain acts as a reverse signal-anchor sequence to localize FAST proteins through the plasma membrane with Necto / Cendo topology. The endodomain typically comprises a membrane- proximal polybasic motif (PBM, or polybasic region (PBR)) and an amphipathic a-helix (AH).

[0008] It is understood that the domains of FAST proteins are modular and can be interchanged between FAST proteins and retain functional activity (e.g., the ability to mediate cell-cell fusion). Therefore, the present disclosure describes modified FAST proteins and engineered chimeras thereof that leverage this feature to generate FAST proteins with improved fusogenicity that can be utilized in drug delivery platforms.

[0009] In some aspects, the present disclosure describes a FAST protein that is modified (e.g., a “variant” FAST protein) by introducing a single mutation or a combination of mutations into the amino acid sequence of a wild type FAST protein. In certain embodiments, the modified FAST protein is a RVB encoded FAST protein and may comprise an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1, 3, and 5-16 (i.e., full length, wild type FAST proteins encoded by a Rotavirus).

[0010] In certain embodiments, the modified FAST protein comprises an ectodomain, a transmembrane domain, and an endodomain, from the same FAST protein, wherein at least one of the three domains comprises one or more mutations. In certain embodiments, the modified FAST protein is a biologically functional FAST protein e.g., facilitates cell-cell fusion). In certain embodiments, the modified FAST protein comprises an endodomain with an amino acid sequence that is at least 80% identical to the endodomain of SEQ ID NOs: 1 or 3, or 5-16. In other embodiments, the modified FAST protein comprises an endodomain with an amino acid sequence that is at least 80% identical to the endodomain of SEQ ID NOs: 1 or 3.

[0011] In another aspect, the modified FAST protein may be a fusion protein comprising additional functional motifs (e.g., a myristoylation or palmitoylation motif, a polybasic motif) from a different FAST protein. In certain embodiments, the FAST protein may be a fusion protein further comprising a linker, an epitope tag, detectable label, effector domain, or a targeting moiety.

[0012] In another aspect, the present disclosure describes a chimeric FAST protein, which comprises domains from two or more different FAST proteins. In certain embodiments the chimeric FAST protein has enhanced fusogenic properties (e.g., cell-cell fusion and syncytium formation, promotesmembrane fusion). Defined herein, a chimeric FAST protein is a protein that comprises domains (e.g., ectodomain, transmembrane domain, or endodomain) from at least two different FAST proteins, to form functional FAST proteins. Alternatively, a chimeric FAST protein is a FAST protein that does not comprise an ectodomain, a transmembrane domain, and an endodomain from the same FAST protein.

[0013] In certain embodiments, a chimeric FAST protein comprises (i) an ectodomain that is derived from a first FAST protein, (ii) a transmembrane domain that is derived from a second FAST protein, and (iii) an endodomain that is derived from a third FAST protein to form a biologically active FAST protein. That is, the ectodomain, the transmembrane domain, and the endodomain are all from different FAST proteins. In other embodiments, a chimeric FAST protein comprises (i) an ectodomain and a transmembrane domain that are derived from the same FAST protein, and (ii) an endodomain that is derived from a second FAST protein. That is, the ectodomain and transmembrane domain are from the same FAST protein. In yet another embodiment, a chimeric FAST protein may comprise (i) an ectodomain that is derived from a first FAST protein, and (ii) a transmembrane domain and an endodomain that are derived from a second FAST protein. That is, the transmembrane domain and endodomain are from the same FAST protein. In another embodiment, a chimeric FAST protein may comprise (i) an ectodomain and an endodomain that are derived from a first FAST protein and (ii) a transmembrane domain that is derived from a second FAST protein. That is, the ectodomain and endodomain are from the same FAST protein. In certain embodiments, the chimeric FAST protein comprises an amino acid sequence provided in Table G (SEQ ID NOs: 311-323 and 337-340).

[0014] In another aspect, the disclosure provides a nucleic acid encoding a FAST protein described herein. In another aspect, the disclosure provides expression vectors comprising such nucleic acids. In yet another aspect, the disclosure provides cells (e.g., transformed cell lines) that comprise a FAST protein described herein, or a nucleic acid or vector encoding FAST proteins described above.

[0015] In certain aspects, the FAST proteins described herein are formulated into particles (e.g., liposomes, lipid nanoparticle) to facilitate membrane fusion. In certain embodiments, the liposomes or particles comprising the one or more FAST proteins described herein may be used as a drug delivery platform for delivery of an agent into a cell (e.g., nucleic acids, polypeptides, ribonucleoproteins, proteins, and / or small molecules).

[0016] Also provided herein are methods of using the FAST proteins, polypeptides, vectors, cells, and particles for drug delivery. In certain embodiments, the method is for delivering an agent to a subject, cell, collection of cells, or tissue. In some embodiments, the method is for delivering an agent to a subject or cell. In certain embodiments, the method is for delivering an agent to a subject. In some embodiments, the method is for delivering an agent to a cell.

[0017] In another aspect, the disclosure provides a pharmaceutical composition comprising: (i) a modified FAST protein and / or chimeric FAST protein described herein, a nucleic acid moleculedescribed herein, an expression vector described herein, a cell described herein, and / or a particle described herein, and (ii) a pharmaceutically acceptable excipient.

[0018] In yet another aspect, the disclosure provides a kit comprising: (i) a modified FAST protein and / or chimeric FAST protein described herein, a nucleic acid molecule described herein, an expression vector described herein, a cell described herein, and / or a particle described herein, and (ii) a set of instructions for delivering a substance or cargo to a cell.

[0019] The foregoing concepts, and additional concepts discussed below, may be arranged in any suitable combination, as the present disclosure is not limited in this respect. Further, other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments when considered in conjunction with the accompanying figures.BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosure and together with the description, provide non-limiting examples.

[0021] Figure 7 is a schematic of a fusion-associated small transmembrane protein (FAST) in proportion to the pre-fusion trimeric structure of a well-known viral fusogen, VSV-G (PDB: 5I2S), on a membrane. A key feature of many other viral fusogens is their tall ectodomains (e.g., VSV-G), which in their metastable pre-fusion state, typically extend more than 10 nm from the membrane. FAST proteins, on the other hand, are all small (~ 100-200 residues), nonstructural viral proteins expressed by virus-infected cells. Figure 1 shows the transmembrane domain serves as a reverse signal-anchor sequence to direct a bitopic Nout / Cintype I topology in the membrane. This topology localizes a very small N-terminal ectodomain (~20-40 residues) external to the plasma membrane and positions the longer (~40-140 residues) C-terminal endodomains in the cytoplasm.

[0022] Figure 2 shows the phylogeny of FAST proteins. FAST proteins of Orthoreovirus, Aquareo virus, and Rotavirus genera are divided into two major clades. One clade consists of the pl 6- FAST of Aquareoviruses C and G and the p22-FAST proteins of Aquareovirus A. Another clade comprises FAST proteins of fusogenic reoviruses of mammals, birds, and reptiles. Baboon orthoreovirus (BRV) pl5-FAST, Broome orthoreovirus pl3-FAST, and reptilian orthoreovirus (RRV) pl4-FAST form a single subcluster, and another subcluster is formed by plO-FAST proteins encoded by avian orthoreovirus (ARV) and Nelson Bay orthoreovirus (NBV). NSP1-1 is the most recently discovered FAST protein and the only FAST protein in the genus Rotavirus. NSP1-1 proteins are encoded by RVB, RVG, and RVI.

[0023] Figure 3 shows the syncytial activity of chimeric FAST proteins (sequences of the chimeric proteins are listed in Table I) versus the syncytial activity of the endogenous wild type FAST protein. More specifically, the Rotavirus chimeric FAST proteins shown in Figure 3 comprise an ectodomainand a transmembrane domain of the same FAST protein, and the endodomain of F100 (amino acids 66-101 from SEQ ID NO: 1), which is referred to herein “FlOOendo” or “endoFlOO.” That is, the FAST protein from which both the ectodomain (“ecto”) and the transmembrane domain (“TMD”) are selected varies, and that the endodomain is kept constant, and only comprises the endodomain of F100. For example, F102, read on the y-axis, is to be interpreted as the chimeric FAST protein comprising the ectodomain and the transmembrane domain of F102 (e.g., “ecto+TMD of F102” or “F102 ecto+TMD”) in combination with the endodomain of F100. While F102, read on the x-axis, is to be interpreted as the wild type FAST protein, Fl 02 which comprises the amino acid sequence of SEQ ID NO: 12. The syncytial activity of each Rotavirus chimeric FAST protein are tabulated in Table K. The sequences of the chimeric FAST proteins can be found in Table G. The sequences of the wild type FAST proteins can be found in Table A.DEFINITIONS

[0024] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5 th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0025] The term “amino acid” refers to organic compounds that serve as the building blocks of proteins. Twenty amino acids play essential roles in protein synthesis and function and differ based on their side chain structures, which contribute to their distinct properties and functions. Amino acids are often grouped by the chemistry of the side chain. These groups are polar-uncharged, polar-charged and non-polar. Eight of the 20 amino acids are polar-uncharged: asparagine (N), cysteine (C), glutamine (Q), histidine (H), serine (S), threonine (T), tryptophan (W) and tyrosine (Y). Eight of the 20 amino acids are non-polar: alanine (A), glycine (G), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), and valine (V). The final four are polar-charged: arginine (R), aspartic acid (D), glutamic acid (E), and lysine (L).

[0026] The terms “administer,” “administering,” and “administration” refer to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a treatment or therapeutic agent, or a composition of treatments or therapeutic agents, in or on a subject.

[0027] The terms “composition” and “formulation” are used interchangeably.

[0028] The term “biomolecule” or “biological molecule” refers to any substance produced by cells or living organisms and includes carbohydrates, lipids, nucleic acids, proteins, and vitamins.

[0029] The terms “condition,” “disease,” and “disorder” are used interchangeably.

[0030] A “cell,” as used herein, may be present in a population of cells (e.g., in a tissue, a sample, a biopsy, an organ, or an organoid). In some embodiments, a population of cells is composed of a plurality of different cell types. Cells for use in the methods and systems of the present disclosure can be present within an organism, a single cell type derived from an organism, or a mixture of cell types. Included are naturally occurring cells and cell populations, genetically engineered cell lines, cells derived from transgenic animals, cells from a subject, etc. In some embodiments, the cells are mammalian cells (e.g., complex cell populations such as naturally occurring tissues). In some embodiments, the cells are from a human. In certain embodiments, the cells are collected from a subject e.g., a human) through a medical procedure, such as a biopsy. Alternatively, the cells may be a cultured population (e.g., a culture derived from a complex population, or a culture derived from a single cell type where the cells have differentiated into multiple lineages). The cells may also be provided in situ in a tissue sample.

[0031] The term “gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5’ non-coding sequences) and following (3’ non- coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” or “chimeric construct” refers to any gene or a construct, not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene or chimeric construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism, but which is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non- native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

[0032] The term “detectable label” refers to a moiety that has at least one element, isotope, or functional group incorporated into the moiety which enables detection of the molecule, e.g., a protein or peptide, or other entity, to which the label is attached. Labels can be directly attached or can be attached via a linker. It will be appreciated that the label may be attached to or incorporated into a molecule, for example, a protein, polypeptide, or other entity, at any position. In general, a detectable label can fall into any one (or more) of five classes: I) a label which contains isotopic moieties, which may be radioactive or heavy isotopes, including, but not limited to,2H,3H,13C,14C,15N,18F,31P, 32P,35S,67Ga,76Br, "mTc (Tc-99m),H 1In,123I,125I,131I, l53Gd,169Yb, and186Re; II) a label which contains an immune moiety, which may be antibodies or antigens, which may be bound to enzymes (e.g., such as horseradish peroxidase); III) a label which is a colored, luminescent, phosphorescent, or fluorescent moieties (e.g., such as the fluorescent label fluorescein-isothiocyanate (FITC); IV) a label which has one or more photo affinity moieties; and V) a label which is a ligand for one or more known bindingpartners (e.g., biotin-streptavidin, FK506-FKBP). In certain embodiments, a FAST protein described herein is modified to comprise a detectable label. In certain embodiments, a label comprises a radioactive isotope, preferably an isotope which emits detectable particles, such as P particles. In certain embodiments, the label comprises a fluorescent moiety. In certain embodiments, the label is the fluorescent label fluorescein-isothiocyanate (FITC). In certain embodiments, the label comprises a ligand moiety with one or more known binding partners. In certain embodiments, the label comprises biotin. In some embodiments, a label is a fluorescent polypeptide (e.g., GFP or a derivative thereof such as enhanced GFP (EGFP)) or a luciferase (e.g., a firefly, Renilla, or Gaussia luciferase). It will be appreciated that, in certain embodiments, a label may react with a suitable substrate (e.g., a luciferin) to generate a detectable signal. Non-limiting examples of fluorescent proteins include GFP and derivatives thereof, proteins comprising fluorophores that emit light of different colors such as red, yellow, and cyan fluorescent proteins. Exemplary fluorescent proteins include, e.g., Sirius, Azurite, EBFP2, TagBFP, mTurquoise, ECFP, Cerulean, TagCFP, mTFPl, mUkGl, mAGl, AcGFPl, TagGFP2, EGFP, mWasabi, EmGFP, TagYPF, EYFP, Topaz, SYFP2, Venus, Citrine, mKO, mK02, mOrange, m0range2, TagRFP, TagRFP-T, mStrawberry, mRuby, mCherry, mRaspberry, mKate2, mPlum, mNeptune, T-Sapphire, mAmetrine, mKeima. See, e.g., Chalfie, M. and Kain, SR (eds.) Green fluorescent protein: properties, applications, and protocols Methods of biochemical analysis, v. 47 Wiley-Interscience, Hoboken, N.J., 2006; and Chudakov, DM, et al., Physiol Rev. 90(3): 1103-63, 2010, for discussion of GFP and numerous other fluorescent or luminescent proteins. In some embodiments, a label comprises a dark quencher, e.g., a substance that absorbs excitation energy from a fluorophore and dissipates the energy as heat.

[0033] The term “peptide tag” refers to a peptide amino acid sequence that is genetically fused to a protein sequence to impart one or more functions onto the proteins that facilitate the manipulation of the protein for various purposes, such as, visualization, purification, solubilization, and separation, etc. Peptide tags can include various types of tags categorized by purpose or function, which may include “affinity tags” (to facilitate protein purification), “solubilization tags” (to assist in proper folding of proteins), “chromatography tags” (to alter chromatographic properties of proteins), “epitope tags” (to bind to high affinity antibodies), “fluorescence tags” (to facilitate visualization of proteins in a cell or in vitro). Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, thioredoxin (Trx) tags, and Strep tags.

[0034] The term “fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the aminoterminal (N-terminal) portion of the fusion protein or at the carboxy-terminal (C-terminal) protein thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively. A protein may comprise different domains, for example, an ectodomain of one FAST protein and an endodomain of another FAST protein. Any of the proteins provided herein may be produced by anymethod known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.

[0035] The term “mutation” as used herein, refers to a substitution, insertion, or deletion of a single residue or a combination of residues within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)).

[0036] The term ‘biologically active” refers to the capacity of a FAST protein described herein to achieve a defined biological effect on a target (e.g., to have fusogenic properties).

[0037] The term “fusogen” refers to a protein or peptide that acts on a membrane to overcome the forces preventing spontaneous membrane fusion and ensure fusion occurs in a controlled and regulated manner. Membrane fusion can refer to fusion of plasma membranes of multiple cells, fusion of a cell membrane or an endosome with a lipid-based delivery vehicle such as a liposome or lipid nanoparticle, fusion of a cell membrane or an endosome with the outer membrane of an enveloped virus or virus-like particle, or fusion of any other combination of two opposed lipid layers. In certain embodiments, membrane fusion can refer to the merger of plasma membranes of multiple cells, merger of a cell membrane or an endosome with a lipid-based delivery vehicle such as a liposome or lipid nanoparticle, merger of a cell membrane or an endosome with the outer membrane of an enveloped virus or virus-like particle, or merger of any other combination of two opposed lipid layers.

[0038] The term “fusion-associated small transmembrane” or “FAST” protein refers to a non- structural, accessory protein that induces cell-cell fusion. FAST proteins are encoded by viruses of the Reoviridae family, specifically found within the genera Aquareovirus, Orthoreo virus, and Rotavirus, to induce syncytium formation. FAST proteins are the smallest viral fusogens, and unlike their enveloped virus counterparts, they have evolved specifically to induce cell-to-cell, not virus-cell, membrane fusion. This distinct evolutionary function is reflected in structural and functional features that distinguish this singular family of viral fusogens from all other protein fusogens. FAST proteins comprise specific combinations of different domains (e.g., an ectodomain, a transmembrane domain, and an endodomain) that form small, modular membrane fusogens. In certain embodiments, a FAST protein is a chimeric FAST protein comprising domains from two or more different FAST proteins (e.g., an ectodomain of a first FAST protein fused with the endodomain of a second FAST protein). Incertain embodiments, the chimeric FAST protein comprises domains from two or more wild type FAST proteins. That is, the domains do not comprise any mutations. In other embodiments, the chimeric FAST protein comprises domains from two or more different FAST proteins, wherein one or more of the domains within the chimeric FAST protein are modified (e.g., comprise one or more mutations). Examples of wild type FAST proteins are found within Table A of the present disclosure. Throughout the disclosure, chimeric FAST proteins that comprise an ectodomain and a transmembrane domain from the same FAST protein may be indicated by the following nomenclature, “ecto+TMD.” For example, to describe a chimeric protein comprising the ectodomain and the transmembrane domain of NSP1-1 (see Figure 1), it may be described as “NSP1-1 ecto+TMD” or “ecto+TMD NSP1-1.” Similarly, the endodomain of a FAST protein may be indicated by the term “endo.” For example. To describe an endodomain of NSP1-1, it may be described as “endo NSP1-1”, “NSP1-1 endo”, or “endo of NSP1-1.”

[0039] The term “syncytium formation” refers to the process of creating a multi-nucleate cell by fusion of neighboring cells.

[0040] The term “fusogenic” refers to the function of “facilitating fusion”, especially relating to cells and pertains to a fusogen. In certain embodiments, a fusogenic peptide may be referred to as “highly fusogenic,” “moderately fusogenic,” or “non-fusogenic.” To classify fusogenicity of each FAST protein, the ratio of the average sum of GFP+ area of splitGFP cells expressing a FAST protein to the average sum of GFP+ of splitGFP cells expressing a reference FAST protein on the same plate (see Examples section). A ratio that is greater than or equal 0.75 is classified as “highly fusogenic,” a ratio between 0.05-0.75 is classified as “moderately fusogenic,” and a ratio less than or equal to 0.05 is classified as “non-fusogenic.”

[0041] The term “protein lipidation” refers to the covalent attachment of lipid molecules to proteins, and there are at least five types of protein lipidations including S-palmitoylation, N-myristoylation, S- prenylation, glycosylphosphatidylinositol (GPI) anchor, and cholesterylation. A “lipidation motif’ refers to the location within a peptide or protein sequence where lipid attachment occurs. Similarly, a “palmitoylation motif’ refers to the location within a peptide or protein sequence where palmitic acid attachment occurs (e.g., at a cysteine residue via a thioester linkage). Likewise, a “myristoylation motif’ refers to the location within a peptide or protein sequence where myristic acid attachment occurs (e.g., the alpha-amino group of an N-terminal glycine residue). In certain embodiments, a “palmitoylation motif’ or a “myristoylation motif’ is incorporated into a FAST protein (e.g., a fusion FAST protein) or into a chimeric FAST protein, wherein the “palmitoylation motif’ and “myristoylation motif’ are the location within a peptide or protein sequence where lipidation occurs. In certain embodiments, a “palmitoylation motif’ comprises a sequence of amino acids that includes at least one cysteine residue and is the location wherein a palmitic acid can be attached. In certain embodiments, a “myristoylation motif’ is a sequence of amino acids that comprises glycine residues, wherein a myristic acid can be attached.

[0042] The term “ectodomain” refers to a domain of a membrane protein that extends into the extracellular space (the space outside a cell). The ectodomain of a FAST protein is also referred to as the N-terminal ectodomain and is often considered to be a small (20-40 amino acid residues), amphiphilic peptide that share function attributes with fusion peptides (FPs), which are specific proteinogenic subsequences within viral fusion proteins that play a key role in interacting and inserting into membranes. In many cases, myristoylation of the ectodomain at a penultimate N- terminal glycine is required for fusion activity. In some embodiments, the hydrophobic residues in the ectodomain may include any non-polar amino acids, such as alanine (A), isoleucine (I), leucine (L), phenylalanine (F), and valine (V) in the extracellular-facing domain of FAST protein.

[0043] The term “transmembrane domain” (TMD) refers to the transmembrane domain of FAST protein which functions as a fusion module, directly participating in the membrane fusion reaction. Specific residues near the N- and C-termini of the TM domain may be particularly important for fusion activity. A TMD of a FAST protein serves as a reverse signal-anchor sequence to direct a bitopic (e.g., a single -pass membrane protein) Nout / Cintype I topology in the membrane. This topology localizes a N-terminal ectodomain (e.g., ~ 20-40 residues) external to the plasma membrane and positions considerably longer (e.g., ~40-140 residues) C-terminal endodomain in the cytoplasm.

[0044] The term “endodomain” refers to the C-terminal cytoplasmic domain of a FAST protein and plays a prominent role in the fusion process and an undefined role in pore formation. The endodomain may comprise a polybasic region (PBR) and / or an amphipathic a-helix (AH), which is sometimes referred to as a hydrophobic patch. For some FAST proteins, fusion activity requires palmitoylation of a juxtamembrane di-cysteine motif in the endodomain. Typically, the polybasic motif consists of positively charged residues, arginine and lysine, in the endodomain and proximal to the transmembrane domain. Positively charged residues can be either consecutive or spaced out within the first 25 residues C-terminal to the transmembrane domain. The amphipathic helix is an alpha helical structure in the endodomain with amphipathic characteristics, as determined by hydrophobic moment. This helical structure is proximal to the transmembrane domain, generally within 30 residues C-terminal to the transmembrane domain.

[0045] The term “particle” refers to a small object, fragment, or piece of a substance that may be a single element, inorganic material, organic material, or mixture thereof. Examples of particles include polymeric particles, single-emulsion particles, double -emulsion particles, coacervates, liposomes, microparticles, nanoparticles (e.g., lipid nanoparticles), each of which have an average characteristic dimension of about less than about 1 mm and at least 1 nm, where the characteristic dimension, or “critical dimension,” of the particle is the smallest cross-sectional dimension of the particle. A particle may be composed of a single substance or multiple substances. In certain embodiments, the particle is not a viral particle. In other embodiments, the particle is not a liposome. In certain embodiments, the particle is not a micelle. In certain embodiments, the particle is substantially solid throughout. In certain embodiments, the average diameter of the particle is at least about 10 nm, at least about 30 nm,at least about 100 nm, at least about 300 nm, at least about 1 pm, at least about 3 pm, at least about 10 pm, at least about 30 pm, at least about 100 pm, at least about 300 pm, or at least about 1 mm. In certain embodiments, the average diameter of the particle is less than about 1 mm, less than about 300 pm, less than about 100 pm, less than about 30 pm less than about 10 pm, less than about 3 pm, less than about 1 pm, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm. Combinations of the above ranges (e.g., at least about 100 nm and less than about 1 pm) are also within the scope of the present invention.

[0046] In certain embodiments, the particle is a nanoparticle (i.e., a particle having a characteristic dimension of less than about 1 micrometer and at least about 1 nanometer, where the characteristic dimension of the particle is the smallest cross-sectional dimension of the particle). In certain embodiments, the particle further comprises one or more of a PEG-lipid, sterol, phospholipid, helper lipid, or stabilizing excipient. In certain embodiments, the particle is a lipid nanoparticle. In certain embodiments, the particle comprises a FAST protein described herein. In certain embodiments, the lipid nanoparticle comprising the FAST protein is used for delivering an agent (e.g., cargo) to a cell, tissue, or subject. In certain embodiments, the FAST protein facilitates the particles incorporation (e.g., fusion) into the cell and / or tissue.

[0047] The term “liposome” refers to a self-assembled (phospho)lipid-based drug vesicles that form a bilayer (uni-lamellar) and / or a concentric series of multiple bilayers (multilamellar) enclosing a central aqueous compartment. The size of liposomes ranges from 30 nm to the micrometer scale, with the phospholipid bilayer being 4-5 nm thick. In certain embodiments, a liposome may comprise a FAST protein and serve as a delivery vehicle for cargos such as small molecular drugs, protein, nucleic acid, and imaging agents. Different administration routes, such as parenteral, pulmonary, oral, transdermal, ophthalmic, and nasal routes, have been developed to improve therapeutic efficacy and patient compliance. Eiposomes can be classified as unilamellar vesicles (UEVs), oligolamellar vesicles (OEVs), multilamellar vesicles (MEVs), and multivesicular liposomes (MVLs) depending on the compartment structure and lamellarity.

[0048] The terms “composition” and “formulation” are used interchangeably.

[0049] The term “prevent,” “preventing,” or “prevention” refers to a prophylactic treatment of a subject who is not and was not with a disease but is at risk of developing the disease or who was with a disease, is not with the disease, but is at risk of regression of the disease. In certain embodiments, the subject is at a higher risk of developing the disease or at a higher risk of regression of the disease than an average healthy member of a population.

[0050] The terms “polynucleotide,” “nucleotide sequence,” “nucleic acid,” “nucleic acid molecule,” “nucleic acid sequence,” and “oligonucleotide” refer to a series of nucleotide bases (also called “nucleotides”) in DNA and RNA and mean any chain of two or more nucleotides. The polynucleotides can be chimeric mixtures or derivatives or modified versions thereof, and singlestranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, orphosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc.

[0051] The term “ribonucleotide” refers to a nucleotide containing ribose as its pentose component. It is considered a molecular precursor of nucleic acids. Nucleotides are the basic building blocks of DNA and RNA. Ribonucleotides themselves are basic monomeric building blocks for RNA. In living organisms, the most common bases for ribonucleotides are adenine (A), guanine (G), cytosine (C), or uracil (U).

[0052] A “protein,” “peptide,” or “polypeptide” comprises a polymer of amino acid residues linked together by peptide bonds. The term refers to proteins, polypeptides, and peptides of any size, structure, or function. Typically, a protein will be at least three amino acids long. A protein may refer to an individual protein or a collection of proteins. Proteins may contain only natural amino acids, although non-natural amino acids (z.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and / or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in a protein may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a lipid (e.g., myristoyl and palmitoyl), a fatty acid group, a linker for conjugation or functionalization, or other modification. A protein may also be a single molecule or may be a multi-molecular complex. A protein may be a fragment of a naturally occurring protein or peptide. A protein may be naturally occurring, recombinant, synthetic, or any combination of these. A protein may also be a therapeutic protein administered as a treatment for a disease or disorder e.g., one that is associated with a change in the RNA expression and / or translation profile of a cell taken from a subject). In certain embodiments, the protein is a FAST protein. In certain embodiments, the protein is a modified FAST protein (e.g., comprising a mutation or a combination of mutations). In certain embodiments, the protein is a fusion protein of one or more FAST proteins. In certain embodiments, the fusion protein is a FAST protein further comprising additional functional motifs of other FAST proteins (e.g., polybasic region). In certain embodiments, the protein is a chimeric FAST protein comprising domains (e.g., ectodomain, transmembrane domain, and endodomain) of two or more FAST proteins.

[0053] The term “linker,” as used herein, refers to a molecule linking two other molecules or moieties. The linker can be an amino acid sequence in the case of a linker joining two polypeptides into a fusion protein. The linker can also be a nucleotide sequence in the case of joining two nucleotide sequences together. In other embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 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, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

[0054] The term “small molecule” refers to molecules, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight. Typically, a small molecule is an organic compound (i.e., it contains carbon). The small molecule may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyl, carbonyls, and heterocyclic rings, etc.). In certain embodiments, the molecular weight of a small molecule is not more than about 1 ,000 g / mol, not more than about 900 g / mol, not more than about 800 g / mol, not more than about 700 g / mol, not more than about 600 g / mol, not more than about 500 g / mol, not more than about 400 g / mol, not more than about 300 g / mol, not more than about 200 g / mol, or not more than about 100 g / mol. In certain embodiments, the molecular weight of a small molecule is at least about 100 g / mol, at least about 200 g / mol, at least about 300 g / mol, at least about 400 g / mol, at least about 500 g / mol, at least about 600 g / mol, at least about 700 g / mol, at least about 800 g / mol, or at least about 900 g / mol, or at least about 1,000 g / mol. Combinations of the above ranges (e.g., at least about 200 g / mol and not more than about 500 g / mol) are also possible. In certain embodiments, the small molecule is a therapeutically active agent such as a drug (e.g., a molecule approved by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations (C.F.R.)). The small molecule may also be complexed with one or more metal atoms and / or metal ions. In this instance, the small molecule is also referred to as a “small organometallic molecule.” Preferred small molecules are biologically active in that they produce a biological effect in animals, preferably mammals, more preferably humans. Small molecules include, but are not limited to, radionuclides and imaging agents. In certain embodiments, the small molecule is a drug. Preferably, though not necessarily, the drug is one that has already been deemed safe and effective for use in humans or animals by the appropriate governmental agency or regulatory body. For example, drugs approved for human use are listed by the FDA under 21 C.F.R. §§ 330.5, 331 through 361, and 440 through 460, incorporated herein by reference; drugs for veterinary use are listed by the FDA under 21 C.F.R. §§ 500 through 589, incorporated herein by reference. All listed drugs are considered acceptable for use in accordance with the present disclosure.

[0055] The term “transcript” or “RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a complementary copy of a DNA sequence, it is referred to as the primary transcript, or it may be an RNA sequence derived from post-transcriptional processing of the primary transcript and is then referred to as the mature RNA. “Messenger RNA” (mRNA) refers to the RNA that is without introns and can be translated into a polypeptide by the cell.

[0056] The term “tissue” refers to any biological tissue of a subject (including a group of cells, a body part, or an organ) or a part thereof, including blood and / or lymph vessels, which is the object to which a compound, particle, and / or composition of the invention is delivered. A tissue may be an abnormal or unhealthy tissue, which may need to be treated. A tissue may also be a normal or healthy tissue that is under a higher than normal risk of becoming abnormal or unhealthy, which may need tobe prevented. In certain embodiments, the tissue is the central nervous system. In certain embodiments, the tissue is the brain.

[0057] The terms “biologic,” “biologic drug,” and “biological product” refer to a wide range of products such as vaccines, blood and blood components, allergenics, somatic cells, gene therapy, tissues, nucleic acids, and proteins. Biologies may include sugars, proteins, or nucleic acids, or complex combinations of these substances, or may be living entities such as cells and tissues. Biologies may be isolated from a variety of natural sources (e.g., human, animal, microorganism) and / or may be produced by biotechnological methods and / or other technologies.

[0058] The term “antibody” refers to a functional component of serum and is often referred to either as a collection of molecules (antibodies or immunoglobulins) or as one molecule (the antibody molecule or immunoglobulin molecule). An antibody is capable of binding to or reacting with a specific antigenic determinant (the antigen or the antigenic epitope), which in turn may lead to induction of immunological effector mechanisms. An individual antibody is usually regarded as monospecific, and a composition of antibodies may be monoclonal (i.e., consisting of identical antibody molecules) or polyclonal (i.e., consisting of two or more different antibodies reacting with the same or different epitopes on the same antigen or even on distinct, different antigens). Each antibody has a unique structure that enables it to bind specifically to its corresponding antigen, and all natural antibodies have the same overall basic structure of two identical light chains and two identical heavy chains. Antibodies are also known collectively as immunoglobulins. An antibody may be of human or non-human (for example, rodent such as murine, dog, camel, etc.) origin (e.g., may have a sequence originally developed in a human or non-human cell or organism), or may be or comprise a chimeric, humanized, reshaped, or reformatted antibody based, e.g., on a such a human or non-human antibody (or, in some embodiments, on an antigen-binding portion thereof).

[0059] A “subject” to which administration is contemplated refers to a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or non-human animal. In some embodiments, the non- human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey) or mouse). The term “patient” refers to a subject in need of treatment of a disease. In some embodiments, the subject is human. In some embodiments, the patient is human. The human may be a male or female at any stage of development. A subject or patient “in need” of treatment of a disease or disorder includes, without limitation, those who exhibit any risk factors or symptoms of a disease or disorder. In some embodiments, a subject is a non-human experimental animal (e.g., a mouse, rat, dog, pig, or non-human primate).

[0060] The term “therapeutic agent,” as used herein, refers to any agent that can be used to treat a disease or disorder, or reduce or alleviate the symptoms of a disease or disorder. In some embodiments, the therapeutic agent is a small molecule, a protein, a peptide, a nucleic acid, a lipid, or a carbohydrate. In some embodiments, the therapeutic agent is a known drug and / or an FDA-approved drug. In certain embodiments, the protein is an antibody. In certain embodiments, the protein is an antibody variant or antibody fragment. In certain embodiments, the protein is a receptor, or a fragment or variant thereof. In certain embodiments, the protein is a cytokine. In certain embodiments, the nucleic acid is an mRNA, an antisense RNA, an miRNA, an siRNA, an RNA aptamer, a double stranded RNA (dsRNA), a short hairpin RNA (shRNA), or an antisense oligonucleotide (ASO).

[0061] The term “agent” or “cargo” refers to an organic molecule, inorganic molecule, protein, peptide, polynucleotide, targeting agent, an isotopically labeled chemical compound, vaccine, an immunological agent, or an agent useful in bioprocessing. Agents that are delivered by the systems (e.g., particles or liposomes comprising a FAST protein) described herein may be therapeutic, prophylactic, diagnostic, cosmetic, or nutraceutical agents. Any chemical compound to be administered to a subject may be delivered using the nanoparticles (e.g., lipid nanoparticles), or liposomes, described herein. Exemplary agents that may be included in a composition (e.g., a particle or liposome for the delivery of the agent) described herein include, but are not limited to, small molecules, organometallic compounds, polynucleotides, proteins, peptides, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, small molecules linked to proteins, glycoproteins, steroids, nucleotides, oligonucleotides, polynucleotides, nucleosides, antisense oligonucleotides, lipids, hormones, vitamins, cells, metals, targeting agents, isotopically labeled chemical compounds, drugs (e.g., compounds approved for human or veterinary use by the U.S. Food and Drug Administration as provided in the Code of Federal Regulations), vaccines, immunological agents, agents useful in bioprocessing, and mixtures thereof. The targeting agents are described in more detail herein. In certain embodiments, the agents are nutraceutical agents. In certain embodiments, the agents are pharmaceutical agents (e.g., a therapeutic or prophylactic agent). In certain embodiments, the agent is an antibiotic agent (e.g., an anti-bacterial, anti-viral, or anti-fungal agent), anesthetic, steroidal agent, anti-proliferative agent, antiinflammatory agent, anti-angiogenesis agent, anti-neoplastic agent, anti-cancer agent, anti-diabetic agent, antigen, vaccine, antibody, decongestant, antihypertensive, sedative, birth control agent, progestational agent, anti-cholinergic, analgesic, immunosuppressant, anti-depressant, anti-psychotic, P-adrenergic blocking agent, diuretic, cardiovascular active agent, vasoactive agent, non-steroidal, nutritional agent, anti-allergic agent, or pain-relieving agent. Vaccines may comprise isolated proteins or peptides, inactivated organisms and viruses, dead organisms and viruses, genetically altered organisms or viruses, polynucleotides (e.g., mRNA), and cell extracts. Therapeutic and prophylactic agents may be combined with interleukins, interferon, cytokines, and adjuvants, such as cholera toxin, alum, Freund’ s adjuvant, etc.

[0062] An “effective amount” of a compound described herein refers to an amount sufficient to elicit the desired biological response. An effective amount of a compound described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound,the condition being treated, the mode of administration, and the age and health of the subject. In certain embodiments, an effective amount is a therapeutically effective amount. In certain embodiments, an effective amount is a prophylactic treatment. In certain embodiments, an effective amount is the amount of a compound described herein in a single dose. In certain embodiments, an effective amount is the combined amounts of a compound described herein in multiple doses.

[0063] A “therapeutically effective amount” of a treatment or therapeutic agent is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a treatment or therapeutic agent means an amount of the therapy, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and / or enhances the therapeutic efficacy of another therapeutic agent.

[0064] The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed (e.g., prophylactically or upon suspicion or risk of disease). In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms in the subject, or family members of the subject). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence. In some embodiments, treatment may be administered after using the methods disclosed herein and observing a change in the RNA expression or translation profile in a cell or tissue in comparison to a healthy cell or tissue.

[0065] As used herein, the term “variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature comprising one or more changes in amino acid residues (i.e., “substitutions”, “insertions, or “deletions”) as compared to a wild type amino acid sequence. The term “variant” encompasses homologous proteins having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a reference sequence and having the same or substantially the same functional activity or activities as the reference sequence. The term also encompasses mutants, truncations, or domains of a reference sequence that display the same or substantially the same functional activity or activities as the reference sequence.

[0066] As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene, or characteristic as it occurs in nature as distinguished from mutant or variant forms.

[0067] Throughout the present disclosure, when a range of values is listed, it is intended to encompass each value and sub-range within the range. Where ranges are given, endpoints are included.

[0068] Unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular.

[0069] The details of certain embodiments of the invention are set forth in the Detailed Description of Certain Embodiments, as described below. Other features, objects, and advantages of the invention will be apparent from the Drawings, Definitions, Examples, and Claims.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0070] The aspects described herein are not limited to specific embodiments, methods, uses, or configurations, and as such can, of course, vary. The terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.

[0071] The present disclosure relates to the modification and engineering of Rotavirus FAST proteins and chimeras thereof. Specifically, the present disclosure utilizes structure-function relationships between different FAST proteins (e.g., different NSP1-1 and NSP1-2 of Rotaviruses) to guide the generation of modified / engineering FAST proteins. In certain embodiments, the modified and / or engineered FAST proteins (e.g., chimeric FAST proteins) display enhanced fusion activity. In certain aspects, the modified / engineered FAST proteins (e.g., a FAST protein comprising an amino acid sequence with one or more mutations) and chimeras (e.g., FAST proteins comprising domains from two or more FAST proteins) are incorporated into liposomes or formulated into particles (e.g., lipid nanoparticles (ENPs)) to facilitate membrane fusion. In certain embodiments, the liposomes or particles comprising the one or more FAST proteins described herein may be used as part of a drug delivery platform for delivering a cargo (e.g., nucleic acids, polypeptides, proteins, ribonucleoproteins, and / or small molecules) to a cell .Modified FAST Proteins

[0072] In one aspect, the present disclosure describes proteins that are modified FAST proteins encoded by Rotaviruses (e.g., wild type FAST proteins comprising at least one amino acid mutation). In some embodiments, the modified FAST protein comprises one of the following amino acid sequences found in Table A. In certain embodiments, the protein comprises a sequence that has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to an amino acid sequence found in Table A. In certain embodiments, the modified FAST protein comprises at least 1, 2, 3, 4, 5, 6, 7 8, 9, 10, 11, 12, 13, 14, or 15 mutations. In certain embodiments, one or more mutations are found in the same domain (e.g., the endodomain) of the FAST protein. In certain embodiments, one or more mutations are found in different domains (e.g., the ectodomain and the endodomain) of the FAST protein. In certain embodiments, the modified FAST protein can comprise up to one, up to two, up to three, up to four, up to five, up to six, up to seven, or up to eight mutations. In someembodiments, the protein is a truncated version of the wild type protein (e.g., a FAST protein comprising only the ectodomain, the endodomain, the ectodomain and transmembrane domain, or the transmembrane domain and the endodomain).Table A. Amino acid sequences of exemplary wild type NSP1-1 and NSP1-2 proteins.Bold indicates the ectodomain of the FAST protein.Underline indicates transmembrane domain of the FAST protein.No effect indicates endodomain of the FAST protein

[0073] In certain embodiments, the ectodomains of the modified FAST proteins and / or chimericFAST proteins comprise an amino acid sequence selected from the following sequences:MGNRQSSAQLNSHLTHINSQNSNLFISDSKTAVFHTQH (SEQ ID NO: 17)MGNRQSSLQSQTHRTDINSHNSNIYLQSASSAEFRTQH (SEQ ID NO: 18)MGSSQSSLQSQVHSTNIHSQHSSIHLQGTSTANFTTQH (SEQ ID NO: 19)MGSHQSSYQVNNQNTIISNSKNLDFKPSTSSSLSQVN (SEQ ID NO: 20)MGNSNSNVQINQQNTHIQASDSKLNLQDQKTTSLESTQ (SEQ ID NO: 21)MGNTYNVHNNQQVSSNTVHGSGQIHSEDQKTSSQITTI (SEQ ID NO: 22)MGGRISQQLSQQNTYHIASGNSQIYSQDQKTNQQVAVA (SEQ ID NO: 23)MGSRQSSLQSQSHRIDIQSHHSNIHLNSNTLADFKTHH (SEQ ID NO: 24)MGNKQSSAQINFRQTNIGSGNHNLRIEDNGSVTFTSTH (SEQ ID NO: 25)MGNRQSSAQLNSHLTHINSQNSNLFISDSKTAVFHTQH (SEQ ID NO: 26)MGSSQSSLQSQIHSTNIHSQHSSIHLQGTSTASFTTQH (SEQ ID NO: 27)MGSRQSSLQSQTPRTDINSHNSNIFLQRAATAEFRTQH (SEQ ID NO: 28)MGNRQSSLQSQTHRTDINSHHSNIYLQSASFAEFKTYH (SEQ ID NO: 29) MGSSQSSLQTQVHSTNIHSQHSSIHLQGTSTATFTTHQ (SEQ ID NO: 30)

[0074] In certain embodiments, the transmembrane domains of the modified FAST proteins and / or chimeric FAST proteins comprise an amino acid sequence selected from the following sequences:ILLAAGVGIIATLLVLLLCSCVL (SEQ ID NO: 31)ILVVAGAALIALLFAFLVSSLVC (SEQ ID NO: 32)IILTVGAALIALLLTSLIFSCIC (SEQ ID NO: 33)LFFVIGAAVGVFLLTVLIISIIL (SEQ ID NO: 34)LLLGIGAIVVVALIILLIFSLIL (SEQ ID NO: 35)VQFSNLSLLFLIALFLFISLLF (SEQ ID NO: 36)FEVCHLSILFIIALALVVHVTC (SEQ ID NO: 37)ILVAAGSAIIALLLAFLISSLIC (SEQ ID NO: 38)IIAVAGAALISILVVSLIFSCIL (SEQ ID NO: 39)ILLAAGVGIVATLLVLLLCSCVL (SEQ ID NO: 40)IIVTVGAVVIALLLTTLIFSCVC (SEQ ID NO: 41)ILITAGAALIALLLAFLISGLVC (SEQ ID NO: 42)LLITVGAALIALLFAFLISSLVC (SEQ ID NO: 43)IIITAGAVLIALLLTSLVFSCIC (SEQ ID NO: 46)

[0075] In certain embodiments, the endodomains of the modified FAST proteins and / or chimeric FAST proteins comprise an amino acid sequence selected from the following sequences:KRTNGVSSLLERNIRQNGSSAKIYVKPVMQSSTIIEEA (SEQ ID NO: 2) NCYLLRRLRNGPRKIYRTGKIQEGSYSSLSKQFIRPDHFV (SEQ ID NO: 47) NCYLYSKLRNGFQTVSQHVRRKERSHTNIPGQQIRPDMYV (SEQ ID NO: 48) NIYLCRRLKNGRKHDGRTSFQSGTTSRHNAKMDEREHLQSNTNV (SEQ ID NO: 49) NCYLCSKLKRKNGYLKRERKISNCRDKGLDKLILSKSDDIASSCV (SEQ ID NO: 50) KCDKNRKKQKWNTREIIEL (SEQ ID NO: 51) SARKRGCAVKSFEKPFLTNV (SEQ ID NO: 52)NCYLIRRLRHGPKRFYRTSQAQERSYSSLPKQPIRSNYTV (SEQ ID NO: 53) NCYFCRRLRRRNGFLSVPQRVYKESGSASDLFIKSNVQSLASCKKTRESPHYNV (SEQ ID NO: 54) NCYLCRKLKRTNGVSSLLERNLRQNGSSAKIYVKPVMQSSTIIEEA (SEQ ID NO: 55) NCYLYSKLRNGIQIVSRNSKGEERHRTTVQRPQLRSDMLV (SEQ ID NO: 56) NCYLLQRLRNGSRKIYRTSKVQEGSYSNLFKQSVRPDHFV (SEQ ID NO: 57) NCYLLRRLRNGPRKVYRAGKVQEGSYSNLSKQFIRSDNFV (SEQ ID NO: 4) NCYLYSKVKNGFQTIPLQFKRKERFDSSIPQPRIQPTQFV (SEQ ID NO: 58)

[0076] Table B shows the nucleic acid sequences that encode the wild type NSP1-1 proteins shown in Table A.Table B. Nucleic acid sequences of exemplary wild type NSP1-1 proteins.Lowercase letters indicate stop codons.

[0077] In certain aspects, a protein described herein comprises an amino acid sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence comprises one or more amino acid substitutions at position(s) selected from the group consisting of positions 2, 5, 6, 7, 9, 13, 17, 19, 22, 32, 33, 34, 36, 38, 39, 44, 51, 55, 58, 62, 63, 64, 65, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 96, and 100 of SEQ ID NO1.

[0078] In some embodiments, a protein described herein may comprises one or more an amino acid substitutions at position(s) selected from the group consisting of positions 2, 5, 6, 7, 9, 13, 17, 19, 22, 32, 34, 36, 38, 39, 44, 51, 55, 58, 62, 63, 64, 65, 71, 74, 96, and 100 of SEQ ID NO 1, wherein the one or more substitutions is any amino acid other than the amino acid found in SEQ ID NO: 1 at those positions.

[0079] In certain embodiments, a protein may comprise an amino acid substitution at position 33 of SEQ ID NO 1 that is any amino acid other than D, S, T, or V. In certain embodiments, the protein may comprise an amino acid substitution at position 67 of SEQ ID NO 1 that is any amino acid other than S, N, or R. In certain embodiments, the protein may comprise an amino acid substitution at position 68 of SEQ ID NO 1 that is any amino acid other than K or R. In certain embodiments, the protein may comprise an amino acid substitution at position 70 of SEQ ID NO 1 that is any aminoacid other than K or R. In certain embodiments, the protein may comprise an amino acid substitution at position 72 of SEQ ID NO 1 that is any amino acid other than T or R. In certain embodiments, the protein may comprise an amino acid substitution at position 73 of SEQ ID NO 1 that is any amino acid other than H or N. In certain embodiments, the protein may comprise an amino acid substitution at position 73 of SEQ ID NO 1 that is any amino acid other than H or N. In certain embodiments, the protein may comprise an amino acid substitution at position 75 of SEQ ID NO 1 that is any amino acid other than P, I, V, or L. In certain embodiments, the protein may comprise an amino acid substitution at position 76 of SEQ ID NO 1 that is any amino acid other than S, Q, or K. In certain embodiments, the protein may comprise an amino acid substitution at position 77 of SEQ ID NO 1 that is any amino acid other than R or S. In certain embodiments, the protein may comprise an amino acid substitution at position 78 of SEQ ID NO 1 that is any amino acid other than F, I, L, or L. In certain embodiments, the protein may comprise an amino acid substitution at position 79 of SEQ ID NO 1 that is any amino acid other than Y, I, L, or L. In certain embodiments, the protein may comprise an amino acid substitution at position 80 of SEQ ID NO 1 that is any amino acid other than R, S, K, or E. In certain embodiments, the protein may comprise an amino acid substitution at position 81 of SEQ ID NO 1 that is any amino acid other than T or R. In certain embodiments, the protein may comprise an amino acid substitution at position 82 of SEQ ID NO 1 that is any amino acid other than S or N. In certain embodiments, the protein may comprise an amino acid substitution at position 83 of SEQ ID NO 1 that is any amino acid other than Q, S, L, or I.

[0080] In some embodiments, the modified FAST proteins described herein comprise an amino acid sequence is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99%, or at least 99.5% identical to the amino acid sequence provided in SEQ ID NO: 1.

[0081] In some embodiments, the modified FAST proteins described herein comprise an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to the amino acid sequence provided in SEQ ID NO: 1.

[0082] In certain embodiments, the protein comprises one or more amino acid substitutions at positions selected from G2, 117, L24, F25, 126, V33, F34, N62, C63, Y64, L65, C66, R67, K68, L69, K70, R71, T72, N73, G74, V75, S76, S77, L78, L79, E80, R81, N82, or 183 relative to SEQ ID NO: 1. In certain embodiments, the protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions relative to SEQ ID NO: 1. In certain embodiments, the protein comprises one or more amino acid substitutions selected from G2A, I17K, I17G, L24G, F25G, F25K, I26G, I26K, V33G, V33K, F34G, N62G, N62A, C63G, C63A, Y64G, Y64A, L65G, L65A, C66A, C66G, R67Q, R67E, R67A, K68Q, K68E, L69G, K70Q, K70E, R71Q, R71E, T72A, N73A, G74A, V75A, V75G, S76A, S77A, L78A, L78G, L79A, L79G, E80A, R81A, N82A, or I83A relative to SEQ ID NO: 1. In some embodiments, the protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions selected from G2A, I17K, I17G, L24G, F25G, F25K, I26G, I26K, V33G, V33K, F34G,N62G, N62A, C63G, C63A, Y64G, Y64A, L65G, L65A, C66A, C66G, R67Q, R67E, R67A, K68Q, K68E, L69G, K70Q, K70E, R71Q, R71E, T72A, N73A, G74A, V75A, V75G, S76A, S77A L78A, L78G, L79A, L79G, E80A, R81A, N82A, or I83A relative to SEQ ID NO: 1.

[0083] In some embodiments, the protein comprises the following amino acid substitutions relative to SEQ ID NO: 1: G2A; I17G, L24G; F25G; I26G; V33G; F34G; F25K; I26K; V33K; F34K; L24G, F25G, and I26G; V33G and F34G; L24G, F25G, I26G, V33G, and F34G; R67Q; R67Q, and K68Q; K70Q and R71Q; R67Q, K68Q, and K70Q; R67Q, K68Q, K70Q, and R71Q; R67E, K68Q, K70Q, and R71Q; R67E, K68Q, K70Q, and R71E; R67E, K68Q, K70E, and R71E; R67E, K68E, K70E, and R71E; N62A, C63A, and Y64A; L65A, C66A, and R67A; T72A, N73A, and G74A; V75A, S76A, and S77A; L78A, L79A, and E80A; R81A, N82A,and I83A; I17G; I17K; L65G, L69G, V75G, L78G, and L79G; N62G, C63G, and Y64G; N62G, C63G, Y64G, L65G, L69G, V75G, L78G, and L79G;V75G, S76G and S77G; or L65G, L69G, V75G, S76G, S77G, L78G, and L79G.

[0084] In some embodiments, the protein comprises the amino acid sequence provided in SEQ ID NO: 1 with one or more amino acid substitutions selected from the group consisting of V33G, V33K, N62A, C63A, Y64A, R67Q, K68Q, K70Q, T72A, N73A, G74A, V75A, S76A, S77A, L78A, L79A, E80A, R81A, N82A, and I83A.

[0085] In certain embodiments, the protein comprises an amino acid sequence is at least 70%. at least 80%, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99%, or 100% identical to the amino acid sequence provided in any one of SEQ ID NOs: 73- 109.

[0086] In certain embodiments, the protein comprises an amino acid sequence that is 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 73-109.

[0087] In some embodiments, the modified FAST protein comprises one of the following amino acid sequences found in Table C. In some embodiments, the modified FAST protein is a biologically active FAST protein (e.g., facilitates cell-cell fusion, promotes membrane fusion).Table C. Amino acid sequences of modified “F100” FAST proteins.Lowercase letters designate the position of the indicated mutation.

[0088] Table D shows the nucleic acid sequences of the modified “F100” FAST proteins shown in Table C.Table D. Nucleic acid sequences of exemplary modified “F100” FAST proteins.Lowercase letters indicate stop codons.

[0089] In other embodiments, a modified FAST protein described herein is a truncation of a wild type FAST protein comprising one or more amino acid substitutions. In certain embodiments, the modified FAST protein is truncated to remove the N-terminal ectodomain. In certain embodiments, the modified FAST protein is truncated to remove the C-terminal endodomain. In certain embodiments, the modified FAST protein is truncated to remove the N-terminal ectodomain and transmembrane domain. In certain embodiments, the modified FAST protein is truncated to remove the transmembrane domain and the C-terminal endodomain. In certain embodiments, only part of a domain is truncated. For example, in some embodiments, the endodomain of a FAST protein is truncated up to but not including the polybasic region. In some embodiments, the protein is a truncation of SEQ ID NO: 1, comprising only the C-terminal endodomain from SEQ ID NO: 1 (e.g., amino acids 61-107). In some embodiments, the protein comprises an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protein comprises the amino acid set forth in SEQ ID NO: 2 further comprising up to one, two, three, four, five, six, or seven mutations.

[0090] In some embodiments, the protein is a truncation of SEQ ID NO: 1, comprising the amino acid sequence (NCYLCRKLKRTNGVSSLLERNIRQNGSSAKIYVKPVMQSSTIIEEA (SEQ ID NO: 44), endodomain from SEQ ID NO: 1).

[0091] In some aspects, a modified FAST protein described herein has one or more mutations in the endodomain. For example, a modified FAST protein may comprise an amino acid sequence, wherein the amino acid sequence comprises one or more amino acid substitutions at position(s) selected from the group consisting of position 62, 63, 64, 65, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 96, and 100 of SEQ ID NO 2. In certain embodiments, the modified FAST protein may comprise one or more amino acid substitutions at position(s) selected from the group consisting of positions 62, 63, 64, 65, 71, 74, 96, and 100 of SEQ ID NO 2, wherein the one or more substitutions is any amino acid other than the amino acid found in SEQ ID NO: 2 at those positions.

[0092] In certain embodiments, the protein may comprise an amino acid substitution at position 67 of SEQ ID NO 2 that is any amino acid other than S, N, or R. In certain embodiments, the protein may comprise an amino acid substitution at position 68 of SEQ ID NO 2 that is any amino acid other thanK or R. In certain embodiments, the protein may comprise an amino acid substitution at position 70 of SEQ ID NO 2 that is any amino acid other than K or R. In certain embodiments, the protein may comprise an amino acid substitution at position 72 of SEQ ID NO 2 that is any amino acid other than T or R. In certain embodiments, the protein may comprise an amino acid substitution at position 73 of SEQ ID NO 2 that is any amino acid other than H or N. In certain embodiments, the protein may comprise an amino acid substitution at position 73 of SEQ ID NO 2 that is any amino acid other than H or N. In certain embodiments, the protein may comprise an amino acid substitution at position 75 of SEQ ID NO 2 that is any amino acid other than P, I, V, or L. In certain embodiments, the protein may comprise an amino acid substitution at position 76 of SEQ ID NO 2 that is any amino acid other than S, Q, or K. In certain embodiments, the protein may comprise an amino acid substitution at position 77 of SEQ ID NO 2 that is any amino acid other than R or S. In certain embodiments, the protein may comprise an amino acid substitution at position 78 of SEQ ID NO 2 that is any amino acid other than F, I, L, or L. In certain embodiments, the protein may comprise an amino acid substitution at position 79 of SEQ ID NO 2 that is any amino acid other than Y, I, L, or L. In certain embodiments, the protein may comprise an amino acid substitution at position 80 of SEQ ID NO 2 that is any amino acid other than R, S, K, or E. In certain embodiments, the protein may comprise an amino acid substitution at position 81 of SEQ ID NO 2 that is any amino acid other than T or R. In certain embodiments, the protein may comprise an amino acid substitution at position 82 of SEQ ID NO 2 that is any amino acid other than S or N. In certain embodiments, the protein may comprise an amino acid substitution at position 83 of SEQ ID NO 2 that is any amino acid other than Q, S, L, or I.

[0093] In another aspect, a modified FAST protein described herein comprises an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 3, wherein the amino acid sequence comprises one or more amino acid substitutions at position(s) selected from the group consisting of positions 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 34, 38, 40, 44, 45, 46, 48, 49, 50, 51, 53, 54, 55, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 83, 84, 90, 93, and 101 of SEQ ID NO 3 (full length, wild type NSP1-1 protein, “F89”).

[0094] In certain embodiments, the modified FAST protein may comprises one or more an amino acid substitutions at position(s) selected from the group consisting of positions 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 26, 27, 28, 29, 30, 32, 34, 38, 40, 44, 45, 46, 48, 49, 50, 51, 53, 54, 55, 57, 58, 59, 60, 61, 62, 63, 65, 66, 68, 69, 72, 74, 77, 78, 83, 84, 90, 93, and 101 of SEQ ID NO 3, wherein the one or more substitutions is any amino acid other than the amino acid found in SEQ ID NO: 3 at those positions.

[0095] In certain embodiments, the protein may comprise an amino acid substitution at position 31 of SEQ ID NO 3 that is any amino acid other than A, L, F, S, or V. In certain embodiments, the protein may comprise an amino acid substitution at position 64 of SEQ ID NO 3 that is any amino acid otherthan F or Y. In certain embodiments, the protein may comprise an amino acid substitution at position 67 of SEQ ID NO 3 that is any amino acid other than R or Q. In certain embodiments, the protein may comprise an amino acid substitution at position 70 of SEQ ID NO 3 that is any amino acid other than R or Q. In certain embodiments, the protein may comprise an amino acid substitution at position 71 of SEQ ID NO 3 that is any amino acid other than H or N. In certain embodiments, the protein may comprise an amino acid substitution at position 73 of SEQ ID NO 3 that is any amino acid other than P or S. In certain embodiments, the protein may comprise an amino acid substitution at position 75 of SEQ ID NO 3 that is any amino acid other than K or E. In certain embodiments, the protein may comprise an amino acid substitution at position 76 of SEQ ID NO 3 that is any amino acid other than I or V.

[0096] In some embodiments, the modified FAST protein comprises an amino acid sequence is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99%, or at least 99.5% identical to the amino acid sequence provided in SEQ ID NO: 3.

[0097] In some embodiments, the modified FAST protein comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to the amino acid sequence provided in SEQ ID NO: 3.

[0098] In certain embodiments, the protein comprises one or more amino acid substitutions at positions selected from G2, R4, Q5, L8, Q9, Qll, H13, R14, T15,D16, 117, S19, H21, S22, N23, 124, L26, S28, A29, S30, F31, A32, F34, T36, Y37, H38, 141, T42, V43, G44, A46, L47, 148, A49, L50, L51, A53, F54, L55, S57, S58, L59, V60, N62, C63, Y64, L65, L66, R67, R68, L69, R70, N71, G72, P73, R74, K75, V76, Y77, and R78 relative to SEQ ID NO: 3. In some embodiments, the protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions relative to SEQ ID NO: 3.

[0099] In certain embodiments, the protein comprises one or more amino acid substitutions selected from G2A, R4T, R4H, R4E, R4A, R4Q, Q5P, L8G, L8K, Q9A, Q9N, Q9V, QI ID, QI IS, QI IP, H13I, H13A, H13V, H13G, H13L, H13T, R14N, R14P, R14L, R14D, T15N, T15E, T15D, D16N, D16E, D16V, D16F, I17G, I17K, I17P, I17V, I17Q, S19Q, S19E, S19N, S19H, S19C, S19Y, H21S, H21S, H21T, H21V, H21I, H21D, S22E, S22Q, S22F, S22D, S22H, S22N, S22P, N23S, N23T, N23Q, N23F, N23A, N23G, , I24K, I24G, I24N, I24L, I24A, I24R, L26G, L26K, L26S, L26N, L26Y, L26E, L26A, L26Q, S28T, S28K, S28I, S28E, A29N, A29L, A29S, A29Q, A29I, A29R, A29T, S30E, S30V, S30D, F31G, F31K, F31D, F31Q, F31S, F31R, F31H, A32G, A32K, A32E, A32N , A32Q, F34G, F34K, F34V, F34D, F34A, F34E, F34L, T36W, T36S, Y37L, H38L, H38F, H38W, H38A, H38Y, H38I, H38T, H38E, H38M, I41L, I41M, I41V, T42A, T42I, T42L, T42F, T42V, V43I, V43C, V43L, V43A, G44S, A46V, A46I, A46L, A46M, A46F, L47V, L47I, I48V, I48C, I48T, I48L, I48S, I48F, A49I, A49T, A49F, A49L, A49G, L50I, L50V, L50M, L51M, L51A, L51V, L51I, A53I, A53L , A53V, A53T, F54A, F54G, F54L, F54S, F54V, L55F, L55A, S57G, S57V, S57A, S57I,S57F, S58A, L59C, L59F, L59M, L59I, V60A, V60I, V60M, N62A, N62G, C63A, C63G, Y64G, L65A, L65G, L66A, L66G, R67Q, R67E, R67A, R68Q, R68E, L69G, R70Q, R70E, N71A, G72A, P73A, R74Q, R74E, K75Q, K75E, V76E, V76A, Y77A, Y77G, R78Q, R78A, or R78G relative to SEQ ID NO: 3.

[0100] In some embodiments, the protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions selected from G2A, R4T, R4H, R4E, R4A, R4Q, Q5P, L8G, L8K, Q9A, Q9N, Q9V, QI ID, QI IS, QI IP, H13I, H13A, H13V, H13G, H13L, H13T, R14N, R14P, R14L, R14D, T15N, T15E, T15D, D16N, D16E, D16V, D16F, I17G, I17K, I17T, I17P, I17V, I17Q, S19Q, S19E, S19N, S19H, S19C, S19Y, H21S, H21T, H21V, H21I, H21D, S22E, S22Q, S22F, S22D, S22H, S22N, S22P, N23S, N23T, N23Q, N23F, N23A, N23G, , I24K, I24G, I24N, I24L, I24A, I24R, L26G, L26K, L26S, L26N, L26Y, L26E, L26A, L26Q, S28T, S28K, S28I, S28E, A29N, A29L, A29S, A29Q, A29I, A29R, A29T, S30E, S30V, S30D, F31G, F31K, F31D, F31Q, F31S, F31R, F31H, A32G, A32K, A32E, A32N , A32Q, F34G, F34K, F34V, F34D, F34A, F34E, F34L, T36W, T36S, Y37L, H38L, H38F, H38W, H38A, H38Y, H38I, H38T, H38E, H38M, I41L, I41M, I41V, T42A, T42I, T42L, T42F, T42V, V43I, V43C, V43L, V43A, G44S, A46V, A46I, A46L, A46M, A46F, L47V, L47I, I48V, I48C, I48T, I48L, I48S, I48F, A49I, A49T, A49F, A49L, A49G, L50I, L50V, L50M, L51M, L51A, L51V, L51I, A53I, A53L , A53V, A53G, A53T, F54A, F54G, F54L, F54S, F54V, L55F, L55A, S57G, S57V, S57A, S57I, S57F, S58A, L59C, L59F, L59M, L59I, V60A, V60I, V60M, N62A, N62G, C63A, C63G, Y64A, Y64G, L65A, L65G, L66A, L66G, R67Q, R67E, R67A, R68Q, R68E, L69G, R70Q, R70E, N71A, G72A, P73A, R74Q, R74E, K75Q, K75E, V76E, V76A, Y77A, Y77G, R78Q, R78E, R78A, or R78G relative to SEQ ID NO: 3.

[0101] In some embodiments, the protein comprises the following amino acid substitutions relative to SEQ ID NO: 3: G2A; I17G; I17K; I24G; L26G; F31G; A32G; F34G; I24K; L26K; F31K; A32K; F34K; L24G and L26G; F31G, A32G, and F34G; I24G, L26G, F31G, A32G, and F34G; I17G, I24G, L26G, F31G, A32G, and F34G; R67Q and R68Q; R67Q, R68Q, R70Q, and R74Q; R67Q, R68Q, R70Q, R74Q, K75Q, and R78Q; R67Q, R68Q, R70E, R74E, K75Q, and R78Q; R67Q, R68E, R70E, R74E, K75E, and R78Q; R67E, R68E, R70E, R74E, K75E, and R78E; N62A, C63A, and Y64; L65A, L66A, and R67A; N71A, G72A, and P73A; V76A, Y77A, and R78A;L8G; L8K; S22E and H38L; S28T and H38F; D16N and H38W; H38A; H38F; F31D and H38L; L26S and H38Y; N23S and T36W; F34A and H38I; A29N and T36W; H38Y; H21S and H38Y; T42I, A46V, L51M, and F54A; T42I, A46I, I48T, L51A, and F54G; V43I, A49I, and L50I; T42A, V43C, A49T, and A53I; T42I, V43I, I48V, and V60A; T42I, I48C, A49F, L50V, and S58A; V43I, A46V, I48T, and L51A; I41L, T42I, F54L, S57G, and L59C; T42I and A46L; A49L and V60I; T42L, A46M, and L51M; T42I, A46I, A53L, and L59F; QI ID, D16E, I17T, S19Q, H21T, N23S, L26S, S28K, A29L, F31Q, and H38L; R4T, H13I, T15N, S19E, S22Q, N23T, L26N, A29S, F31S, and H38T; H13I, I17P, S22F, N23Q, I24N, L26Y, A29Q, A32E, F34V, and H38Y; Q9A, H13A, T15E, D16E, H21V, S22D, N23F, I24A, A29I, A32N, F34D, and H38L; Q5P, QI IS, H13V, S19E, H21I, N23A, I24L, L26E, A32N,and H38L; R4H, Q9N, H13G, R14N, I17G, S22H, L26A, A29R, S30V, F34E, Y37L, and H38L; R4E, QI IP, R14P, T15D, I17P, S19N, S22Q, I24K, S28E, A29N, and S30E; H13T, R14N, T15D, D16V, S22N, L26A, S28I, A29Q, S30D, A32Q, and H38E; R4A, Q9V, H13L, R14L, H21S, N23G, L26N, S28I, A29T, F31R, and H38M; Q9N, D16F, I17V, S19H, S22P, N23Q, I24R, L26N, F31H, F34L, and H38F; Q9A, Q11P, R14N, S19C, H21D, N23T, L26A, T36S, and H38I; R4Q, Q11S, R14D, T15D, I17Q, S19Y, S22E, L26Q, S28I, A29S, and A32N; T42L, L47V, I48V, A49L, L50M, S57V, and L59F; I41M, T42I, L55F, S57V, L59M, and V60I; T42F, A46F, L47I, I48L, A49I, L50I, A53L, and F54L; I41L, T42I, V43L, A46I, A49G, F54S, and V60I; T42I, G44S, A49L, L50V, L51V, A53V, and V60A; I41L, T42I, A46V, I48S, A49L, L51A, L55A, S57A, and L59C; T42V, V43I, I48F, L51V, A53I, F54L, L59F, and V60M; T42V, A46F, I48V, A49L, L50I, A53L, and S57I; T42L, V43A, A46L, L47I, A49G, L50I, L55F, S57F, L59I, and V60I; I41M, T42I, V43A, A46I, A49L, F54A, and S57A; T42I, A46I, I48F, L50I, L51I, A53G, F54L, and V60I; 141 V, T42I, V43A, L47I, A49L, L50I, L51A, A53T, F54V, and S57V; L65G, L66G, and L69G; N62G, C63G, and Y64G; N62G, C63G, Y64G, L65G, L66G, and L69G; V76G, Y77G, and R78G; or V76G, Y77G, R78G, L65G, L66G, and L69G.

[0102] In some embodiments, the protein comprises the amino acid sequence provided in SEQ ID NO: 3 with one or more amino acid substitutions selected from the group consisting of F31K, A32G, N62A, C63A, Y64A, L65A, L66A, R67A, R67Q, R68Q, R70E, N71A, G72A, P73A, R74E, K75Q, V76A, Y77A, and R78A.

[0103] In certain embodiments, the protein comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to an amino acid sequence provided in any one of SEQ ID NOs: 147-228.

[0104] In certain embodiments, the protein comprises an amino acid sequence that is 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 147-228.

[0105] In some embodiments, the FAST protein comprises one of the following amino acid sequences found in Table E. In some embodiments, the FAST protein is a biologically active FAST protein (e.g., facilitates cell-cell fusion, promotes membrane fusion).Table E. Amino acid sequences of exemplary modified “F89” FAST proteins.Lower case letters in Table C designate the position of the indicated mutation.

[0106] Table F shows the nucleic acid sequences of the modified “F89” FAST proteins shown in Table E.Table F. Nucleic acid sequences of exemplary modified “F89” FAST proteins.Lowercase letters indicate stop codons.

[0107] In some embodiments, the modified FAST protein is a truncation of a wild type FAST protein comprising one or more amino acid substitutions. In certain embodiments, the modified FAST protein is truncated to remove the N-terminal ectodomain. In certain embodiments, the modified FAST protein is truncated to remove the C-terminal endodomain. In certain embodiments, the modified FAST protein is truncated to remove the N-terminal ectodomain and transmembrane domain. In certain embodiments, the modified FAST protein is truncated to remove the transmembrane domain and the C-terminal endodomain. In some embodiments, the protein is a truncation of SEQ ID NO: 3, comprising only the endodomain from SEQ ID NO: 3 (e.g., amino acids 60-101). In some embodiments, the protein comprises an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4. In some embodiments, the protein comprises an amino acid set forth in SEQ ID NO: 4 further comprising up to one, two, three, four, five, six, or seven mutations.

[0108] In some embodiments, the protein is a truncation of SEQ ID NO: 3, comprising the amino acid sequence (LRRLRNGPRKVYRAGKVQEGSYSNLSKQFIRSDNFV, endodomain from SEQ ID NO: 3).

[0109] In some aspects, a modified FAST protein described herein has one or more mutations in the endodomain. For example, a modified FAST protein may comprise an amino acid sequence wherein the amino acid sequence comprises one or more amino acid substitutions at positions selected from the group consisting of position 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 83, 84, 90, 93, and 101 of SEQ ID NO 4. In other embodiments, the modified FAST protein may comprise one or more amino acid substitutions at position(s) selected from the group consisting of positions 61, 62, 63, 65, 66, 68, 69, 72, 74, 77, 78, 83, 84, 90, 93, and 101 of SEQ ID NO 4, wherein the one or more substitutions is any amino acid other than the amino acid found in SEQ ID NO: 2 at those positions.

[0110] In certain embodiments, the protein may comprise an amino acid substitution at position 31 of SEQ ID NO 4 that is any amino acid other than A, L, F, S, or V. In certain embodiments, the protein may comprise an amino acid substitution at position 64 of SEQ ID NO 4 that is any amino acid other than F or Y. In certain embodiments, the protein may comprise an amino acid substitution at position 67 of SEQ ID NO 4 that is any amino acid other than R or Q. In certain embodiments, the protein may comprise an amino acid substitution at position 70 of SEQ ID NO 4 that is any amino acid other than R or Q. In certain embodiments, the protein may comprise an amino acid substitution at position 71 of SEQ ID NO 4 that is any amino acid other than H or N. In certain embodiments, the protein may comprise an amino acid substitution at position 73 of SEQ ID NO 4 that is any amino acid other than P or S. In certain embodiments, the protein may comprise an amino acid substitution at position 75 of SEQ ID NO 4 that is any amino acid other than K or E. In certain embodiments, the protein may comprise an amino acid substitution at position 76 of SEQ ID NO 3 that is any amino acid other than I or V.

[0111] In another aspect, the present disclosure describes a modified FAST protein that is a fusion protein. For instance, the modified FAST protein may be a fusion protein that further comprises additional effector domains (e.g., a myristoylation or palmitoylation motif, a polybasic motif) from other FAST proteins. In other embodiments, the fusion protein may comprise an ectodomain, a transmembrane domain, and an endodomain, from the same FAST protein, wherein at least one of the three domains comprises one or more mutations. In certain embodiments, the fusion protein comprises an ectodomain, a transmembrane domain, and an endodomain from at least two or more different FAST proteins. In certain embodiments, the fusion protein is a biologically functional (e.g., facilitates cell-cell fusion) FAST protein. In certain embodiments, the modified FAST protein is incorporated (e.g., formulated) into a particle (e.g., nano particle, lipid nanoparticle) or liposome. In certain embodiments, the FAST protein incorporated in the particle or liposome facilitates the fusion of the particle or liposome into a cell. In certain embodiments, the fusion protein comprises an endodomain with an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the endodomain of SEQ ID NOs: 1 or 3.Chimeric FAST Proteins

[0112] In certain aspects, the present disclosure describes chimeric FAST proteins, which combine the domains (e.g., ectodomain, transmembrane domain, or endodomain) from at least two different FAST proteins, such as the NSP1-1 and NSP1-2 peptides from Rotavirus, to form functional FAST proteins. For example, as shown in Figure 3, a number of different chimeric FAST proteins have been generated. In certain embodiments, a chimeric FAST protein comprises (1) an ectodomain, or a functional portion thereof, (2) a transmembrane domain, or a functional portion thereof, and (3) an endodomain, or a functional portion thereof, from at least two different NSP1-1 FAST proteins.

[0113] In certain embodiments, a chimeric FAST protein comprises (1) an ectodomain, or a function portion thereof, from a first FAST protein, (2) a transmembrane domain, or a functional portion thereof, from a second FAST protein, and (3) an endodomain, or functional portion thereof, from a third FAST protein (e.g., a NSP1-1 FAST protein) to form a biologically active FAST protein. That is, all three domains are from different FAST proteins.

[0114] In certain embodiments, a chimeric FAST protein comprises an (1) ectodomain, or a functional portion thereof, from a first FAST protein; (2) a transmembrane domain, or a functional portion thereof, from the same first FAST protein; and (3) an endodomain, or function fragment thereof, from a second FAST protein. That is, the ectodomain and transmembrane domain are from the same FAST protein.

[0115] In certain embodiments, a chimeric FAST protein comprises (1) an ectodomain, or a functional portion thereof, from a first FAST protein; (2) a transmembrane domain, or a functional portion thereof, from a second FAST protein; and (3) an endodomain, or function fragment thereof, from the same second FAST protein. That is, the transmembrane domain and the endodomain are from the same FAST protein.

[0116] In certain embodiments, a chimeric FAST protein may have one of the following structures: NH2- [ectodomain from a first FAST protein] -[transmembrane domain from a second FAST protein] -[endodomain from a third FAST protein]-COOH;NH2- [ectodomain from a first FAST protein] -[transmembrane domain from a first FAST protein] -[endodomain from a second FAST protein] -COOH;NH2- [ectodomain from a first FAST protein] -[transmembrane domain from a second FAST protein] -[endodomain from a second FAST protein] -COOH;NH2- [ectodomain from a first FAST protein] -[transmembrane domain from a second FAST protein] -[endodomain from a first FAST protein]-COOH;NH2- [ectodomain from a first FAST protein] -[transmembrane domain from a first FAST protein] -[polybasic region from a first FAST protein] -[amphipathic helix from a first FAST protein] -[endodomain from a second FAST protein] -COOH; orNH2- [ectodomain from a first FAST protein] -[transmembrane domain from a first FAST protein] -[polybasic region from a first FAST protein] -[amphipathic helix from a second FAST protein] -[endodomain from a second FAST protein] -COOH, wherein “]-[“ represent a direct bond or a linker. In certain embodiments, the linker comprises 1-50 amino acids.

[0117] In certain embodiments, the ectodomain of a chimeric FAST protein comprises an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to an ectodomain from any one of SEQ ID NOs: 1, 3, and 5-16. In certain embodiments, the ectodomain of a chimeric FAST protein comprises an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or atleast 99% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 17-30. In certain embodiments, the ectodomain of a chimeric FAST protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 17-30 further comprising one or more amino acid mutations. In certain embodiments, the ectodomain of a chimeric FAST protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 17-30 further comprising up to one, two, three, four, five, six, or seven mutations. In certain embodiments, the ectodomain of a chimeric FAST is identical to the amino acid sequence set forth any one of SEQ ID NOs: 17-30.

[0118] In certain embodiments, the transmembrane domain of a chimeric FAST protein comprises an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to a transmembrane domain from any one of SEQ ID NOs: 1, 3, and 5-16. In certain embodiments, the transmembrane domain of a chimeric FAST protein comprises an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 31-46. In certain embodiments, the transmembrane of a chimeric FAST protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 31-46 further comprising one or more amino acid mutations. In certain embodiments, the transmembrane domain of a chimeric FAST protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 31-46 further comprising up to one, two, three, four, five, six, or seven mutations. In certain embodiments, the transmembrane domain of a chimeric FAST is identical to the amino acid sequence set forth any one of SEQ ID NOs: 31-46.

[0119] In certain embodiments, the endodomain of a chimeric FAST protein comprises an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to an endodomain from any one of SEQ ID NOs: 1, 3, and 5-16. In certain embodiments, the endodomain of a chimeric FAST protein comprises an amino acid sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, and 47-58. In certain embodiments, the endodomain of a chimeric FAST protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, and 47-58 further comprising one or more amino acid mutations. In certain embodiments, the endodomain of a chimeric FAST protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, and 47-58 further comprising up to one, two, three, four, five, six, or seven mutations. In certain embodiments, the endodomain of a chimeric FAST is identical to the amino acid sequence set forth any one of SEQ ID NOs: 2, 4, and 47-58.

[0120] In certain embodiments, a chimeric FAST protein comprises an ectodomain and transmembrane domain from the same FAST protein and an endodomain from SEQ ID NO: 1. In certain embodiments, the chimeric FAST protein comprises an ectodomain and a transmembrane domain that are at least 80%, least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% identical, or 100% identical to the ectodomain and transmembrane domain from a sequenceselected from SEQ ID NOs: 5-16, and an endodomain that is at least 80%, least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% identical, or 100% identical to SEQ ID NO 2 (endodomain of F100 (SEQ ID NO:1)).

[0121] In certain embodiments, a chimeric FAST protein comprises an ectodomain and transmembrane domain from the same FAST protein and an endodomain from SEQ ID NO: 3. In certain embodiments, the chimeric FAST protein comprises an ectodomain and a transmembrane domain that are at least 80%, least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% identical, or 100% identical to the ectodomain and transmembrane domain from a sequence selected from SEQ ID NOs: 5-16, and an endodomain that is at least 80%, least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% identical, or 100% identical to SEQ ID NO 4 (endodomain of F89 (SEQ ID NO:3)).

[0122] In certain embodiments, the chimeric FAST protein may have one of the following structures: NH2- [ectodomain of F84]- [transmembrane domain of F84]- [endodomain of F100]-COOH; NH2- [ectodomain of F85]-[transmembrane domain of F85]-[endodomain of F100]-COOH; NH2- [ectodomain of F86]-[transmembrane domain of F86] -[endodomain of F100]-COOH; NH2- [ectodomain of F87]- [transmembrane domain of F87]- [endodomain of F100]-COOH; NH2- [ectodomain of F88]-[transmembrane domain of F88] -[endodomain of F100]-COOH; NH2- [ectodomain of F89]- [transmembrane domain of F89]- [endodomain of F100]-COOH; NH2- [ectodomain of F90]- [transmembrane domain of F90]- [endodomain of F100]-COOH;NH2- [ectodomain of F101 (-[transmembrane domain of F101] -[endodomain of F100]-COOH; NH2- [ectodomain of Fl 02] -[transmembrane domain of Fl 02] -[endodomain of F100]-COOH; NH2- [ectodomain of Fl 03] -[transmembrane domain of Fl 03] -[endodomain of F100]-COOH; NH2- [ectodomain of Fl 04] -[transmembrane domain of Fl 04] -[endodomain of F100]-COOH; NH2- [ectodomain of Fl 05] -[transmembrane domain of Fl 05] -[endodomain of F100]-COOH; andNH2- [ectodomain of Fl 06] -[transmembrane domain of Fl 06] -[endodomain of F100]-COOH, wherein “]-[“ represent a direct bond or a linker. In certain embodiments, the linker comprises 1-50 amino acids.

[0123] In certain embodiments, the chimeric FAST protein may have one of the following structures: NH2- [ectodomain of F84]- [transmembrane domain of F84]- [endodomain of F89]-COOH; NH2- [ectodomain of F85]-[transmembrane domain of F85]-[endodomain of F89]-COOH; NH2- [ectodomain of F86]-[transmembrane domain of F86] -[endodomain of F89]-COOH; NH2- [ectodomain of F87]- [transmembrane domain of F87]- [endodomain of F89]-COOH; NH2- [ectodomain of F88]-[transmembrane domain of F88] -[endodomain of F89]-COOH; NH2- [ectodomain of F90]- [transmembrane domain of F90]- [endodomain of F89]-COOH; NH2- [ectodomain of Fl 00] -[transmembrane domain of Fl 00] -[endodomain of F89]-COOH; NH2- [ectodomain of F101]-[transmembrane domain of F101] -[endodomain of F89]-COOH;NH2- [ectodomain of Fl 02] -[transmembrane domain of Fl 02] -[endodomain of F89]-COOH; NH2- [ectodomain of Fl 03] -[transmembrane domain of Fl 03] -[endodomain of F89]-COOH; NH2- [ectodomain of Fl 04] -[transmembrane domain of Fl 04] -[endodomain of F89]-COOH; NH2- [ectodomain of Fl 05] -[transmembrane domain of Fl 05] -[endodomain of F89]-COOH; andNH2- [ectodomain of Fl 06] -[transmembrane domain of Fl 06] -[endodomain of F100]-COOH, wherein “]-[“ represent a direct bond or a linker. In certain embodiments, the linker comprises 1-50 amino acids.

[0124] In certain embodiments, the chimeric FAST protein comprises an amino acid sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence provided in any one of SEQ ID NOs: 311-323 and 337-340.

[0125] In certain embodiments, the chimeric FAST protein comprises an amino acid sequence that is 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 311-323 and 337- 34O.In some embodiments, the chimeric FAST protein comprises an amino acid sequence provided in Table G. In certain embodiments, the chimeric FAST protein comprising an amino acid sequence provided in Table G is a biologically active (e.g., facilitates cell-cell fusion, promotes membrane fusion) FAST protein.Table G. Amino acid sequences of exemplary chimeric FAST proteins comprising varying ecto+TM domains from the same FAST protein and the F100 endodomain.

[0126] Table H shows the nucleic acid sequences of chimeric “F100” FAST proteins shown in Table G.Table H. Nucleic acid sequences of exemplary chimeric “F100” FAST proteins comprising varying ecto+TMD and F100 endodomain.Lowercase letters indicate stop codons.

[0127] In certain embodiments, the chimeric FAST protein further comprises a myristoylation motif. In certain embodiments, chimeric FAST protein further comprises a palmitoylation motif. A myristoylation motif conforms to the following: M-G-X-X-X-S, where M is an N-terminal methionine, G is glycine, X is any amino acid, and S is serine. Myristic acid is attached to the glycine.

[0128] Palmitoylation motif consists of di-cysteine residues in the endodomain, usually the two residues C-terminal to the transmembrane domain.

[0129] In certain embodiments, the myristoylation motif is located in the ectodomain of the FAST protein. In certain embodiments, the palmitoylation motif is located in the endodomain of the FAST protein. In certain embodiments, the myristoylation motif is located at the N-terminus of the ectodomain of a FAST protein. In certain embodiments, the palmitoylation motif is located at the endodomain of a FAST protein, proximal to the transmembrane domain. In certain embodiments, the chimeric FAST protein may comprise an amino acid sequence comprising a polybasic region (PBR).

[0130] An exemplary chimeric FAST protein comprising a myristoylation motif is F296 (Fl 05 (Ecto+TMD) with the endodomain of Fl 00) having the amino acid sequence: MGNTYSVHNNQQVSSNTVHGSGQIHSEDQKTSSQITTIVQFSNLSLLFLIALFLFISLLFNCYLC RKLKRTNGVSSLLERNIRQNGSSAKIYVKPVMQSSTIIEEA (SEQ ID NO: 337). Myristoylation motif is underlined.

[0131] An exemplary chimeric FAST protein comprising a myristoylation motif and a polybasic region is F347 (F105 (Ecto+TMD+PBR) with endodomain of F100) having the amino acid sequence: MGNTYSVHNNQQVSSNTVHGSGQIHSEDQKTSSQITTIVQFSNLSLLFLIALFLFISLLFKCDK NRKKQKNCYLCRKLKRTNGVSSLLERNIRQNGSSAKIYVKPVMQSSTIIEEA (SEQ ID NO: 338). Myristoylation motif is underlined.

[0132] In certain embodiments, a chimeric FAST protein chimera is categorized as non-fusogenic, moderately fusogenic, or highly fusogenic compared to a reference FAST protein (Example 3).

[0133] In another aspect, the disclosure provides a nucleic acid molecule encoding a chimeric FAST protein described above and provided in this disclosure. In certain embodiments, the nucleic acid molecule comprises a sequence that is least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95%, identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical to a nucleic acid sequence set forth in any one of SEQ ID NOs: 59-72, 110-146, 229-310, 324-336, or 341-344. In certain embodiments, the nucleic acid molecule comprises a sequence that is 100% identical to a nucleic acid sequence set forth in any one of SEQ ID NOs: 59-72, 110-146, 229-310, 324-336, or 341-344. In another aspect, the disclosure provides an expression vector comprising the above nucleic acid molecule. In yet another aspect, the disclosure provides cells (e.g., transformed cell lines) that comprise the chimeric FAST proteins described above.Cells

[0134] Cells that may contain any of the compositions described herein include prokaryotic cells and eukaryotic cells. The methods described herein are used to deliver a FAST protein, a nucleic acid encoding a FAST protein, and / or a vector encoding a FAST protein into a eukaryotic cell (e.g., a mammalian cell, such as a human cell). In some embodiments, the cell is in vitro e.g., cultured cell. In some embodiments, the cell is in vivo (e.g., in a subject such as a human subject). In some embodiments, the cell is ex vivo (e.g., isolated from a subject and may be administered back to the same or a different subject).

[0135] Mammalian cells of the present disclosure include human cells, primate cells (e.g., vero cells), rat cells (e.g., GH3 cells, OC23 cells) or mouse cells (e.g., MC3T3 cells). There are a variety of human cell lines, including, without limitation, human embryonic kidney (HEK) cells, HeLa cells, cancer cells from the National Cancer Institute's 60 cancer cell lines (NCI60), DU145 (prostate cancer) cells, Lncap (prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-438 (breast cancer) cells, PC3 (prostate cancer) cells, T47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, SHSY5Y human neuroblastoma cells (cloned from a myeloma) and Saos-2 (bone cancer) cells.

[0136] A stem cell refers to a cell with the ability to divide for indefinite periods in culture and to give rise to specialized cells. A pluripotent stem cell refers to a type of stem cell that is capable of differentiating into all tissues of an organism, but not alone capable of sustaining full organismal development. A human induced pluripotent stem cell refers to a somatic (e.g., mature or adult) cell that has been reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells see, e.g., Takahashi and Yamanaka, Cell 126 (4): 663-76, 2006, incorporated by reference herein). Human induced pluripotent stem cell cells express stem cell markers and are capable of generating cells characteristic of all three germ layers (ectoderm, endoderm, mesoderm).

[0137] Additional non-limiting examples of cell lines that may be used in accordance with the present disclosure include 293-T, 293-T, 3T3, 4T1, 721, 9L, A-549, A172, A20, A253, A2780, A2780ADR, A2780cis, A431, ALC, B16, B35, BCP-1, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C2C12, C3H-10T1 / 2, C6, C6 / 36, Cal-27, CGR8, CHO, CML Tl, CMT, COR-L23, CGR-L23 / 5010, COR-L23 / CPR, COR-L23 / R23, COS-7, COV-434, CT26, D17, DH82, DU145, DuCaP, E14Tg2a, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, Hepalclc7, High Five cells, HL-60, HMEC, HT-29, HUVEC, J558L cells, Jurkat, JY cells, K562 cells, KCL22, KG1, Ku812, KYO1, LNCap, Ma-Mel 1, 2, 3 ...48, MC-38, MCF-10A, MCF-7, MDA-MB-231, MDA-MB-435, MDA-MB-468, MDCK II, MG63, MONO-MAC 6, MOR / 0.2R, MRC5, MTD-1A, My End, NALM-1, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NW- 145, OPCN / OPCT Peer, PNT-1A / PNT 2, PTK2, Raji, RBL cells, RenCa, RIN-5F, RMA / RMAS, S2,Saos-2 cells, Sf21, Sf9, SiHa, SKBR3, SKOV-3, T-47D, T2, T84, THP1, U373, U87, U937, VCaP, WM39, WT-49, X63, YAC-1 and YAR cells.

[0138] Some aspects of this disclosure provide cells comprising any of the constructs disclosed herein. In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line.Vectors

[0139] In some aspects, a vector used herein may encode a FAST protein, or any of the components thereof (e.g., ectodomain, transmembrane domain, endodomain). The vectors may be capable of driving expression of one or more coding sequences in a cell. In some embodiments, the cell may be a prokaryotic cell, such as, e.g., a bacterial cell. In some embodiments, the cell may be a eukaryotic cell, such as, e.g., a yeast, plant, insect, or mammalian cell. In some embodiments, the eukaryotic cell may be a mammalian cell. In some embodiments, the eukaryotic cell may be a rodent cell. In some embodiments, the eukaryotic cell may be a human cell. Suitable promoters to drive expression in different types of cells are known in the art. In some embodiments, the promoter may be wild type. In other embodiments, the promoter may be modified for more efficient or efficacious expression. In yet other embodiments, the promoter may be truncated yet retain its function. For example, the promoter may have a normal size or a reduced size that is suitable for proper packaging of the vector into a virus.

[0140] In some embodiments, the vector of the present disclosure comprises one or more regulatory elements to control the expression of the heterologous nucleic acid region (e.g., promoters, transcriptional terminators, and / or other regulatory elements). In some embodiments, the first and / or second nucleotide sequence is operably linked to one or more (e.g., 1, 2, 3, 4, 5, or more) transcriptional terminators.

[0141] In some embodiments, the promoters that may be used in the expression vectors may be constitutive, inducible, or tissue-specific. In some embodiments, the promoters may be constitutive promoters. Non-limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-alpha (EFla) promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, a functional fragment thereof, or a combination of any of the foregoing. In some embodiments, the promoter may be a CMV promoter. In some embodiments, the promoter may be a truncated CMV promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary inducible promoters include thoseinducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low basal (non-induced) expression level, such as, e.g., the Tet-On® promoter (Clontech).

[0142] In some embodiments, the promoter may be a tissue-specific promoter. In some embodiments, the tissue-specific promoter is exclusively or predominantly expressed in liver tissue. Non-limiting exemplary tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase- 1 promoter, endoglin promoter, fibronectin promoter, Fit- 1 promoter, GFAP promoter, GPIIb promoter, ICAM- 2 promoter, INF-P promoter, Mb promoter, Nphsl promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.Targeting Agents

[0143] Since it is often desirable to target a particular cell, collection of cells, or tissue, the particles (e.g., microparticles and nanoparticles) and liposomes described herein, may be modified to include targeting moieties. For example, a FAST protein described herein may be further modified to include a targeting moiety.

[0144] A variety of agents or regions that target particular cells are known in the art. See, e.g., Cotten et al., Methods Enzym. 217:618, 1993. The targeting agent may be a protein, peptide, carbohydrate, glycoprotein, lipid, small molecule, or polynucleotide, etc. The targeting agent may be used to target specific cells or tissues or may be used to promote endocytosis or phagocytosis of the particle.

[0145] Examples of targeting agents include, but are not limited to, antibodies, fragments of antibodies, proteins, peptides, carbohydrates, receptor ligands, sialic acid, and aptamers, etc. If the targeting agent is included throughout a particle, the targeting agent may be included in the mixture that is used to form the particle. If the targeting agent is only on the surface of a particle, the targeting agent may be associated with (e.g., by covalent or non-covalent (e.g., electrostatic, hydrophobic, hydrogen bonding, van der Waals, 71-71 stacking) interactions) the formed particle using standard chemical techniques.

[0146] In some embodiments, a FAST protein is modified to comprise a targeting agent. In certain embodiments, a particle comprises one or more FAST proteins and targeting agents, wherein the FAST proteins and targeting agents are directly attached to one another. In certain embodiments, a particle comprises one or more FAST proteins and targeting agents, wherein the FAST proteins and targeting agents are not directly attached to one another.Particles

[0147] In certain aspects, a FAST protein provided herein and an agent (e.g., one or more of a PEG- lipid, sterol, phospholipid, helper lipid, or stabilizing excipient) may be in the form of a particle. In some embodiments, the particle may be a lipid particle.

[0148] In certain embodiments, the particle is a microparticle (i.e., particle having a characteristic dimension of less than about 1 millimeter and at least about 1 micrometer, where the characteristic dimension of the particle is the smallest cross-sectional dimension of the particle).

[0149] In certain embodiments, the particle is a nanoparticle (i.e., a particle having a characteristic dimension of less than about 1 micrometer and at least about 1 nanometer, where the characteristic dimension of the particle is the smallest cross- sectional dimension of the particle). In certain embodiments, the average diameter of the particle is at least about 10 nm, at least about 30 nm, at least about 100 nm, at least about 300 nm, at least about 1 pm, at least about 3 pm, at least about 10 pm, at least about 30 pm, at least about 100 pm, at least about 300 pm, or at least about 1 mm. In certain embodiments, the average diameter of the particle is less than about 1 mm, less than about 300 pm, less than about 100 pm, less than about 30 pm less than about 10 pm, less than about 3 pm, less than about 1 pm, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm. Combinations of the above ranges (e.g., at least about 100 nm and less than about 1 pm) are also within the scope of the present invention.

[0150] The particles described herein may include additional materials, such as polymers (e.g., synthetic polymers (e.g., PEG, PLGA) and natural polymers (e.g., phospholipids)). In certain embodiments, the additional materials are approved by a regulatory agency, such as the U.S. FDA, for human and veterinary use.

[0151] In certain embodiments, the particle further comprises one or more of a PEG-lipid, sterol, phospholipid, helper lipid, or stabilizing excipient. In certain embodiments, the particle comprises a PEG-lipid, sterol, and phospholipid. In certain embodiments, the particle comprises a PEG-lipid, sterol, phospholipid, and helper lipid. In certain embodiments, the particle comprises a PEG-lipid, sterol, phospholipid, and stabilizing excipient. In certain embodiments, the particle comprises a PEG- lipid, sterol, phospholipid, helper lipid, and stabilizing excipient. In certain embodiments, the particle comprises a PEG-lipid. In some embodiments, the particle comprises a sterol. In certain embodiments, the particle comprises a phospholipid. In some embodiments, the particle comprises a helper lipid. In some embodiments, the particle comprises a stabilizing excipient. In certain embodiments, the particle comprises one or more of a PEG-lipid, sterol, phospholipid, helper lipid, or stabilizing excipient. In some embodiments, the particle comprises one or more of a PEG-lipid, sterol, phospholipid, helper lipid, or stabilizing excipient and is formulated as a nanoparticle or microparticle. In certain embodiments, a particle comprises one or more of a PEG-lipid, sterol, phospholipid, helper lipid, or stabilizing excipient and is formulated as a lipid nanoparticle.

[0152] In certain embodiments, a modified or engineered FAST protein or chimeric FAST protein is incorporated into a lipid nanoparticle to promote intracellular delivery of substance. In certain embodiments, the cargo is, for example, a nucleic acid, a polypeptide, a protein, or a small molecule. Additionally, the instant disclosure provides methods of delivering a substance to a target cell, the method comprising contacting the target cell with the lipid nanoparticle described above and elsewhere in the disclosure.Liposomes

[0153] A FAST protein provided herein, and an agent may be in the form of a particle, wherein the particle is a liposome or liposome nanoparticle. Liposomes are particularly useful in delivering an agent, such as a hydrophobic agent. In certain embodiments, a liposome comprising a FAST protein is complexed with (e.g., encapsulates or covers) a cargo (e.g., a nucleic acid, a protein, or small molecule). In certain embodiments, a FAST protein is formulated into liposome membranes in order to induce liposome-cell and / or liposome-liposome fusion. In certain embodiments, the liposome comprising the FAST protein is used to deliver a cargo (e.g., a nucleic acid, a protein, a small molecule) to a cell. The instant disclosure provides methods of delivering a substance to a target cell, the method comprising contacting the target cell with a liposome described above and elsewhere in the disclosure.

[0154] Many techniques for preparing micelles and liposomes are known in the art, and any such method may be used herein to make micelles and liposomes. The following scientific papers described other methods for preparing liposomes and micelles: Narang et al., “Cationic Lipids with Increased DNA Binding Affinity for Nonviral Gene Transfer in Dividing and Nondividing Cells,” Bioconjugate Chem.l6:156- 68, 2005; Hofland et al., “Formation of stable cationic lipid / DNA complexes for gene transfer,” Proc. Natl. Acad. Sci. USA 93:7305-7309, July 1996; Byk et al., “Synthesis, Activity, and Structure - Activity Relationship Studies of Novel Cationic Lipids for DNA Transfer,” J. Med. Chem.41(2):224-235, 1998; Wu et al., “Cationic Lipid Polymerization as a Novel Approach for Constructing New DNA Delivery Agents f Bioconjugate Chem. 12:251-57, 2001; Lukyanov et al., “Micelles from lipid derivatives of water-soluble polymers as delivery systems for poorly soluble drugs,” Advanced Drug Delivery Reviews 56:1273-1289, 2004; Tranchant et al., “Physicochemical optimisation of plasmid delivery by cationic lipids,” J. Gene Med.6:S24-S35, 2004; van Balen et al., “Liposome / Water Lipophilicity: Methods, Information Content, and Pharmaceutical Applications,” Medicinal Research Rev.24(3):299-324, 2004; each of which is incorporated herein by reference.

[0155] In certain embodiments, a modified or engineered FAST protein or chimeric FAST protein is formulated into a liposome nanoparticle to promote intracellular delivery of cargo. In certain embodiments, the cargo is, for example, a nucleic acid, a polypeptide, a protein, or a small molecule.

[0156] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.Pharmaceutical Compositions

[0157] Other aspects of the present disclosure relate to pharmaceutical compositions comprising the FAST proteins, nucleic acids, vectors, cells, and particles (e.g., lipid nanoparticles and liposomes) described herein. The term “pharmaceutical composition”, as used herein, refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g. for specific delivery, increasing half-life, or other therapeutic compounds). Pharmaceutical compositions may optionally comprise one or more additional therapeutically active substances.

[0158] In some embodiments, compositions provided herein are administered to a subject, for example, to a human subject, in order to effect a targeted genomic modification within the subject. In some embodiments, cells are obtained from the subject and contacted with a any of the pharmaceutical compositions provided herein. In some embodiments, cells removed from a subject and contacted ex vivo with a pharmaceutical composition are re-introduced into the subject, optionally after the desired genomic modification has been effected or detected in the cells. Methods of delivering pharmaceutical compositions comprising nucleases are known, and are described, for example, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, the disclosures of all of which are incorporated by reference herein in their entireties. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals or organisms of all sorts.Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and / or other primates; mammals, domesticated animals, pets, and commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant birds such as chickens, ducks, geese, and / or turkeys.

[0159] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient(s) into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0160] Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington’s The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated in its entirety herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

[0161] In some embodiments, compositions in accordance with the present invention may be used for treatment of any of a variety of diseases, disorders, and / or conditions, including but not limited to one or more of the following: autoimmune disorders (e.g. diabetes, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis); inflammatory disorders (e.g. arthritis, pelvic inflammatory disease); infectious diseases (e.g. viral infections e.g., HIV, HCV, RSV), bacterial infections, fungal infections, sepsis); neurological disorders (e.g. Alzheimer’s disease, Huntington’s disease; autism; Duchenne muscular dystrophy); cardiovascular disorders (e.g. atherosclerosis, hypercholesterolemia, thrombosis, clotting disorders, angiogenic disorders such as macular degeneration); proliferative disorders (e.g. cancer, benign neoplasms); respiratory disorders (e.g. chronic obstructive pulmonary disease); digestive disorders (e.g. inflammatory bowel disease, ulcers); musculoskeletal disorders (e.g. fibromyalgia, arthritis); endocrine, metabolic, and nutritional disorders (e.g. diabetes, osteoporosis); urological disorders (e.g. renal disease); psychological disorders (e.g. depression, schizophrenia); skin disorders (e.g. wounds, eczema); blood and lymphatic disorders (e.g. anemia, hemophilia); etc.Kits

[0162] The compositions (e.g., pharmaceutical compositions) of the present disclosure may be assembled into kits. In some embodiments, the kit comprises nucleic acid vectors for the expression of the FAST proteins described herein. In other embodiments, the kit further comprises particles comprising a FAST protein described herein. In certain embodiments, the kit comprises the FAST proteins described herein, the nucleic acids described herein, the expression vectors described herein, the cells described herein, and / or the particles described herein, with a set of instructions. In some embodiments, the instructions are for delivering the FAST proteins described herein, the nucleic acidsdescribed herein, the expression vectors described herein, the cells described herein, and / or the particles described herein for delivering a substance or cargo to a cell.

[0163] The kit described herein may include one or more containers housing components for performing the methods described herein and optionally instructions for use. Any of the kit described herein may further comprise components needed for performing the assay methods. Each component of the kits, where applicable, may be provided in liquid form (e.g., in solution) or in solid form, (e.g., a dry powder). In certain cases, some of the components may be reconstitutable or otherwise processible (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water), which may or may not be provided with the kit.

[0164] In some embodiments, the kits may optionally include instructions and / or promotion for use of the components provided. As used herein, “instructions” can define a component of instruction and / or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit.Methods of Use

[0165] In some aspects, the invention provides methods of use for delivering an agent or cargo incorporated in a particle (e.g., lipid nanoparticle or liposome) that further comprise a FAST protein. In certain embodiments, the method is for delivering an agent to a subject, cell, collection of cells, or tissue. In some embodiments, the method is for delivering an agent to a subject or cell. In certain embodiments, the method is for delivering an agent to a subject. In some embodiments, the method is for delivering an agent to a cell. In some embodiments, the agent is any agent provided herein.

[0166] Specifically, the present disclosure describes improved methods for intracellular delivery of bioactive compounds via the liposomes or lipid nanoparticles described herein. That is, the improvement is due to the incorporation of one or more of the disclosed FAST proteins into the liposomes or lipid nanoparticles because the FAST proteins can promote membrane fusion in a diversity of cell types from different species.EXAMPLES

[0167] In order that the present disclosure may be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compounds, compositions, and methods provided herein and are not to be construed in any way as limiting in their scope.

[0168] The mechanisms of membrane fusion and determinants of host range for pathogens remain poorly understood. Improved understanding of these concepts could open new areas for therapeutic development. The examples provided herein modify and / or engineer chimeric FAST proteinscomprising various domains from naturally occurring NSP1-1 or NSP1-2 proteins encoded by Rotavirus species B and G (RVB and RVG, respectively). In certain embodiments, the domains are further modified to include one or more mutations (e.g., substitutions, insertions, or deletions of residues).

[0169] The examples below indicate how domain diversity between different NSP1-1 or NSP1-2 proteins (e.g., wild type sequences given in Table A) influence functional activity of FAST proteins. For example, a selection of the generated chimeric FAST proteins can induce syncytia formation in at least some cell types (indicated by terms “moderately fusogenic” and highly fusogenic”), while other chimeric FAST proteins are not active (e.g., “non-fusogenic”).Example 1: Materials and MethodsCloning

[0170] Wild type Rotavirus FAST proteins (accession numbers in Table A), modified (i.e., variant) Rotavirus FAST proteins, or chimeric FAST proteins were synthesized. A gene fragment encoding a Kozak consensus sequence was inserted upstream to mCherry (mCh), encephalomyocarditis virus (EMCV), and internal ribosome entry site (IRES) in pcDNA3.1 at Hindlll / EcoRI cut sites. Unique restriction cut sites, EcoRI and Agel, were introduced downstream of the IRES sequence to facilitate the insertion of sequences encoding the FAST proteins. Truncations were introduced with primers. Plasmids were generated for FAST protein expression in mammalian cells (e.g., pcDNA-mCh-IRES- FAST).

[0171] A gene fragment encoding a Kozak consensus sequence was inserted upstream to mCh and FLAG epitope of pcDNA3.1 using Hindlll / EcoRI cut sites to generate a plasmid (pcDNA-mCh) for use as negative control in syncytial assay. Likewise, a gene fragment encoding FLAG epitope tag (DYKDDDDK (SEQ ID NO: 345)) and a leucine zipper (bJun, LAAANSKAERKRMRNRIAASKCRKRKLERIARLEEKVKTLKAQNSELASTANMLREQVAQL KQKVMNHVNSGCQLMLTQQLQTFSRRSI (SEQ ID NO: 346)) was inserted upstream of a 3xGGGS (SEQ ID NO: 347) linker and GFP11 (RDHMVLHEYVNAAGIT (SEQ ID NO: 348)) in the pLVX-IRES-Puro plasmid (Takara) at Spel / EcoRI cut sites to generate a plasmid (pLVX-bJun- GFP11) to quantify syncytial activity of FAST proteins, and to use as a positive control in syncytial assay. Similarly, a gene fragment encoding HA epitope tag (YPYDVPDYA (SEQ ID NO: 349)) and a leucine zipper (bFos, LMAMEARIRGRAQSIGRRGKVEQLSPEEEEKRRIRRERNKMAAAKCRNRRRELTDTLQAETD QLEDEKSALQTEIANLLKEKEKLEFILAAHRPACKIPNDLG (SEQ ID NO: 350)) was inserted upstream of a 3xGGGS (SEQ ID NO: 347) linker and GFP1-10(SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLKFICTTGKLPVPWPTLVTTL TYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGKYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFTVRHNVEDGSVQLADHYQQNT PIGDGPVLLPDNHYLSTQTVLSKDPNEK (SEQ ID NO: 351)) in the pLVX-IRES-Puro plasmid (Takara) at Spel / EcoRI cut sites to generate a plasmid (pLVX-bFos-GFPl-10) to quantify syncytial activity of FAST proteins.Transduction

[0172] To generate lentivirus, Lenti-X 293T cells (Takara) were transfected with pLVX-bJun-GFPl l and pLVX-bFos-GFPl-10 with fourth-generation packaging plasmids with Lenti-X Packaging Single Shots (Takara). Lentivirus is concentrated with Lenti-X concentrator (Takara) and resuspended in 575 pl of Opti-Mem. 293T cells were transduced with the lentivirus-containing supernatant together with polybrene to generate two cell lines. Two days post transduction, cells were selected and maintained with 1.5ug / ml puromycin.

[0173] 70% confluent well in a 6 well plate of 293T cells (ATCC CRL-3216) were transduced with the lentivirus-containing supernatant together with 8 pg / mL polybrene to generate two cell lines. 2 days post transduction, cells were selected and maintained with 1.5 pg / ml puromycin (Thermo). The day before transfection, cells stably expressing pLVX-bJun-GFPl 1 and pLVX-bFos-GFPl-10 were passaged, such that they were growing at 60-80% confluency the next day.

[0174] Cells expressing pLVX-bJun-GFPl l were mixed with cells expressing pLVX-bFos-GFPl-10 at 50:50 ratio and transfected with TransIT-293 Transfection Reagent (Minis Bio) according to manufacturer’s instructions. Specifically, for each well in a 96 well plate, 6.8x104 cells of each cell line is mixed, resuspended in CO2 independent media (Leibovitz's L-15 media, 10% FBS, 1% Penicillin-Streptomycin), and transfected with 0.3 pl of TransIT-293, 50 ng of pUC19 plasmid and 50 ng of pcDNA-mCh-IRES-FAST plasmids. Transfected cells are plated in 96 well coverslip #1.5 bottom plate (Cellvis) pre-treated with 20 pg / ml fibronectin. For each condition, 8 wells of cells are transfected and plated. Negative control and positive controls, where pcDNA-mCherry-IRES-FAST plasmid is replaced with pcDNA mCherry and pLVX-bJun-GFPl 1 respectively, were included in each plate. Plates were incubated at 37 °C, 0% CO2.Imaging

[0175] At 16 and 24 hours post transfection, cells were removed from the incubator and imaged using a widefield fluorescence microscope (Biotek Cytation CIO) outfitted with Sony CMOS 16-bit camera, and a 4x objective (Olympus, Plan Fluorite, NA 0.13), GFP (EX 469 / 35 nm, EM 525 / 39 nm, dichroic mirror 497nm, 465nm LED) and Texas-Red ( EX 586 / 15 nm, EM 647 / 57 nm, dichroic mirror 605nm, 590nm LED) filter cube / LED set. Camera gain and exposure time was held constant and set to ensure there were no saturated pixels.

[0176] Images were processed using a rolling ball algorithm and deconvolved based on point spread function of the objective. GFP+ objects were masked by thresholding on fluorescence intensity,particle size, and background flattening. The cumulative area of GFP+ objects was quantified and reported as “Syncytial activity / Sum of GFP+ area”.Example 2: Syncytial activity of wild-type FAST proteins

[0177] To classify fusogenicity of each FAST protein, the ratio of the average sum of GFP+ area of splitGFP cells expressing FAST protein to the average sum of GFP+ of splitGFP cells expressing a reference FAST protein on the same plate. Ratio that is greater than or equal 0.75 is classified as “highly fusogenic.” Ratio between 0.05-0.75 is classified as “moderately fusogenic.” Ratio less than or equal to 0.05 is classified as “non-fusogenic.”

[0178] Table I. Syncytial activity of wild type FAST proteins.

[0179] Table J. Syncytial activity of variant (e.g., modified) “F100” FAST proteins

[0180] Table K. Syncytial activity of endoFl 00 chimeras

[0181] Table L. Syncytial activity of variant (e.g., modified) “F89” FAST proteinsEQUIVALENTS AND SCOPE

[0182] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0183] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is / are referred to as comprising particular elements and / or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein.

[0184] It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0185] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0186] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, butrather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

Claims

CLAIMSWhat is claimed is:

1. A protein comprising an amino acid sequence that is at least 80% identical to the amino acid sequence provided in SEQ ID NO: 1, wherein the amino acid sequence comprises one or more amino acid substitutions at position(s) selected from the group consisting of positions 2, 5, 6, 7, 9, 13, 17, 19, 22, 32, 33, 34, 36, 38, 39, 44, 51, 55, 58, 62, 63, 64, 65, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 96, and 100 of SEQ ID NO: 1.

2. The protein of claim 1 wherein the one or more amino acid substitutions is selected from the group consisting of at position 2 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 5 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 6 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 7 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 9 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 13 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 17 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 19 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 22 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 32 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 33 a substitution of any amino acid other than D, S, T, or V, at position 34 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 36 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 38 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 39 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 44 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 51 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 55 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 58 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 62 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 63 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 64 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 65 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 67 a substitution of any amino acid other than S, N, or R, at position 68 a substitution of any amino acid other than K or R,at position 70 a substitution of any amino acid other than K or R, at position 71 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 72 a substitution of any amino acid other than T or R, at position 73 a substitution of any amino acid other than H or N, at position 74 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, at position 75 a substitution of any amino acid other than P, I, V, or L, at position 76 a substitution of any amino acid other than S, Q, or K, at position 77 a substitution of any amino acid other than R or S, at position 78 a substitution of any amino acid other than F, I, L, or L, at position 79 a substitution of any amino acid other than Y, I, L, or L, at position 80 a substitution of any amino acid other than R, S, K, or E, at position 81 a substitution of any amino acid other than T or R, at position 82 a substitution of any amino acid other than S or N, at position 83 a substitution of any amino acid other than Q, S, L, or I, at position 96 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1, and at position 100 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 1.

3. The protein of any one of claims 1-2, wherein the amino acid sequence is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99%, or at least 99.5% identical to the amino acid sequence provided in SEQ ID NO: 1.

4. The protein of any one of claims 1-3, wherein the amino acid sequence is at least 80% identical to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence comprises one or more amino acid substitutions selected from the group consisting of V33G, V33K, N62A, C63A,Y64A, R67Q, K68Q, K70Q, T71A, N72A, G73A, V75A, S76A, S77A, L78A, L79A, E80A, R81A, N82A, and I83A.

5. The protein of any one of claims 1-4, wherein the amino acid sequence comprises amino acid substitutions:(a) V33G;(b) V33K;(c) R67Q;(d) R67Q and K68Q;(e) K70Q and R71Q;(f) R67Q, K68Q, and K70Q;(g) N62A, C63A, and Y64A;(f) T71A, N72A, and G73A;(g) V75A, S76A, and S77A;(h) L78A, L79A, and E80A; or(g) R81A, N82A, and I83A.

6. The protein of any one of claims 1-5, wherein the amino acid sequence is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence provided in any one of SEQ ID NOs: 73-109.

7. A protein comprising an amino acid sequence that is at least 80% identical to the amino acid sequence provided in SEQ ID NO: 2, wherein the amino acid sequence comprises one or more amino acid substitutions at position(s) selected from the group consisting of positions 62, 63, 64, 65, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 96, and 100 of SEQ ID NO: 2.

8. The protein of claim 7, wherein the one or more amino acid substitutions is selected from the group consisting of at position 62 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 2, at position 63 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 2, at position 64 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 2, at position 65 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 2, at position 67 a substitution of any amino acid other than S, N, or R, at position 68 a substitution of any amino acid other than K or R, at position 70 a substitution of any amino acid other than K or R, at position 71 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 2, at position 72 a substitution of any amino acid other than T or R, at position 73 a substitution of any amino acid other than H or N, at position 74 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 2, at position 75 a substitution of any amino acid other than P, I, V, or L, at position 76 a substitution of any amino acid other than S, Q, or K, at position 77 a substitution of any amino acid other than R or S, at position 78 a substitution of any amino acid other than F, I, L, or L, at position 79 a substitution of any amino acid other than Y, I, L, or L, at position 80 a substitution of any amino acid other than R, S, K, or E, at position 81 a substitution of any amino acid other than T or R, at position 82 a substitution of any amino acid other than S or N, at position 83 a substitution of any amino acid other than Q, S, L, or I, at position 96 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 2, and at position 100 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 2.

9. The protein of claim 7 or 8, wherein the amino acid sequence is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical to the amino acid sequence provided in any one of SEQ ID NOs: 311-323 and 337-340.

10. A protein comprising an amino acid sequence that is at least 80% identical to the amino acid sequence provided in SEQ ID NO: 3, wherein the amino acid sequence comprises one or more amino acid substitutions at position(s) selected from the group consisting of positions 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 34, 38, 40, 44, 45, 46, 48, 49, 50, 51, 53, 54, 55, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 83, 84, 90, 93, and 101 of SEQ ID NO: 3.

11. The protein of claim 10, wherein the one or more amino acid substitutions is selected from the group consisting of at position 4 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 5 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 6 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 7 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 9 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 10 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position I l a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 12 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 13 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 14 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 15 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 16 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 17 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 18 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 19 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 20 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 22 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 23 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 24 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 26 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 27 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 28 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 29 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3,at position 30 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 31 a substitution of any amino acid other than A, L, F, S, or V, at position 32 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 34 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 38 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 40 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 44 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 45 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 46 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 48 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 49 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 50 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 51 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 53 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 54 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 55 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 57 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 58 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 59 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 60 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 61 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 62 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 63 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 64 a substitution of any amino acid other than F or Y, at position 65 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 66 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 67 a substitution of any amino acid other than R or Q, at position 68 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 69 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 70 a substitution of any amino acid other than or R or K, at position 71 a substitution of any amino acid other than H or N, at position 72 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 73 a substitution of any amino acid other than P or S, at position 74 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 75 a substitution of any amino acid other than K or E, at position 76 a substitution of any amino acid other than I or V, at position 77 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3,at position 78 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 83 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 84 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 90 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, at position 93 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3, and at position 101 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 3.

12. The protein of claims 10 or 11, wherein the amino acid sequence is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99%, or at least 99.5% identical to the amino acid sequence provided in SEQ ID NO: 3.

13. The protein of any one of claims 10-12, wherein the amino acid sequence is at least 80% identical to the amino acid sequence of SEQ ID NO: 3, wherein the amino acid sequence comprises one or more amino acid substitutions selected from the group consisting of F31K, A32G, N62A, C63A, Y64A, L65A, L66A, R67A, R67Q, R68Q, R70E, N71A, G72A, P73A, R74E, K75Q, V76A, Y77A, and R78A.

14. The protein of any one of claims 10-13, wherein the amino acid sequence comprises amino acid substitutions:(a) F31K;(b) A32G;(c) R67Q and R68Q;(d) R67Q, R68Q, R70E, R74E, K75Q, and R78Q;(e) N62A, C63A, and Y64A;(f) L65A, L66A, and R67A;(g) N71A, G72A, and P73A; or(f) V76A, Y77A, and R78A.

15. The protein of any one of claims 10-14, wherein the amino acid sequence is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99%, or 100% identical to the amino acid sequence provided in any one of SEQ ID NOs: 147-228.

16. A protein comprising an amino acid sequence that is at least 80% identical to the amino acid sequence provided in SEQ ID NO: 4, wherein the amino acid sequence comprises one or more amino acid substitutions selected from the group consisting of residues 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 83, 84, 90, 93, and 101 of SEQ ID NO: 4.

17. The protein of claim 16 wherein the one or more amino acid substitutions is selected from the group consisting of at position 61 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 4, at position 62 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 4, at position 63 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 4, at position 64 a substitution of any amino acid other than F or Y, at position 65 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 4, at position 66 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 4, at position 67 a substitution of any amino acid other than R or Q, at position 68 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 4, at position 69 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 4, at position 70 a substitution of any amino acid other than or R or K, at position 71 a substitution of any amino acid other than H or N, at position 72 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 4, at position 73 a substitution of any amino acid other than P or S, at position 74 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 4, at position 75 a substitution of any amino acid other than K or E, at position 76 a substitution of any amino acid other than I or V, at position 77 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 4, at position 78 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 4, at position 83 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 4,' at position 84 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 4, at position 90 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 4, at position 93 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 4, and at position 101 a substitution of any amino acid other than the amino acid found in SEQ ID NO: 4.

18. A protein comprising a chimera of:(a) an ectodomain;(b) a transmembrane domain; and(c) an endodomain; wherein the ectodomain of (a), the transmembrane of (b), and the endodomain of (c) are derived from at least two different fusion-associated small transmembrane (FAST) proteins.

19. The protein of claim 18, wherein the chimera is a biologically-active FAST protein.

20. A protein comprising a chimera of:(a) an ectodomain, wherein the ectodomain comprises an amino acid sequence that is at least 80% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 17-30;(b) a transmembrane domain, wherein the transmembrane domain (TMD) comprises an amino acid sequence that is at least 80% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 31-46; and(c) an endodomain, wherein the endodomain comprises an amino acid sequence that is at least 80% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 47-58, wherein the ectodomain of (a), the transmembrane domain of (b), and the endodomain of (c) are derived from at least two different FAST proteins.

21. The protein of claim 20, wherein the chimera is a biologically-active FAST protein.

22. A protein comprising a fusion of:(a) an ectodomain, wherein the ectodomain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 17-30;(b) a transmembrane domain, wherein the transmembrane domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 31-46; and(c) an endodomain, wherein the endodomain comprises any one of the proteins of claims 6-7 and 13-14.

23. The protein of claim 22, wherein the fusion is a biologically-active FAST protein.

24. The protein of any one of claims 20-23, wherein the ectodomain comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 17-30.

25. The protein of any one of claims 20-23, wherein the ectodomain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 17-30 further comprising at least one, two, three, four, five, six, or seven amino acid substitutions.

26. The protein of any one of claims 20-23, wherein the ectodomain comprises an amino acid sequence that is identical to the amino acid sequence set forth in any one of SEQ ID NOs: 17-30.

27. The protein of any one of claims 20-26, wherein the transmembrane domain comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 31-46.

28. The protein of any one of claims 20-26, wherein the transmembrane domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 31-46 further comprising at least one, two, three, four, five, six, or seven amino acid substitutions.

29. The protein of any one of claims 20-26, wherein the transmembrane domain comprises an amino acid sequence that is identical to the amino acid sequence set forth in any one of SEQ ID NOs: 31-46.

30. The protein of any one of claims 20-21 or 24-29, wherein the endodomain comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, and 47-58.

31. The protein of any one of claims 20-21 or 24-29, wherein the endodomain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, and 47-58 further comprising at least one, two, three, four, five, six, or seven amino acid substitutions.

32. The protein of any one of claims 20-21 or 24-29, wherein the endodomain comprises an amino acid sequence that is identical to the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, and 47-58.

33. The protein of claim 20, wherein the endodomain comprises an amino acid sequence set forth in SEQ ID NO: 2.

34. The protein of claim 20, wherein the endodomain comprises an amino acid sequence set forth in SEQ ID NO: 4.

35. The protein of any one of claims 18-35, wherein (i) the ectodomain is derived from a first FAST protein, (ii) the transmembrane domain is derived from a second FAST protein, and (iii) the endodomain is derived from a third FAST protein.

36. The protein of any one of claims 18-35, wherein (i) the ectodomain and transmembrane domain are derived from a first FAST protein, and (ii) the endodomain is derived from a second FAST protein.

37. The protein of any one of claims 18-35, wherein (i) the ectodomain and the endodomain are derived from a first FAST protein, and (ii) the transmembrane domain is derived from a second FAST protein.

38. The protein of any one of claims 18-35, wherein (i) the ectodomain is derived from a first FAST protein, and (ii) the transmembrane domain and endodomain are derived from a second FAST protein.

39. The protein of any one of claims 18-38, wherein the protein comprises the structure NH2- [ectodomain] - [transmembrane domain] - [endodomain] -COOH wherein “]-[“ is optionally a linker.

40. The protein of any one of claims 18-39 further comprising a lipidation motif.

41. The protein of claim 40, wherein the lipidation motif is a myristoylation motif located at the N-terminus of the ectodomain.

42. The protein of claim 40, wherein the lipidation motif is a palmitoylation motif located in the endodomain.

43. The protein of any one of claims 18-42, wherein the protein further comprises a linker, an epitope tag, detectable label, effector domain, or a targeting moiety.

44. A nucleic acid encoding the protein of any one of claims 1-43.

45. An expression vector comprising the nucleic acid of claim 44.

46. A cell comprising the expression vector of claim 45, the nucleic acid of claim 44, and / or the protein of any one of claims 1-43.

47. A particle comprising a NSP1 fusion-associated small transmembrane (FAST) protein from the genus Rotavirus.

48. A particle comprising a protein of any one of claims 1-43.

49. The particle of claim 47 or 48 further comprising a cargo.

50. The particle of claim 49, wherein the cargo is a nucleic acid, a protein, or small molecule.

51. The particle of any one of claims 47-50, wherein the particle is a lipid nanoparticle or a liposome.

52. A method of delivering a cargo to a target cell, the method comprising contacting the target cell with the particle of any one of claims 47-51.

53. Use of the particle of any one of claims 47-51 for delivery of a cargo to a cell.

54. The use of claim 53, wherein the cargo is a nucleic acid, a protein, or small molecule.

55. A pharmaceutical composition comprising: (i) the protein of any one of claims 1-43, the nucleic acid of claim 44, the expression vector of claim 45, the cell of claim 46, and / or the particle of any one of claims 47-51; and (ii) a pharmaceutically acceptable excipient.

56. A kit comprising: (i) the protein of any one of claims 1-43, the nucleic acid of claim 44, the expression vector of claim 45, the cell of claim 46, and / or the particle of any one of claims 47-51; and (ii) a set of instructions for delivering a substance to a cell.

Images