PNMA2-Based Capsids and Uses Thereof

Abstract

Disclosed herein, in certain embodiments, are recombinant PNMA2 and endogenous Gag polypeptides, capsids comprising recombinant PNMA2 or endogenous Gag polypeptides, and methods of making and using recombinant PNMA2 and endogenous Gag polypeptides.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 142,441, filed January 27, 2021, and U.S. Provisional Application No. 63 / 152,653, filed February 23, 2021, each of which is incorporated by reference in its entirety herein. Description of sequence listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. This ASCII copy, created on January 26, 2022, is named 54838_709_601_SL.txt and is 94,168 bytes in size. [Background technology]

[0002] Precise administration of diagnostic or therapeutic agents to the desired site remains an ongoing challenge. Available methods of delivering nucleic acids to cells suffer from multiple limitations. For example, AAV viral vectors, which are often used in gene therapy, are immunogenic, have limited payload capacity, have poor biodistribution, can only be administered by direct injection, and carry the risk of disrupting host genes upon integration. Some studies suggest that a significant proportion of the population (e.g., 50% or more) has pre-existing immunity to viral vectors such as AAV, which may severely limit the effectiveness of viral vectors in therapeutic applications. Summary of the Invention [Problem to be solved by the invention]

[0003] Thus, there is a need for new and improved compositions and methods for delivering therapeutic payloads. [Means for solving the problem]

[0004] The capsids (or virus-like particles, VLPs) disclosed herein have the potential to address many of these shortcomings. In certain embodiments, endogenous retroviral capsid polypeptides are disclosed herein. In some embodiments, endogenous Gag (endo-Gag) polypeptides of the disclosure, such as PNMA2 polypeptides, assemble into capsids for delivery of cargo of interest. The endo-Gag polypeptides of the disclosure exhibit surprising and unexpected advantages over existing and alternative encapsidation polypeptides, such as improved efficiency in capsid assembly, capsid disassembly, and / or capsid reassembly, e.g., reassembly with heterologous cargo. In additional embodiments, capsids, e.g., PNMA2-based or endo-Gag-based capsids, for delivery of cargo of interest are described herein. Also disclosed herein are engineered endo-Gag polypeptides, such as PNMA2 polypeptides, with additional modifications that have certain advantages and usefulness. In some aspects, disclosed herein are capsids comprising engineered endogenous retroviral capsid polypeptides, which are capable of disassembling into a non-capsid state with at least 1% efficiency and reassembling into a capsid state with at least 1% efficiency.

[0005] In some embodiments, the efficiency of disassembly is determined by quantifying the amount of endogenous retroviral capsid polypeptides in solution after treating purified capsids with a disassembly buffer. In some embodiments, the efficiency of reassembly is determined by comparing the amount of engineered endogenous retroviral capsid polypeptides in the capsid peak with the amount of engineered endogenous retroviral capsid polypeptides in the monomer peak, and the capsid peak and monomer peak are identified by multi-angle dynamic light scattering or size exclusion chromatography. In some embodiments, the efficiency of reassembly is determined by quantifying the amount of retroviral capsid polypeptides in a non-capsid state before reassembly and quantifying the amount of retroviral capsid polypeptides in a capsid state after reassembly by size exclusion chromatography. In some embodiments, the capsids can be disassembled to a non-capsid state with at least about 20% efficiency. In some embodiments, the capsids can be reassembled to a capsid state with at least about 20% efficiency. In some embodiments, the engineered endogenous retroviral capsid polypeptide is an engineered endo-Gag polypeptide. In some embodiments, the engineered endogenous retroviral capsid polypeptide comprises a sequence of an endo-Gag polypeptide. In some embodiments, the engineered endogenous retroviral capsid polypeptide is an engineered PNMA protein. In some embodiments, the engineered endogenous retroviral capsid polypeptide is an engineered PNMA2 protein. In some embodiments, the reassembly is performed in a buffer containing less than 500 mOsm / kg salt. In some embodiments, the capsid further comprises a heterologous cargo that is not naturally associated with the endogenous retroviral capsid polypeptide. In some embodiments, the heterologous cargo comprises a nucleic acid. In some embodiments, the heterologous cargo comprises RNA. In some embodiments, the heterologous cargo comprises DNA. In some embodiments, the heterologous cargo comprises or encodes a gene editing system or a component thereof.In some embodiments, the heterologous cargo comprises or encodes a CRISPR / Cas system or a component thereof. In some embodiments, the heterologous cargo comprises or encodes a zinc finger nuclease system or a component thereof. In some embodiments, the heterologous cargo comprises or encodes a TALEN system or a component thereof. In some embodiments, the heterologous cargo comprises a therapeutic agent. In some embodiments, the heterologous cargo comprises a polypeptide. In some embodiments, the heterologous cargo comprises an antibody or an antigen-binding fragment thereof, a peptidomimetic, a nucleotide mimetic, a drug, a diagnostic tool, an imaging tool, a small molecule, or a combination thereof. In some embodiments, at least 50% of the heterologous cargo is inside the capsid. In some embodiments, at least 50% of the heterologous cargo is outside the capsid. In some embodiments, the heterologous cargo is inside at least 50% of the capsid. In some embodiments, the heterologous cargo is outside at least 1% of the capsid. In some embodiments, the engineered PNMA2 polypeptide comprises the amino acid sequence of a mammalian PNMA2. In some embodiments, the engineered PNMA2 polypeptide comprises an amino acid sequence of human PNMA2. In some embodiments, the engineered PNMA2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to a sequence comprising at least 100 contiguous amino acids of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:1. In some embodiments, the PNMA2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to a sequence comprising at least 250 contiguous amino acids of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:1. In some embodiments, the engineered PNMA2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:1. In some embodiments, the engineered PNMA2 polypeptide comprises an amino acid sequence that is SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:1.

[0006] In some aspects, disclosed herein are isolated engineered endogenous retroviral capsid polypeptides capable of assembling to form capsids with an efficiency of at least 5%.

[0007] In some embodiments, the efficiency of assembly is determined by a size exclusion chromatography assay to quantify the percentage of endogenous retroviral capsid polypeptides present in a capsid state. In some embodiments, when isolated, the engineered endogenous retroviral capsid polypeptides are capable of assembling to form capsids with an efficiency of at least about 20%. In some embodiments, when isolated, the engineered endogenous retroviral capsid polypeptides are capable of assembling to form capsids with an efficiency of at least about 50%. In some embodiments, the assembly is performed in a buffer containing less than 500 mOsm / kg salt. In some embodiments, the engineered endogenous retroviral capsid polypeptide is an endo-Gag polypeptide. In some embodiments, the engineered endogenous retroviral capsid polypeptide is an engineered PNMA protein. In some embodiments, the engineered endogenous retroviral capsid polypeptide is an engineered PNMA2 protein. In some embodiments, the engineered PNMA2 protein comprises a sequence modification to SEQ ID NO:1 or SEQ ID NO:7. In some embodiments, the sequence modification comprises an amino acid insertion. In some embodiments, the sequence modification comprises an amino acid deletion. In some embodiments, the sequence modification comprises an amino acid substitution. In some embodiments, the sequence modification comprises a cargo binding domain. In some embodiments, the sequence modification comprises a nucleic acid binding domain. In some embodiments, the sequence modification comprises an RNA binding domain. In some embodiments, the sequence modification comprises a DNA binding domain. In some embodiments, the sequence modification comprises a zinc finger domain. In some embodiments, the sequence modification comprises a subcellular localization signal. In some embodiments, the sequence modification comprises a nuclear localization signal (NLS). In some embodiments, the sequence modification comprises an antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment comprises a single chain variable fragment (scFv) or a single domain antibody. In some embodiments, the sequence modification comprises a domain that binds to a cell surface molecule.In some embodiments, the sequence modification comprises an arginine-rich domain. In some embodiments, the sequence modification is at the N-terminus of the engineered PNMA2 polypeptide. In some embodiments, the sequence modification is at the C-terminus of the engineered PNMA2 polypeptide. In some embodiments, the sequence modification is within the PNMA2 polypeptide. In some embodiments, the sequence modification comprises the addition of a cysteine ​​residue not present in native PNMA2 or the loss of a cysteine ​​residue present in native PNMA2.

[0008] In some aspects, disclosed herein are methods for producing a capsid, the methods comprising: (a) expressing endogenous retroviral capsid polypeptides in a host cell or a cell-free expression system; and (b) isolating the endogenous retroviral capsid polypeptides, wherein at least about 5% of the isolated endogenous retroviral capsid polypeptides assemble to form a capsid.

[0009] In some aspects, disclosed herein are methods of producing a capsid comprising a cargo, the methods comprising: (a) expressing an endogenous retroviral capsid polypeptide in a host cell or a cell-free expression system; (b) isolating a capsid comprising the endogenous retroviral capsid polypeptide; (c) disassembling the capsid, thereby producing a disassembled endogenous retroviral capsid polypeptide; (d) contacting the disassembled endogenous retroviral capsid polypeptide with a cargo; and (e) reassembling the disassembled endogenous retroviral capsid polypeptide, thereby producing a capsid comprising the cargo.

[0010] In some embodiments, the percentage of isolated endogenous retroviral capsids that assemble to form capsids is determined by size exclusion chromatography. In some embodiments, at least 20% of the isolated endogenous retroviral capsid polypeptides assemble to form capsids.

[0011] In some aspects, disclosed herein are methods for producing capsids comprising a heterologous cargo not naturally associated with the capsid, the methods comprising: (a) expressing endogenous retroviral capsid polypeptides in a host cell or a cell-free expression system; (b) isolating the endogenous retroviral capsid polypeptides; (c) treating the isolated endogenous retroviral capsid polypeptides with a disassembly buffer, thereby producing disassembled endogenous retroviral capsid polypeptides; and (d) reassembling the disassembled endogenous retroviral capsid polypeptides in a reassembly buffer in the presence of the heterologous cargo, thereby producing a capsid comprising the heterologous cargo.

[0012] In some embodiments, the isolated PNMA2 polypeptide is present at least partially in capsid form prior to step (c). In some embodiments, at least about 5% of the PNMA2 polypeptide present in capsid form prior to step (c) is disassembled after step (c). In some embodiments, at least about 5% of the PNMA2 polypeptide present in disassembled form after step (c) is present in capsid form after step (d).

[0013] In some aspects, disclosed herein are methods of loading endogenous retroviral capsids, the methods comprising: (a) disassembling an endogenous retroviral capsid into a composition comprising endogenous retroviral capsid monomers; (b) contacting a cargo with the composition comprising endogenous retroviral capsid monomers; and (c) reassembling the composition comprising endogenous retroviral capsid monomers into an endogenous retroviral capsid, whereby the cargo is loaded inside the endogenous retroviral capsid.

[0014] In some embodiments, the endogenous retroviral capsid polypeptide is a native endo-Gag polypeptide or an engineered endo-Gag polypeptide. In some embodiments, the endogenous retroviral capsid polypeptide is a native PNMA protein or an engineered PNMA protein. In some embodiments, the endogenous retroviral capsid polypeptide is a native PNMA2 protein or an engineered PNMA2 protein. In some embodiments, the method produces assembled capsids of at least about 50% purity as measured by SDS-PAGE. In some embodiments, the method produces assembled capsids with at least about 50% particle uniformity as measured by multi-angle dynamic light scattering. In some embodiments, the disassembly occurs in a disassembly buffer comprising a reducing agent. In some embodiments, the disassembly buffer comprises a reducing agent. In some embodiments, the reducing agent comprises reduced glutathione (GSH), beta mercaptoethanol (β-ME), dithiothreitol (DTT), or tris(2-carboxyethyl)phosphine (TCEP). In some embodiments, the disassembly buffer comprises a non-denaturing detergent. In some embodiments, the disassembly occurs in a disassembly buffer that includes a non-denaturing detergent. In some embodiments, the non-denaturing detergent is CHAPS. In some embodiments, the reassembly occurs in a reassembly buffer that includes less than 500 mOsm / kg salt. In some embodiments, the reassembly occurs in a reassembly buffer that includes about 270-330 mOsm / kg salt.

[0015] In some aspects, disclosed herein are engineered PNMA2 polypeptides that contain sequence modifications relative to SEQ ID NO:1 or SEQ ID NO:7.

[0016] In some embodiments, the sequence modification comprises an amino acid insertion. In some embodiments, the sequence modification comprises an amino acid deletion. In some embodiments, the sequence modification comprises an amino acid substitution. In some embodiments, the sequence modification comprises a cargo binding domain. In some embodiments, the sequence modification comprises a nucleic acid binding domain. In some embodiments, the sequence modification comprises an RNA binding domain. In some embodiments, the sequence modification comprises a DNA binding domain. In some embodiments, the sequence modification comprises a zinc finger domain. In some embodiments, the sequence modification comprises a subcellular localization signal. In some embodiments, the sequence modification comprises a nuclear localization signal (NLS). In some embodiments, the sequence modification comprises an antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment comprises a single chain variable fragment (scFv) or a single domain antibody. In some embodiments, the sequence modification comprises a domain that binds to a cell surface molecule. In some embodiments, the sequence modification comprises an arginine-rich domain. In some embodiments, the sequence modification is at the N-terminus of the engineered PNMA2 polypeptide. In some embodiments, the sequence modification is at the C-terminus of the engineered PNMA2 polypeptide. In some embodiments, the sequence modification is within the PNMA2 polypeptide. In some embodiments, the sequence modification comprises a cysteine ​​residue that is not present in native PNMA2.

[0017] In some aspects, disclosed herein are capsids comprising a PNMA2 polypeptide and a heterologous cargo that is not naturally associated with a capsid comprising PNMA2, wherein the PNMA2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:1.

[0018] In some aspects, disclosed herein is a capsid comprising an engineered PNMA2 polypeptide of any one of the preceding embodiments.

[0019] A capsid comprising: (a) a first endogenous retroviral capsid polypeptide comprising the amino acid sequence of a native PNMA2 polypeptide or an engineered PNMA2 polypeptide; and (b) a second endogenous retroviral capsid polypeptide; wherein the amino acid sequence of the first endogenous retroviral capsid polypeptide is not identical to the amino acid sequence of the second endogenous retroviral capsid polypeptide.

[0020] In some aspects, disclosed herein is a capsid comprising: (a) a first endogenous retroviral capsid polypeptide that is an engineered PNMA2 polypeptide of any one of the preceding embodiments; and (b) a second endogenous retroviral capsid polypeptide; wherein the amino acid sequence of the first endogenous retroviral capsid polypeptide is not identical to the amino acid sequence of the second endogenous retroviral capsid polypeptide.

[0021] In some embodiments, the second endogenous retroviral capsid polypeptide comprises the amino acid sequence of a native PNMA polypeptide or an engineered PNMA polypeptide. In some embodiments, the second endogenous retroviral capsid polypeptide comprises the amino acid sequence of a native PNMA2 polypeptide or an engineered PNMA2 polypeptide. In some embodiments, the second endogenous retroviral capsid polypeptide is not a PNMA2 polypeptide. In some embodiments, the capsid further comprises a heterologous cargo that is not naturally associated with the capsid comprising PNMA2. In some embodiments, the heterologous cargo comprises a nucleic acid. In some embodiments, the heterologous cargo comprises RNA. In some embodiments, the heterologous cargo comprises DNA. In some embodiments, the heterologous cargo comprises or encodes a gene editing system or a component thereof. In some embodiments, the heterologous cargo comprises or encodes a CRISPR / Cas system or a component thereof. In some embodiments, the heterologous cargo comprises or encodes a zinc finger nuclease system or a component thereof. In some embodiments, the heterologous cargo comprises or encodes a TALEN system or a component thereof. In some embodiments, the heterologous cargo comprises a therapeutic agent. In some embodiments, the heterologous cargo comprises a polypeptide. In some embodiments, the heterologous cargo comprises an antibody or antigen-binding fragment thereof, a peptidomimetic, a nucleotide mimetic, a drug, a diagnostic tool, an imaging tool, a small molecule, or a combination thereof. In some embodiments, at least 50% of the heterologous cargo is inside the capsid. In some embodiments, at least 50% of the heterologous cargo is outside the capsid. In some embodiments, the heterologous cargo is inside at least 50% of the capsid. In some embodiments, the heterologous cargo is outside at least 1% of the capsid. In some embodiments, the PNMA2 polypeptide comprises the amino acid sequence of a mammalian PNMA2. In some embodiments, the PNMA2 polypeptide comprises the amino acid sequence of a human PNMA2.In some embodiments, the PNMA2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to a sequence comprising at least 100 contiguous amino acids of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:1. In some embodiments, the PNMA2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to a sequence comprising at least 250 contiguous amino acids of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:1. In some embodiments, the PNMA2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:1. In some embodiments, the PNMA2 polypeptide comprises an amino acid sequence that is SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:1. In some embodiments, the capsid comprises disulfide bonds.

[0022] In some aspects, disclosed herein are compositions comprising a plurality of capsids comprising a PNMA2 polypeptide, wherein the capsids are at least 50% pure as measured by SDS-PAGE.

[0023] In some aspects, disclosed herein is a composition comprising a plurality of capsids of any one of the preceding embodiments, wherein the capsids are at least 50% pure as measured by SDS-PAGE.

[0024] In some aspects, disclosed herein are compositions comprising a plurality of capsids comprising a PNMA2 polypeptide, wherein the capsids comprise at least 50% particle uniformity as measured by multi-angle dynamic light scattering.

[0025] In some aspects, disclosed herein is a composition comprising a plurality of capsids comprising a PNMA2 polypeptide, wherein the composition comprises at least 50% particle uniformity as measured by multi-angle dynamic light scattering.

[0026] In some aspects, disclosed herein is a composition comprising a plurality of capsids of any one of the preceding embodiments, wherein the capsids comprise at least 50% particle uniformity as measured by multi-angle dynamic light scattering.

[0027] In some aspects, disclosed herein is a composition comprising a plurality of capsids of any one of the preceding embodiments, wherein the composition comprises a particle uniformity of at least 50% as measured by multi-angle dynamic light scattering.

[0028] In some embodiments, the composition further comprises a delivery component. In some embodiments, the delivery component comprises a lipid. In some embodiments, the delivery component comprises a polypeptide. In some embodiments, the delivery component comprises a polymer. In some embodiments, the delivery component comprises a cationic lipid. In some embodiments, the delivery component comprises a cationic peptide. In some embodiments, the delivery component comprises a cationic polymer. In some embodiments, the delivery component comprises a cell penetrating peptide. In some embodiments, the delivery component comprises a fusion protein. In some embodiments, the delivery component comprises an endogenous retroviral envelope protein. In some embodiments, the delivery component comprises a liposome. In some aspects, disclosed herein is a nucleic acid encoding an engineered PNMA2 polypeptide of any one of the preceding embodiments. In some aspects, disclosed herein is a vector comprising the nucleic acid. Disclosed herein is a cell comprising the nucleic acid.

[0029] In some aspects, disclosed herein is a method of delivering a heterologous cargo to a cell, comprising contacting the cell with a composition or capsid of any one of the preceding embodiments.

[0030] In some aspects, disclosed herein is a method of delivering a cargo to a cell, the method comprising contacting the cell with a composition comprising a plurality of isolated capsids, each capsid of the plurality of isolated capsids comprising a polypeptide comprising a PNMA2 sequence and a cargo.

[0031] In some aspects, disclosed herein are methods for delivering cargo to a cell, the methods comprising contacting a cell with at least about 0.001 pg / mL of a capsid comprising a capsid polypeptide comprising at least 100 consecutive amino acids of SEQ ID NO:1 and a heterologous cargo that is not naturally associated with the polypeptide comprising at least 100 consecutive amino acids of SEQ ID NO:1.

[0032] In some aspects, disclosed herein are methods for delivering cargo to a cell, the methods comprising contacting a cell with at least about 0.001 pg / mL of a capsid comprising a native or engineered PNMA2 polypeptide and a heterologous cargo that is not naturally associated with an endogenous retroviral capsid polypeptide.

[0033] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is contacted with the capsid at a concentration of at least about 0.01 pg / mL, at least about 0.1 pg / mL, at least about 1 pg / mL, at least about 10 pg / mL, at least about 100 pg / mL, at least about 1 ng / mL, at least about 10 ng / mL, at least about 100 ng / mL, at least about 1 μg / mL, at least about 10 μg / mL, at least about 100 μg / mL, at least about 1 mg / mL, at least about 10 mg / mL, or at least about 100 mg / mL. In some embodiments, the contacting is in vivo. In some embodiments, the contacting is in vitro or ex vivo. In some embodiments, the heterologous cargo comprises a nucleic acid and the cell expresses a gene encoded by the nucleic acid after delivery. In some embodiments, the heterologous cargo comprises a protein, a peptide, an antibody or a binding fragment thereof, a peptidomimetic, a nucleotide mimetic, a drug, a diagnostic tool, an imaging tool, a small molecule, or a combination thereof. In some embodiments, the heterologous cargo comprises or encodes a gene editing system or a component thereof. In some embodiments, the heterologous cargo comprises a CRISPR / Cas system or a component thereof. In some embodiments, the heterologous cargo comprises a zinc finger nuclease system or a component thereof. In some embodiments, the heterologous cargo comprises a TALEN system or a component thereof.

[0034] In some aspects, disclosed herein are capsids comprising a recombinant PNMA2 polypeptide and a heterologous cargo.

[0035] In some embodiments, the heterologous cargo is a nucleic acid. In some embodiments, the heterologous cargo is DNA. In some embodiments, the heterologous cargo is RNA. In some embodiments, the heterologous cargo is a therapeutic agent. In some embodiments, the heterologous cargo is a protein, peptide, antibody or binding fragment thereof, peptidomimetic, nucleotide mimetic, drug, diagnostic tool, imaging tool, or small molecule. In some embodiments, the recombinant PNMA2 polypeptide is mammalian PNMA2. In some embodiments, the recombinant PNMA2 polypeptide is human PNMA2. In some embodiments, the recombinant PNMA2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:1. In some embodiments, the PNMA2 polypeptide comprises an amino acid sequence comprising at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:1. In some embodiments, the PNMA2 polypeptide comprises the amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:1. In some embodiments, the recombinant PNMA2 polypeptide comprises a sequence modification relative to SEQ ID NO:1. In some embodiments, the capsid comprises a disulfide bond. In some embodiments, the capsid comprises a disulfide bond comprising a cysteine ​​at position 10, 136, 233, or 310 of the recombinant PNMA2 polypeptide relative to SEQ ID NO:1. In some embodiments, the capsid comprises a disulfide bond comprising a cysteine ​​at position 136 of the recombinant PNMA2 polypeptide relative to SEQ ID NO:1. In some embodiments, the capsid comprises a disulfide bond comprising a cysteine ​​at position 310 of the recombinant PNMA2 polypeptide relative to SEQ ID NO:1.In some aspects, the disclosure provides a composition comprising a plurality of capsids of any one of the preceding embodiments, wherein the capsids are at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% pure as measured by SDS-PAGE. In some aspects, the disclosure provides a composition comprising a plurality of capsids of any one of the preceding embodiments, wherein the capsids are at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% particle uniformity as measured by multi-angle dynamic light scattering.

[0036] In some aspects, disclosed herein are engineered PNMA2 polypeptides that contain sequence modifications relative to SEQ ID NO:1.

[0037] In some embodiments, the sequence modification comprises an amino acid deletion. In some embodiments, the sequence modification comprises an amino acid insertion. In some embodiments, the sequence modification comprises an amino acid substitution. In some embodiments, the sequence modification comprises a cargo binding domain. In some embodiments, the sequence modification comprises a nucleic acid binding domain. In some embodiments, the sequence modification comprises a DNA binding domain. In some embodiments, the sequence modification comprises an RNA binding domain. In some embodiments, the sequence modification comprises a zinc finger domain. In some embodiments, the sequence modification comprises a subcellular localization signal. In some embodiments, the sequence modification comprises a nuclear localization signal (NLS). In some embodiments, the sequence modification comprises an arginine-rich domain. In some embodiments, the sequence modification is at the N-terminus of SEQ ID NO:1. In some embodiments, the sequence modification is at the C-terminus of SEQ ID NO:1. In some embodiments, the sequence modification is within SEQ ID NO:1. In some aspects, the disclosure provides a nucleic acid encoding a PNMA2 polypeptide of any one of the preceding embodiments. In some embodiments, the disclosure provides a vector comprising the nucleic acid. In some embodiments, the disclosure provides a cell comprising the nucleic acid.

[0038] In some aspects, disclosed herein is a method of delivering a heterologous cargo to a cell, comprising contacting the cell with a capsid of any one of the preceding embodiments.

[0039] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a vertebrate cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is contacted with the capsid at a concentration of at least about 0.001 pg / mL, at least about 0.01 pg / mL, at least about 0.1 pg / mL, at least about 1 pg / mL, at least about 10 pg / mL, at least about 100 pg / mL, at least about 1 ng / mL, at least about 10 ng / mL, at least about 100 ng / mL, at least about 1 μg / mL, at least about 10 μg / mL, at least about 100 μg / mL, at least about 1 mg / mL, at least about 10 mg / mL, or at least about 100 mg / mL. In some embodiments, the contacting is in vivo. In some embodiments, the contacting is in vitro or ex vivo. In some embodiments, the heterologous cargo comprises a nucleic acid and the cell expresses a gene encoded by the nucleic acid after delivery. In some embodiments, the heterologous cargo is a protein, peptide, antibody or binding fragment thereof, peptidomimetic, nucleotide mimetic, drug, diagnostic tool, imaging tool, or small molecule.

[0040] In some aspects, disclosed herein are engineered endogenous retroviral capsid polypeptides that contain cysteine ​​residues, wherein, when isolated, the engineered endogenous retroviral capsid polypeptides are capable of assembling to form capsids with an efficiency of at least 5%.

[0041] In some embodiments, when isolated, the engineered endogenous retroviral capsid polypeptide can assemble to form a capsid with an efficiency of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. In some embodiments, the engineered endogenous retroviral capsid polypeptide is an endo-Gag polypeptide. In some embodiments, the engineered endogenous retroviral capsid polypeptide is an engineered PNMA protein. In some embodiments, the engineered endogenous retroviral capsid polypeptide is an engineered PNMA2 protein. In some embodiments, the assembling is performed in a buffer containing less than 2000 mOsm / kg, less than 1000 mOsm / kg, less than 900 mOsm / kg, less than 800 mOsm / kg, less than 700 mOsm / kg, less than 600 mOsm / kg, less than 500 mOsm / kg, less than 400 mOsm / kg, less than 300 mOsm / kg, or less than 200 mOsm / kg of salt.

[0042] In some aspects, disclosed herein are capsids comprising engineered endogenous retroviral capsid polypeptides, which are capable of disassembling into a non-capsid state with at least 1% efficiency and reassembling into a capsid state with at least 1% efficiency.

[0043] In some embodiments, the capsid may be disassembled into a non-capsid state with at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% efficiency. In some embodiments, when in a disassembled non-capsid state, the capsid may be reassembled into a capsid state with at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% efficiency. In some embodiments, the engineered endogenous retroviral capsid polypeptide is an endo-Gag polypeptide. In some embodiments, the engineered endogenous retroviral capsid polypeptide is an engineered PNMA protein. In some embodiments, the engineered endogenous retroviral capsid polypeptide is an engineered PNMA2 protein. In some embodiments, the efficiency of reassortment is determined in a buffer containing less than 2000 mOsm / kg, less than 1000 mOsm / kg, less than 900 mOsm / kg, less than 800 mOsm / kg, less than 700 mOsm / kg, less than 600 mOsm / kg, less than 500 mOsm / kg, less than 400 mOsm / kg, less than 300 mOsm / kg, or less than 200 mOsm / kg of salt.

[0044] In some aspects, disclosed herein are methods for producing a capsid, the methods comprising: (a) expressing a PNMA2 polypeptide in a host cell or a cell-free expression system; and (b) isolating the PNMA2 polypeptide, wherein the isolated PNMA2 polypeptide assembles to form a capsid.

[0045] In some embodiments, at least about 5% of the isolated PNMA2 polypeptides are assembled to form capsids. In some embodiments, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the isolated PNMA2 polypeptides are assembled to form capsids. In some embodiments, the percentage of the isolated PNMA2 polypeptides present in capsid form is measured by multi-angle dynamic light scattering assay. In some embodiments, the method produces assembled capsids of a purity of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% as measured by SDS-PAGE. In some embodiments, the method produces assembled capsids with a particle uniformity of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%%, at least about 70%, at least about 80%, or at least about 90% as measured by multi-angle dynamic light scattering. In some embodiments, the isolated PNMA2 polypeptide is assembled in a buffer containing less than 2000 mOsm / kg, less than 1000 mOsm / kg, less than 900 mOsm / kg, less than 800 mOsm / kg, less than 700 mOsm / kg, less than 600 mOsm / kg, less than 500 mOsm / kg, less than 400 mOsm / kg, less than 300 mOsm / kg, or less than 200 mOsm / kg salt.

[0046] In some aspects, disclosed herein is a method of making a capsid of any one of the preceding embodiments, the method comprising: (a) expressing a PNMA2 polypeptide in a host cell or a cell-free expression system; (b) isolating the PNMA2 polypeptide; (c) treating the isolated PNMA2 polypeptide with a disassembly buffer, thereby producing a disassembled form of the PNMA2 polypeptide; and (d) incubating the disassembled PNMA2 polypeptide in a reassembly buffer in the presence of a heterologous cargo, thereby producing a capsid comprising the heterologous cargo.

[0047] In some embodiments, the isolated PNMA2 polypeptide is at least partially present in capsid form prior to step (c). In some embodiments, the PNMA2 polypeptide is a recombinant PNMA2 polypeptide of any one of the preceding embodiments. In some embodiments, the PNMA2 polypeptide is an engineered PNMA2 polypeptide of any one of the preceding embodiments. In some embodiments, at least about 5% of the PNMA2 polypeptide present in capsid form prior to step (c) is disassembled after step (c). In some embodiments, at least about 5% of the PNMA2 polypeptide present in disassembled form after step (c) is present in capsid form after step (d). In some embodiments, the percentage of PNMA2 polypeptide present in capsid form or disassembled form is measured by a multi-angle dynamic light scattering assay. In some embodiments, the disassembly buffer comprises a reducing agent. In some embodiments, the reducing agent is GSH or TCEP. In some embodiments, the disassembly buffer comprises a non-denaturing detergent. In some embodiments, the non-denaturing detergent is CHAPS. In some embodiments, the reassembly buffer comprises less than 2000 mOsm / kg, less than 1000 mOsm / kg, less than 900 mOsm / kg, less than 800 mOsm / kg, less than 700 mOsm / kg, less than 600 mOsm / kg, less than 500 mOsm / kg, less than 400 mOsm / kg, less than 300 mOsm / kg, or less than 200 mOsm / kg of salt.

[0048] In some embodiments, disclosed herein are methods for loading PNMA capsids, the methods comprising: disassembling the PNMA capsid into a composition comprising PNMA monomers; contacting a cargo with the composition comprising PNMA monomers; and reassembling the composition comprising PNMA monomers into a PNMA capsid, thereby loading the cargo into the interior of the PNMA capsid.

[0049] In some embodiments, disassembling comprises reducing disulfide bonds. In some embodiments, reassembling comprises incubating a composition comprising cargo and PNMA monomers in a physiological buffer. In some embodiments, reassembling comprises incubating a composition comprising cargo and PNMA monomers in a buffer comprising about 270-330 mOsm / kg salt.

[0050] Various aspects of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings, in which: [Brief description of the drawings]

[0051] [Figure 1A] An exemplary alignment of the N-terminal 237 amino acids of PNMA2 with other PNMA family members is provided, with selected cysteine- and lysine- and arginine-rich KRs regions boxed. [Figure 1B] An exemplary alignment of amino acids 238-364 of PNMA2 with other PNMA family members is provided, with the cysteine ​​at position 310 of PNMA2 and the polyglutamic acid-rich region (PolyE) boxed. [Figure 2A] 1 shows a schematic diagram of an exemplary PNMA2 polypeptide construct including a 6xHis tag (SEQ ID NO:27), a TEV cleavage site, a linker, and a PNMA2 sequence, disclosed herein as "GSGSGS" as SEQ ID NO:58. [Figure 2B] 1 shows an outline of the purification procedure for recombinant 6xHis tagged PNMA2 ("6xHis tag" disclosed as SEQ ID NO:27). [Figure 2C] 1 shows an exemplary polyacrylamide gel demonstrating the purity of recombinant PNMA2. [Figure 2D] 1 shows an exemplary electron microscopy image showing concentrated PNMA2 capsids. [Figure 2E] 1 shows a multi-angle dynamic light scattering (MADLS) profile of purified PNMA2 capsid. [Figure 3A] FIG. 1 shows a schematic diagram of an exemplary untagged PNMA2 polypeptide construct comprising a PNMA2 sequence. [Figure 3B] 1 shows an outline of the purification procedure for untagged recombinant PNMA2. [Figure 3C] 1 shows an exemplary polyacrylamide gel demonstrating the purity of untagged recombinant PNMA2. [Figure 3D] 1 shows an exemplary electron microscopy image showing enriched PNMA2 capsids containing untagged PNMA2 polypeptides. [Figure 3E] 1 shows a multi-angle dynamic light scattering (MADLS) profile of purified PNMA2 capsid containing an untagged PNMA2 polypeptide. [Figure 4] 1 shows a schematic diagram of an exemplary engineered PNMA2 polypeptide construct with functional elements at the N- or C-terminus of the polypeptide. [Figure 5A] 1 shows a schematic diagram of an exemplary engineered PNMA2 polypeptide construct including a 6xHis tag (SEQ ID NO:27), a TEV cleavage site, a linker, an engineered RNA binding domain, and a PNMA2 sequence, disclosed herein as "GSGSGS" as SEQ ID NO:58. [Figure 5B] 1 shows an outline of the purification procedure for recombinantly engineered 6xHis-tagged PNMA2 with an RNA binding domain ("6xHis tag" disclosed as SEQ ID NO:27). [Figure 5C]1 provides an exemplary polyacrylamide gel showing the purity of recombinantly engineered 6xHis tagged PNMA2 with an RNA binding domain ("6xHis tag" disclosed as SEQ ID NO:27). [Figure 6A] FIG. 1 shows a schematic diagram of an exemplary engineered, tag-free PNMA2 polypeptide construct comprising an engineered RNA-binding domain and a PNMA2 sequence. [Figure 6B] FIG. 1 shows an outline of the purification procedure for recombinant engineered untagged PNMA2 with an RNA-binding domain. [Figure 6C] 1 provides an exemplary polyacrylamide gel showing the purity of recombinantly engineered, untagged PNMA2 with an RNA binding domain. [Figure 7A] An alignment of the cysteine ​​residues present in PNMA2 with the equivalent residues in PNMA1, PNMA3, PNMA4, PNMA5, and PNMA6A is shown, showing that some cysteines are conserved among PNMA family members (e.g., C10, C233) while others are not (e.g., C136, C310). [Figure 7B] Results of SDS-PAGE and Western blot assays are shown, demonstrating that dimers, trimers, tetramers, and many higher order interactions were observed for wild-type PNMA2, and that PNMA2 multimers were disrupted when disulfide bonds were reduced by TCEP treatment. C10S, C136S, C233S, and C310S mutant forms of PNMA2 showed reduced multimerization. [Figure 8A] Exemplary transmission electron microscopy images (top panel) and multi-angle dynamic light scattering (MADLS) profiles (bottom panel) of initially purified PNMA2 capsids are shown. [Figure 8B] Exemplary transmission electron microscopy images (top panel) and multi-angle dynamic light scattering (MADLS) profiles (bottom panel) of chemically disassembled PNMA2 capsids are shown. [Figure 8C] Exemplary transmission electron microscopy images (top panel) and multi-angle dynamic light scattering (MADLS) profiles (bottom panel) of reassembled PNMA2 capsids are shown. [Figure 9A] 1 is a MonoQ chromatograph showing the association of RNA with PNMA2 capsids. [Figure 9B] We quantified RNA extracted from the PNMA2 capsid peak of the MonoQ chromatograph shown in Fig. 9A and show that the ∼116 nt RNA is tightly associated with the capsid, while the PNMA2 capsid is separated from the bulk of the RNA. [Figure 10A] FIG. 1 is a schematic showing packaging of RNA cargo in PNMA2 capsids during reassembly. [Figure 10B] 1 shows the results of a gel shift assay demonstrating the association of a 67 nt RNA with reassembled PNMA2 capsids. The upshift is associated with the reassembled PNMA2 capsid RNA. [Figure 11A] FIG. 1 shows a schematic representation of the reassembly process of PNMA2 constructs with different engineered RNA-binding domains into the corresponding RNA-loaded capsids. [Figure 11B] Exemplary transmission electron microscopy images of the corresponding reassembled RNA- or DNA-loaded PNMA2 capsids are shown. [Figure 11C] Representative images of gel shift assays showing the association of various RNA or DNA cargoes with MonoQ-purified (nucleic acid-free) PNMA2 polypeptides containing various RNA-binding domains in reassembled capsids are shown. [Figure 12A] Representative images of gel shift assays are shown demonstrating that various amounts of RNA cargo packaged into reassembled PNMA2 capsids are protected from degradation by benzonase. [Figure 12B] Representative images of gel shift assays are shown, demonstrating that various amounts of RNA cargo packaged into reassembled PNMA2 capsids are protected from degradation by RNase A. [Figure 13A] A schematic diagram of two PNMA2 constructs, with and without an N-terminal 6xHis tag, is shown (SEQ ID NO:27). [Figure 13B]1 shows a schematic of the process of reassembly of hybrid capsids containing a mixture of untagged and 6xHis-tagged PNMA2 subunits ("6xHis tag" disclosed as SEQ ID NO:27). [Figure 13C] Representative transmission electron microscopy images of reassembled hybrid PNMA2 capsids are shown. [Figure 13D] FIG. 1 shows an exemplary image of a Coomassie R-250 stained denaturing polyacrylamide gel showing the subunit composition of hybrid PNMA2 capsids containing various amounts of untagged and 6xHis tagged PNMA2 polypeptides after reassembly and affinity purification on Ni-NTA agarose ("6xHis tag" disclosed as SEQ ID NO:27). [Figure 14A] Shown are epifluorescence microscopy images of mock-treated neurons (incubated without PNMA2) and subsequently stained with anti-PNMA2 antibody. The dotted line encircles the position of the nucleus. [Figure 14B] Fluorescence microscopy images of neurons incubated with PNMA2 capsids, washed, and then stained with anti-PNMA2 antibody are shown. A punctate staining pattern is observed, suggesting that the PNMA2 capsids were internalized by the neurons. The dotted line encircles the location of the nucleus. [Figure 14C] 1 shows epifluorescence microscopy images of tissue from a C57BL6 mouse injected intramuscularly in the hind leg with 10 μg of PNMA2 capsid, and stained muscle tissue from the contralateral (uninjected) hind leg. [Figure 14D] 1 shows epifluorescence microscopy images of tissue from a C57BL6 mouse that was injected intramuscularly in the rear with 10 μg of PNMA2 capsid, showing stained muscle tissue from the injected side. [Figure 15A] The top panel shows a schematic of the wild-type and ΔCYS PNMA2 proteins, indicating the location of the original cysteine ​​residues in the wild-type PNMA2 polypeptide, and the bottom panel shows a transmission electron microscopy image of the PNMA2 ΔCYS capsid showing that cysteines are not essential for capsid assembly. [Figure 15B] The top panel shows a schematic of the PNMA2 ΔCYS+6xGS-CYS construct with Alexa647 dye conjugated to one cysteine ​​C-terminal residue via maleimide chemistry ("6xGS-CYS" disclosed as SEQ ID NO: 59). The bottom panel shows PNMA2 ΔCYS+6xGS-CYS linked to Alexa647 run on a denaturing SDS-polyacrylamide gel and stained with Coomassie R-250 or visualized with Alexa657 fluorescence. [Figure 15C] 1 shows intracellular detection of Alexa647-labeled PNMA2 capsids by fluorescence microscopy in HCT116 colon cancer cells. [Figure 16A] FIG. 1 shows a diagram of potential modifications to the PNMA2 protein to enhance cellular targeting. [Figure 16B] FIG. 2. Diagram of camelid heavy chain only antibodies (HCAbs). [Figure 16C] 1 shows the addition of a single domain camelid antibody heavy chain variable region fragment (VH) to the N-terminus of the PNMA2 construct (PNMA2-VH). [Figure 16D] Figure 1 shows cellular targeting of PNMA2 capsids in A431 cancer cells, an epidermoid carcinoma with increased expression of EGFR. The left two images show cells treated with PNMA2 capsids, and the right two images show cells treated with capsids containing 10% PNMA2-VH (anti-EGFR). The top two images show the staining pattern from the anti-PNMA2 primary antibody, and the bottom two images show the fluorescent signal from the anti-EGFR primary antibody. [Figure 17A] The left panel shows the unmodified PNMA2 protein structure, and the right panel shows immunofluorescence staining of PNMA2 following treatment of HeLa cells with unmodified PNMA2. [Figure 17B] The left panel shows a mixture of PNMA2 capsids with a positively charged peptide consisting of nine arginines (R9) (SEQ ID NO: 56), and the right panel shows immunofluorescence staining of PNMA2 after treatment of HeLa cells with the PNMA2 capsid / peptide mixture. [Figure 17C]The left panel shows the addition of a cell penetrating peptide (CPP) to the N-terminus of the PNMA2 protein, and the right panel shows immunofluorescence staining of PNMA2 following treatment of HeLa cells with PNMA2-CPP. [Figure 18A] FIG. 1 shows the experimental setup for administration of Cre mRNA-loaded PNMA2 capsids onto U87MG cells constitutively expressing a LoxP-GFP-stop-LoxP-RFP (GFP / RFP switch) cassette. [Figure 18B] RFP expression in cells after incubation with Cre mRNA-loaded PNMA2 capsids or unpackaged Cre mRNA (after nuclease treatment) is shown. [Figure 19A] FIG. 1 shows the experimental setup for treatment of GFP / RFP switched cells with Cre mRNA-loaded PNMA2-RBD capsids. [Figure 19B] RFP expression in GFP / RFP switched cells is shown after treatment with Cre mRNA-loaded PNMA2-RBD capsids (upper panel) or Cre mRNA alone (lower panel). [Figure 20A] FIG. 1 shows the experimental setup for treatment of GFP / RFP switched cells with Cre mRNA-loaded PNMA2-RBD capsids supplemented with cationic lipids. [Figure 20B] RFP expression in cells following incubation with Cre mRNA-loaded PNMA2-RBD capsids (upper panel) or Cre mRNA alone (lower panel) after nuclease treatment and addition of cationic lipids is shown. [Figure 20C] Graph of the percentage of "GFP+" pixels that are also "RFP+", which is used as a surrogate readout for the percentage of cells into which Cre mRNA was successfully delivered to induce RFP expression. [Figure 21A] FIG. 1 shows the experimental setup for treatment of GFP / RFP switched cells with Cre mRNA-loaded PNMA2-RBD capsids supplemented with cationic peptide. [Figure 21B]RFP expression in cells following incubation with Cre mRNA-loaded PNMA2-RBD capsids (upper panel) or Cre mRNA alone (lower panel) after nuclease treatment and addition of cationic peptide is shown. [Figure 22A] 1 shows the experimental setup for treatment of THP-1 reporter cells with hairpin RNA (hpRNA)-loaded PNMA2-RBD capsids, optionally supplemented with a transduction enhancer. [Figure 22B] 1 shows a cell-based assay for detecting delivery of hpRNA into THP-1 cells. THP-1 monocytes express a secreted form of luciferase upon hpRNA-inducible interferon (INF) stimulation. [Figure 22C] 13 is a plot of luminescence from culture medium of THP-1 cells treated with either hpRNA-loaded PNMA2-RBD capsids alone, capsids loaded with added cationic polymer, or capsids loaded with added cationic lipid. [Figure 23A] FIG. 1 shows the experimental set-up for treating GFP / RFP switched cells with preformed RNMA2-RBD capsids that are incubated with Cre mRNA prior to nuclease treatment and addition of cationic lipids. [Figure 23B] Expression of RFP in cells after incubation with preformed PNMA2-RBD capsids mixed with Cre mRNA is shown with (top panel) or without (bottom panel) the addition of nuclease. [Figure 24A] The experimental set-up for administration of Cre mRNA-loaded PNMA2-RBD capsids into Ai14 mice genetically modified to express strong tdTomato fluorescence after Cre-mediated recombination is shown. [Figure 24B] Shows tdTomato expression in skeletal muscle-associated cells following intramuscular (IM) injection of Cre mRNA-loaded PNMA2-RBD capsids. [Figure 25A] 1 shows a multi-angle dynamic light scattering (MADLS) profile of disassembled PNMA2 capsids. [Figure 25B] 1 shows an exemplary electron microscopy image showing disassembled PNMA2 capsids. [Figure 25C] Multi-angle dynamic light scattering (MADLS) profiles of reassembled PNMA2 capsids are shown. [Figure 25D] 1 shows an exemplary electron microscopy image showing reassembled PNMA2 capsids. [Figure 26] 1 shows size-exclusion chromatography traces showing separation of assembled PNMA2 capsids from disassembled monomers. Vertical axis is mAU280. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] Precise administration of diagnostic or therapeutic agents to the desired site remains an ongoing challenge. Available methods of delivering nucleic acids to cells suffer from multiple limitations. For example, AAV viral vectors, which are often used in gene therapy, are immunogenic, have limited payload capacity, have poor biodistribution, can only be administered by direct injection, and have the risk of host gene disruption upon integration. Some studies suggest that a significant percentage of the population (e.g., 50% or more) has pre-existing immunity to viral vectors such as AAV, which can severely limit the effectiveness of viral vectors in therapeutic applications. The utility of existing non-viral methods is also limited by multiple drawbacks. Liposomes can be delivered primarily to the liver. Extracellular vesicles have limitations in payload amount, scalability, and can be difficult to purify. Thus, there is a need for new and improved compositions and methods for delivering therapeutic payloads. The capsids (or virus-like particles, VLPs) disclosed herein have the potential to address many of these drawbacks.

[0053] In certain embodiments, endogenous retroviral capsid polypeptides, such as endogenous Gag (endo-Gag) polypeptides, are disclosed herein. In some embodiments, the endo-Gag polypeptides of the disclosure, such as the PNMA2 polypeptide, are assembled into capsids for delivery of a cargo of interest. The endo-Gag polypeptides of the disclosure exhibit surprising and unexpected advantages over existing and alternative encapsidation polypeptides, such as improved efficiency in capsid assembly, capsid disassembly, and / or capsid reassembly, e.g., with heterologous cargo. In additional embodiments, capsids, e.g., PNMA2-based or endo-Gag-based capsids, for delivery of a cargo of interest are described herein. Also disclosed herein are engineered endo-Gag polypeptides, such as the PNMA2 polypeptide, with additional modifications that have certain advantages and usefulness.

[0054] Endogenous Gag and PNMA2 Polypeptides Endogenous Gag (endo-Gag) protein is a eukaryotic protein that has predicted and annotated similarity to viral Gag proteins. As described herein, in some embodiments, endo-Gag protein can assemble into a capsid (VLP) state or form. The endo-Gag protein of the present disclosure can be useful, for example, for packaging and delivering cargo (e.g., heterologous cargo) to cells.

[0055] The paraneoplastic Ma (PNMA) family of Endo-Gag proteins includes 15 members: PNMA1, PNMA2, PNMA3, PNMA4 (MOAP1), PNMA5, PNMA6A, PNMA6B / 6D, PNMA6E, PNMA6F, PNMA7A, PNMA7B, PNMA8A, PNMA8B, PNMA8C, and CCDC8. Members of the PNMA family share sequence homology with Gag proteins of long terminal repeat (LTR) retrotransposons. The PNMA2 protein is discussed in Pang et al., (2018) PNMA family: Protein interaction network and cell signalling pathways implicated in cancer and apoptosis. Cellular signalling, 45, 54-62, which is incorporated herein by reference for such disclosure.

[0056] The PNMA polypeptide contains several domains. Most PNMA family members share high sequence homology in the N-terminal conserved domain (NCD) and the central conserved domain (CCD). In some family members, a unique protein sequence or domain (UPD) is located between the NCD and CCD domains. A region rich in lysine and arginine basic residues, called KRs, is also found in the protein sequences of most members of the PNMA family. Many PNMA family members share sequence homology in a polyglutamic acid-rich region (PolyE) near the C-terminus. The polyglutamic acid-rich region has a C-terminus of variable length and low sequence homology (variable C-terminal sequence, VCS) that can be identified in the protein sequences between PNMA family members. Homologs of the PNMA protein are also present in other mammalian species, such as chimpanzees, monkeys, rats, and mice. In some family members, electrostatic interactions between the KRs and PolyE contribute to the three-dimensional structure of the protein.

[0057] PNMA2 is one of the members of the PNMA family. An exemplary PNMA2 is human PNMA2, which can include 364 amino acids, for example, as shown in SEQ ID NO:1. PNMA2 includes a central conserved domain (CCD), for example, at residues 202-204 of SEQ ID NO:1, and a polyglutamic acid-rich region (PolyE) near the C-terminus, for example, at residues 333-338 of SEQ ID NO:1. PNMA2 can form heterodimers with PNMA1 and PNMA4 (MOAP1), for example, to modulate (e.g., inhibit) apoptosis signaling. PNMA2 can also include cysteine ​​residues with the potential to form disulfide bonds, for example, at positions 10, 136, 233, and 310 of SEQ ID NO:1. An exemplary alignment of PNMA2 to other PNMA family members is shown in Figures 1A and 1B. The full sequences of exemplary PNMA family members are shown in Table 1.

[0058] [Table 1-1] [Table 1-2]

[0059] Further examples of Endo-Gag proteins are disclosed in Campillos et al., (2006) Computational characterization of multiple Gag-like human proteins, Trends Genet 22(11):585-9, the disclosure of which is incorporated herein by reference. Arc (activity-regulated cytoskeleton-associated protein) is another example of an endo-Gag protein. Arc regulates the endocytic trafficking of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors. Arc activity leads to synaptic strength and neuronal plasticity. The phenotype of Arc loss in experimental mouse models includes defects in long-term memory formation and reduced neuronal activity and plasticity.

[0060] In certain embodiments, endo-Gag polypeptides are disclosed herein. In some embodiments, it is understood that the endo-Gag sequence is any substitution of the PNMA2 sequence to form the engineered PNMA2 polypeptide disclosed herein. For example, in some embodiments, a sequence from a PNMA family member other than PNMA2 or a sequence from another endo-Gag polypeptide can be substituted for the PNMA2 sequence to form the engineered PNMA2 polypeptide of the present disclosure.

[0061] In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not Arc or do not include an amino acid sequence derived from Arc. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not PNMA5 or do not include an amino acid sequence derived from PNMA5. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not PNMA6 or do not include an amino acid sequence derived from PNMA6. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not PNMA6A or do not include an amino acid sequence derived from PNMA6A. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not PNMA6B or do not include an amino acid sequence derived from PNMA6B. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not a protein derived from the retrotransposon Gag-like family or do not include an amino acid sequence derived from a protein derived from the retrotransposon Gag-like family. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not PEG10(RTL2) or do not contain an amino acid sequence derived from PEG10(RTL2). In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not RTL1 or do not contain an amino acid sequence derived from RTL1. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not RTL3 or do not contain an amino acid sequence derived from RTL3. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not RTL6 or do not contain an amino acid sequence derived from RTL6. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not RTL8A or do not contain an amino acid sequence derived from RTL8A.In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not RTL8B or do not include an amino acid sequence from RTL8B. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not BOP(RTL10) or do not include an amino acid sequence from BOP(RTL10). In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not LDOC1(RTL7) or do not include an amino acid sequence from LDOC1(RTL7). In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not MOAP1(PNMA4) or do not include an amino acid sequence from MOAP1(PNMA4). In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not ZNF18 or do not include an amino acid sequence from ZNF18. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not Asprv1 or do not include an amino acid sequence from Asprv1. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not CCDC8 or do not include an amino acid sequence from CCDC8. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not PGBD1 or do not include an amino acid sequence from PGBD1. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not PNMA1 or do not include an amino acid sequence from PNMA1. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not PNMA3 or do not include an amino acid sequence from PNMA3. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not PNMA5 or do not include an amino acid sequence from PNMA5.In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not PNMA6B / 6D or do not include an amino acid sequence derived from PNMA6B / 6D. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not PNMA6E or do not include an amino acid sequence derived from PNMA6E. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not PNMA6F or do not include an amino acid sequence derived from PNMA6F. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not PNMA7A or do not include an amino acid sequence derived from PNMA7A. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not PNMA7B or do not include an amino acid sequence derived from PNMA7B. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not PNMA8A or do not include an amino acid sequence derived from PNMA8A. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not PNMA8B or do not include an amino acid sequence derived from PNMA8B. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not PNMA8C or do not include an amino acid sequence derived from PNMA8C. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not SCAN or do not include an amino acid sequence derived from SCAN. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not ZCCHC12 or do not include an amino acid sequence derived from ZCCHC12. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not ZCCHC18 or do not include an amino acid sequence derived from ZCCHC18.In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not ZNF274 or do not contain an amino acid sequence from ZNF274. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not SUSHI family members or do not contain an amino acid sequence from a SUSHI family member. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not SCAN family members or do not contain an amino acid sequence from a SCAN family member. In some embodiments, the endo-Gag or PNMA polypeptides disclosed herein are not PNMA2 or do not contain an amino acid sequence from PNMA2.

[0062] In some cases, the PNMA2 polypeptide comprises a full-length PNMA2 polypeptide. In other cases, the PNMA2 polypeptide comprises a fragment of PNMA2, such as a truncated PNMA2 polypeptide that is involved in capsid formation. In further cases, the PNMA2 polypeptide is a recombinant PNMA2 polypeptide.

[0063] In some cases, the endo-Gag polypeptide comprises a full-length endo-Gag protein. In other cases, the endo-Gag polypeptide comprises a fragment of an endo-Gag protein, such as a truncated endo-Gag polypeptide, that can participate in capsid formation. In further cases, the endo-Gag polypeptide is a recombinant endo-Gag polypeptide.

[0064] In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: NCD, UPD, CCD, KRs, PolyE, and VCS.

[0065] In some embodiments, one or more non-essential regions that are not involved in encapsidation are removed from the PNMA2 or endo-Gag protein to generate an engineered PNMA2 or endo-Gag polypeptide. In such cases, one or more non-essential regions, such as the N-terminal region (e.g., up to 10 amino acids, up to 15 amino acids, up to 20 amino acids, up to 25 amino acids, up to 30 amino acids, up to 40 amino acids, or up to 50 amino acids), the C-terminal region (e.g., up to 10 amino acids, up to 15 amino acids, up to 20 amino acids, up to 25 amino acids, up to 30 amino acids, up to 40 amino acids, or up to 50 amino acids), or a combination thereof, are deleted from the PNMA2 or endo-Gag protein (e.g., a native or wild-type PNMA2 or endo-Gag protein) to generate an engineered PNMA2 or endo-Gag polypeptide.

[0066] In some embodiments, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acids are removed from the N-terminus of a PNMA2 or endo-Gag protein (e.g., a native or wild-type PNMA2 or endo-Gag protein) to generate an engineered PNMA2 or endo-Gag polypeptide. In some embodiments, up to 5, up to 10, up to 15, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, or up to 50 amino acids are removed from the N-terminus of a PNMA2 or endo-Gag protein (e.g., a native or wild-type PNMA2 or endo-Gag protein) to generate an engineered PNMA2 or endo-Gag polypeptide. In some embodiments, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 amino acids are removed from the N-terminus of a PNMA2 or endo-Gag protein (e.g., a native or wild-type PNMA2 or endo-Gag protein) to generate an engineered PNMA2 or endo-Gag polypeptide. In some embodiments, about 1 to about 50, about 1 to about 40, about 1 to about 35, about 1 to about 30, about 1 to about 27, about 1 to about 25, about 1 to about 23, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, about 5 to 50, about 5 to 40, about 5 to 35, about 5 to 30, about 5 to 27, about 5 to 25, about 5 to 23, about 5 to 20, about 5 to 15, about 5 to 10, about 10 to 50, about 10 to 40, about 10 to 35, about 10 to 30, about 10 to 27, about 10 to 25, about 10 to 23, about 10 to 20, about 10 to 15, about 15 About 50, about 15-40, about 15-35, about 15-30, about 15-27, about 15-25, about 15-23, about 15-20, about 20-50, about 20-40, about 20-35, about 20-30, about 20-27, about 20-25, or about 20-23 amino acids are removed from the N-terminus of a PNMA2 or endo-Gag protein (e.g., a native or wild-type PNMA2 or endo-Gag protein) to generate an engineered PNMA2 or endo-Gag polypeptide.

[0067] In some embodiments, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acids are removed from the C-terminus of a PNMA2 or endo-Gag protein (e.g., a native or wild-type PNMA2 or endo-Gag protein) to generate an engineered PNMA2 or endo-Gag polypeptide. In some embodiments, up to 5, up to 10, up to 15, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, or up to 50 amino acids are removed from the C-terminus of a PNMA2 or endo-Gag protein (e.g., a native or wild-type PNMA2 or endo-Gag protein) to generate an engineered PNMA2 or endo-Gag polypeptide. In some embodiments, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 amino acids are removed from the C-terminus of a PNMA2 or endo-Gag protein (e.g., a native or wild-type PNMA2 or endo-Gag protein) to generate an engineered PNMA2 or endo-Gag polypeptide. In some embodiments, about 1 to about 50, about 1 to about 40, about 1 to about 35, about 1 to about 30, about 1 to about 27, about 1 to about 25, about 1 to about 23, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 1 to about 5, about 5 to 50, about 5 to 40, about 5 to 35, about 5 to 30, about 5 to 27, about 5 to 25, about 5 to 23, about 5 to 20, about 5 to 15, about 5 to 10, about 10 to 50, about 10 to 40, about 10 to 35, about 10 to 30, about 10 to 27, about 10 to 25, about 10 to 23, about 10 to 20, about 10 to 15, about 15 About 50, about 15-40, about 15-35, about 15-30, about 15-27, about 15-25, about 15-23, about 15-20, about 20-50, about 20-40, about 20-35, about 20-30, about 20-27, about 20-25, or about 20-23 amino acids are removed from the C-terminus of a PNMA2 or endo-Gag protein (e.g., a native or wild-type PNMA2 or endo-Gag protein) to generate an engineered PNMA2 or endo-Gag polypeptide.

[0068] In some embodiments, at least 15 amino acids are removed from native or wild-type PNMA2 to generate an engineered PNMA2 polypeptide. In some embodiments, up to 25 amino acids are removed from the C-terminus of native or wild-type PNMA2 to generate an engineered PNMA2 polypeptide. In some embodiments, about 25 amino acids are removed from native or wild-type PNMA2 to generate an engineered PNMA2 polypeptide. In some embodiments, about 10-30 amino acids are removed from native or wild-type PNMA2 to generate an engineered PNMA2 polypeptide. In some embodiments, about 20-27 amino acids are removed from native or wild-type PNMA2 to generate an engineered PNMA2 polypeptide. In some embodiments, about 24-26 amino acids are removed from native or wild-type PNMA2 to generate an engineered PNMA2 polypeptide.

[0069] In some cases, only the essential regions involved in capsid assembly / formation and cargo binding remain in the PNMA2 or endo-Gag polypeptide. In further cases, only the essential regions involved in capsid assembly / formation remain in the PNMA2 polypeptide. In some embodiments, the PNMA2 or endo-Gag polypeptide contains truncations or modifications of domains involved in encapsidation, nucleic acid binding, or delivery.

[0070] In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: UPD, CCD, KRs, PolyE, and VCS. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: NCD, CCD, KRs, PolyE, and VCS. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: NCD, UPD, KRs, PolyE, and VCS. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: NCD, UPD, CCD, PolyE, and VCS. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: NCD, UPD, CCD, KRs, and VCS. In some cases, the PNMA2 or endo-Gag polypeptide comprises the following domains from N-terminus to C-terminus: NCD, UPD, CCD, KRs, and PolyE. In some cases, each of the domains is fused directly or indirectly to each of the two adjacent domains. In some embodiments, the PNMA2 or endo-Gag polypeptide disclosed herein comprises a tag, e.g., an epitope tag, or an affinity tag, such as a polyhistidine tag. In some embodiments, the PNMA2 or endo-Gag polypeptide disclosed herein does not have a tag, e.g., does not include an epitope tag, does not include an affinity tag, or does not include a polyhistidine tag. In some embodiments, the tag is initially present but is removed, e.g., by enzymatic digestion. In some embodiments, the PNMA2 or endo-Gag polypeptide disclosed herein comprises an N-terminal methionine. In some embodiments, the PNMA2 or endo-Gag polypeptide disclosed herein does not comprise an N-terminal methionine. In some embodiments, the N-terminal methionine is removed cotranslationally and / or via enzymatic digestion.

[0071] In some cases, the domains are arranged in an order that does not interfere with capsid assembly and cargo binding, in some cases, each of the domains is fused directly or indirectly to each of the two adjacent domains.

[0072] In some cases, the PNMA2 or endo-Gag polypeptide is an engineered PNMA2 or endo-Gag polypeptide. As used herein, an engineered polypeptide is a recombinant polypeptide that is not identical in sequence to the full-length wild-type polypeptide. An engineered PNMA2 or endo-Gag polypeptide can be functionally different from a native PNMA2 or endo-Gag polypeptide, for example, includes additional functions than the native polypeptide or lacks a function that the native polypeptide has. In some cases, the PNMA2 or endo-Gag polypeptide is a native PNMA2 or endo-Gag polypeptide, for example, includes, consists essentially of, or consists of a wild-type amino acid sequence.

[0073] In some cases, the PNMA2 or endo-Gag polypeptide comprises a cargo-binding domain. In some embodiments, the PNMA2 or endo-Gag polypeptide is engineered to contain a cargo-binding domain, for example, via insertion of the cargo-binding domain or modification of a cargo-binding domain that is endogenous to the PNMA2 or endo-Gag polypeptide. In some embodiments, the cargo-binding domain is endogenous to the PNMA2 or endo-Gag polypeptide.

[0074] The cargo-binding domain can be covalently or non-covalently linked to the cargo, for example, via a linker disclosed herein. The linker can be a peptide linker, for example, a peptide linker that links the cargo-binding domain to a peptide or polypeptide cargo. The linker can be a non-peptide linker disclosed herein that links to the cargo via a covalent or non-covalent bond disclosed herein. The cargo-binding domain can be covalently or non-covalently linked to the PNMA2 or endo-Gag polypeptide, for example, via a linker disclosed herein.

[0075] In some embodiments, the cargo-binding domain binds a heterologous cargo, for example, a cargo that is not native to the capsid naturally formed from the PNMA2 or endo-Gag polypeptide.

[0076] In some cases, the cargo binding domain comprises a nucleic acid binding domain, an RNA binding domain, a DNA binding domain, a protein binding domain, a peptide binding domain, an antibody binding domain, a small molecule binding domain, or a peptidomimetic / nucleotide mimetic binding domain. Exemplary cargo binding domains include, but are not limited to, synthetic nucleic acid (e.g., RNA and / or DNA) binding domains, zinc finger domains, arginine-rich domains, domains derived from GPCRs, antibodies or binding fragments thereof, lipoproteins, integrins, tyrosine kinases, DNA binding proteins, RNA binding proteins, nucleases, ligases, proteases, integrases, isomerases, phosphatases, GTPases, aromatases, esterases, adaptor proteins, G proteins, GEFs, cytokines, interleukins, interleukin receptors, interferons, interferon receptors, caspases, transcription factors, neurotrophic factors and their receptors, growth factors and their receptors, signal recognition particle and receptor components, extracellular matrix proteins, integral components of membranes, ribosomal proteins, translation elongation factors, translation initiation factors, GPI-anchored proteins, tissue factor, dystrophin, utrophin, dystrobrevin, any fusion, combination, subunit, derivative, or domain thereof. In some embodiments, the cargo-binding domain comprises a domain derived from an endogenous Gag or PNMA polypeptide that is heterologous with respect to the remainder of the protein or the portion of the protein that induces encapsidation. For example, a PNMA2 polypeptide can be engineered to contain a cargo-binding domain from or derived from a heterologous PNMA protein, such as a heterologous endo-Gag polypeptide, such as PNMA3 or another endo-Gag protein.

[0077] In some embodiments, the cargo-binding domain or nucleic acid-binding domain is from or derived from a viral protein, such as the TAT protein of HIV. In some embodiments, the cargo-binding domain is inverted, e.g., compared to a wild-type version of the cargo-binding domain.

[0078] The cargo-binding domain can be a synthetic cargo-binding domain, for example, a synthetic nucleic acid-binding domain. In some embodiments, the synthetic cargo-binding domain is designed to include an extension of the C-terminal alpha helix of a PNMA2 polypeptide or an endo-Gag polypeptide. In some embodiments, the synthetic cargo-binding domain is designed to orient the cargo-binding domain inside the assembled PNMA2 or endo-Gag capsid.

[0079] The nucleic acid binding domain may be from or derived from an RNA binding protein. The nucleic acid binding domain may be from or derived from a DNA binding protein.

[0080] In some embodiments, the cargo-binding domain is lysine-rich, e.g., comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% lysine residues. In some embodiments, the cargo-binding domain is arginine-rich, e.g., comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% arginine residues. In some embodiments, the cargo-binding domain comprises one or more Lys / Arg(RK) repeats, e.g., at least 1, at least 2, at least 3, at least 4, at least 5, up to 2, up to 3, up to 4, up to 5, about 1, about 2, about 3, about 4, or about 5 Lys / Arg repeats.

[0081] Non-limiting examples of cargo binding domains that are nucleic acid binding domains are the amino acid sequences of SEQ ID NO:39, SEQ ID NO:42, and SEQ ID NO:45, as well as amino acid sequences that comprise at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any one of SEQ ID NOs:39, 72, and 45.

[0082] In some embodiments, the cargo binding domain binds to RNA, e.g., mRNA, hairpin RNA, guide RNA, shRNA, siRNA, mRNA, tRNA, rRNA, snRNA, microRNA, or non-coding RNA. In some embodiments, the cargo binding domain binds to DNA, e.g., ssDNA, dsDNA, or oligonucleotides. In some embodiments, the cargo binding domain binds to RNA and DNA. In some embodiments, the cargo binding domain binds preferentially to RNA over DNA. In some embodiments, the cargo binding domain binds preferentially to DNA over RNA. In some embodiments, the cargo binding domain binds preferentially to ssDNA. In some embodiments, the cargo binding domain specifically or preferentially binds to a particular nucleic acid structural motif, e.g., a hairpin, such as a bulge hairpin. In some embodiments, the cargo binding domain binds non-specifically to nucleic acid, RNA, and / or DNA, e.g., ssDNA.

[0083] In some embodiments, the PNMA2 is an engineered PNMA2 polypeptide having at least an RNA binding domain inserted and / or modified to bind a heterologous cargo that is not native to the capsid naturally formed from the PNMA2 polypeptide. In some cases, the PNMA2 polypeptide comprises a full-length PNMA2 polypeptide having at least an RNA binding domain inserted and / or modified to bind a heterologous cargo that is not native to the capsid naturally formed from the PNMA2 protein. In further cases, the engineered PNMA2 polypeptide comprises one or more domains of a PNMA2 polypeptide, at least one of which is involved in the formation of a capsid, and the RNA binding domain inserted and / or modified to bind a heterologous cargo that is not bound by the native PNMA2 protein.

[0084] In some embodiments, the endo-Gag (e.g., a PNMA family member) is an engineered endo-Gag polypeptide in which at least the RNA binding domain has been inserted and / or modified to bind a heterologous cargo that is not native to the capsid formed from the endo-Gag protein in nature. In some cases, the endo-Gag polypeptide comprises a full-length endo-Gag polypeptide, in which at least the RNA binding domain has been modified to bind a heterologous cargo that is not native to the capsid formed from the endo-Gag protein in nature. In other cases, the endo-Gag polypeptide comprises an engineered endo-Gag fragment that includes modification(s) in at least its RNA binding domain to bind a heterologous cargo that is not bound by the native endo-Gag protein. In further cases, the endo-Gag polypeptide comprises one or more domains of an engineered endo-Gag polypeptide, in which at least one of the domains is involved in the formation of a capsid, and in which the RNA binding domain has been modified to bind a heterologous cargo that is not native to the capsid formed from the endo-Gag protein in nature.

[0085] In some embodiments, the cargo-binding domain is at the C-terminus of an endo-Gag or PNMA2 polypeptide disclosed herein. In some embodiments, the cargo-binding domain is at the N-terminus of an endo-Gag or PNMA2 polypeptide disclosed herein.

[0086] In some embodiments, the cargo-binding domain may be inserted adjacent to a domain of the PNMA2 or endo-Gag polypeptide. For example, in some embodiments, the cargo-binding domain may be inserted adjacent (e.g., N- or C-terminally) to one or more of the NCD, CCD, UPD, KRs, PolyE, or VCS domains.

[0087] In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: a cargo-binding domain and PolyE. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: PolyE and a cargo-binding domain.

[0088] In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: a cargo-binding domain and KRs. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: a KRs and a cargo-binding domain.

[0089] In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: a cargo binding domain, a KRs, and PolyE. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: a KRs, a cargo binding domain, and PolyE. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: a KRs, PolyE, and a cargo binding domain. In some cases, each of the domains is fused directly or indirectly to each of the two adjacent domains.

[0090] In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: a cargo binding domain, an NCD, a UPD, a CCD, a KRs, a PolyE, and a VCS. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: an NCD, a cargo binding domain, an UPD, a CCD, a KRs, a PolyE, and a VCS. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: an NCD, a UPD, a cargo binding domain, a CCD, a KRs, a PolyE, and a VCS. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: an NCD, a UPD, a cargo binding domain, a CCD, a KRs, a PolyE, and a VCS. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: NCD, UPD, CCD, KRs, a cargo binding domain, PolyE, and a VCS. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: NCD, UPD, CCD, KRs, PolyE, a cargo binding domain, and a VCS. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: NCD, UPD, CCD, KRs, PolyE, a VCS, and a cargo binding domain. In some cases, each of the domains is fused directly or indirectly to each of the two adjacent domains.

[0091] In certain embodiments, domains of the PNMA2 or endo-Gag polypeptide are replaced, partially replaced, or serve as sites for insertion of a cargo-binding domain. For example, in some embodiments, one or more of the NCD, CCD, UPD, KRs, PolyE, or VCS domains can be replaced, partially replaced, or serve as sites for insertion of a cargo-binding domain.

[0092] In some embodiments, the engineered PNMA2 or endo-Gag polypeptide is modified to include a foreign polypeptide sequence, for example, in addition to the cargo-binding domain. The foreign polypeptide sequence can be fused to the PNMA2 or endo-Gag polypeptide. The foreign polypeptide sequence can be covalently or non-covalently attached to the PNMA2 or endo-Gag polypeptide, for example, via a linker as disclosed herein.

[0093] In some embodiments, the foreign polypeptide sequence may include an intracellular localization signal, such as a nuclear localization signal (NLS), or a sequence that targets the polypeptide to a membrane (e.g., an arginine-rich domain). In some embodiments, the foreign polypeptide sequence includes a domain that binds to a cell surface molecule, such as an antigen, a polypeptide, a receptor, a lipid (e.g., a phospholipid), a lipoprotein, a glycoprotein, and the like. In some embodiments, the foreign polypeptide recognizes and binds to a receptor displayed on the surface of a target cell. Upon reaching the cell of interest, the cargo is optionally further delivered to an intracellular target. For example, a therapeutic RNA may be translated into a protein when contacted with a ribosome in the cytoplasm of the cell.

[0094] In some embodiments, the cell surface molecule is a cell surface protein. In some cases, the cell surface molecule is an antigen expressed by a cancerous cell. In some cases, the cell surface molecule is a neoepitope. In some cases, the cell surface molecule comprises one or more mutations compared to a wild-type protein. Exemplary cancer antigens that may be bound by the exogenous polypeptide sequence include, but are not limited to, alpha-fetoprotein, ASLG659, B7-H3, BAFF-R, brevican, CA125 (MUC16), CA15-3, CA19-9, carcinoembryonic antigen (CEA), CA242, CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor), CTLA-4, CXCR5, E16 (LAT1, SLC7A5), FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase anchor protein 1a), SPAP1B, SPAP1C), epidermal growth factor, ETBR, Fc receptor-like protein 1 (FCRH1), G EDA, HLA-DOB (β subunit of MHC class II molecule (Ia antigen), human chorionic gonadotropin, ICOS, IL-2 receptor, IL20Rα, immunoglobulin superfamily receptor translocation associated 2 (IRTA2), L6, LewisY, LewisX, MAGE-1, MAGE-2, MAGE-3, MAGE4, ​​MART1, mesothelin, MDP, MPF (SMR, MSLN), MCP1 (CCL2), macrophage inhibitory factor (MIF), MPG, MSG783, mucin, MUC1-KLH, Napi3b (SLC34A2), nectin-4, Neu oncogene product, NCA, placental alkaline phosphatase, prostate-specific membrane antigen (PMSA), prostatic acid phosphatase, PSCA hlg, anti-transferrin receptor, p97, purinergic receptor P2X ligand-gated ion channel 5 (P2X5), LY64 (lymphocyte antigen 64 (RP105), gp100, P21, six-transmembrane epithelial antigen of the prostate (STEAP1)), STEAP2, Sema 5b, tumor-associated glycoprotein 72 (TAG-72), TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4), etc.

[0095] In some cases, the cell surface molecule bound by the exogenous polypeptide sequence is a cluster of differentiation (CD) cell surface marker, e.g., CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD5 8, CD59, CDw60, CD61, CD62E, CD62L (L-selectin), CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, These include CD66e, CD71, CD79 (e.g., CD79a, CD79b), CD90, CD95 (Fas), CD103, CD104, CD125 (IL5RA), CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), CD221, CD274, CD279 (PD-1), CD319 (SLAMF7), CD326 (EpCAM), and the like.

[0096] In some cases, the foreign polypeptide sequence is or is derived from a protein (e.g., a human protein), an antibody or binding fragment thereof, a viral protein, a Gag-like protein (e.g., a human Gag-like protein), or a de novo engineered protein designed to bind to a target receptor of interest. In some cases, the antibody or binding fragment thereof comprises a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a multispecific antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv, (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide-stabilized Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a camelid antibody or binding fragment thereof (e.g., a VHH domain), or a chemically modified derivative thereof. In some cases, the foreign polypeptide sequence directs delivery of the capsid formed by the engineered PNMA2 polypeptide to a desired target site.

[0097] The antibody or antigen-binding fragment thereof disclosed herein (e.g., as a foreign polypeptide sequence or cargo) can comprise a complementarity determining region (CDR). In some embodiments, the CDR determines or substantially determines the binding specificity and / or affinity of the antibody or antigen-binding fragment. For example, the CDR can be grafted into another suitable framework or the framework region can be modified (e.g., via amino acid substitution, deletion, and / or insertion) and the antigen-binding fragment or domain can retain binding to the target and the extracellular binding domain retains function despite the modification of the CDR exterior. The CDR can be identified by various methods, including, but not limited to, the Kabat method, the Chothia method, the IMGT method, the AHO method, and the Paratome method. An antigen-binding site of an antibody having a heavy chain and a light chain or variable regions thereof comprises six CDRs, three of which are in the hypervariable region of the light chain variable region and three of which are in the hypervariable region of the heavy chain variable region. The CDRs in the light chain are referred to as L1, L2, and L3, and the CDRs in the heavy chain are referred to as H1, H2, and H3. The CDRs can also be referred to as LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3, respectively. Certain antibodies or antigen-binding domains contain fewer than six CDRs. For example, certain antibodies lack a light chain and can be referred to as heavy chain-only antibodies (HCAbs). HCAbs have three CDRs in the variable region, referred to as VHH. Single domain antibodies or nanobodies can be generated from such VHH regions of heavy chain-only antibodies.

[0098] Non-limiting examples of exogenous polypeptide sequences include, but are not limited to, those encoding zinc finger domains, arginine-rich domains, domains from GPCRs, antibodies or binding fragments thereof, lipoproteins, integrins, tyrosine kinases, DNA binding proteins, RNA binding proteins, nucleases, ligases, proteases, integrases, isomerases, phosphatases, GTPases, aromatases, esterases, adaptor proteins, G proteins, GEFs, cytokines, interleukins, interleukin receptors, interferons, interferon receptors, caspases, transcription factors, neurotrophic factors and their receptors, growth factors and their receptors, signal recognition particle and receptor components, extracellular matrix proteins, integral components of membranes, ribosomal proteins, translation elongation factors, translation initiation factors, GPI-anchored proteins, tissue factors, dystrophin, utrophin, dystrobrevin, cell penetrating peptides, fusion proteins, viral envelope proteins, endogenous retroviral envelope proteins, any fusion, combination, subunit, derivative, or domain thereof.

[0099] The foreign polypeptide sequence may be or may include a cell-penetrating peptide (CPP). A cell-penetrating peptide (CPP) may facilitate the uptake of macromolecules through the cell membrane and enhance the delivery of CPP-modified molecules to the cell interior. The cell-penetrating peptide may include or may be a cationic CPP (e.g., TAT, R8, DPV3, DPV6, penetratin, R9-TAT), an amphipathic CPP (e.g., pVEC, ARF, MPG, MAP, transportan), or a hydrophobic CPP (e.g., Bip4, C105Y, Melttin, gH625). The CPP may be a protein-derived CPP, a synthetic CPP, or a chimeric CPP. The CPP may include amphipathic helical peptides such as transportan and MAP, in which lysine residues are the main contributors to the positive charge, as well as Arg-rich peptides such as TATp, antennapedia, or penetratin. Other CPPs may include: the minimal protein transduction domain of Antennapedia, a Drosophila homeoprotein called penetratin, which is a 16mer peptide (residues 43-58) present in the third helix of the homeodomain; a 27 amino acid long chimeric CPP, which contains a peptide sequence from the amino terminus of the neuropeptide galanin linked via a Lys residue, mastoparan, a wasp venom peptide; VP22, which is a major structural component of HSV-1 that facilitates intracellular trafficking, and transportan (18-mer), an amphipathic model peptide that translocates the plasma membrane of mast cells and endothelial cells by both energy-dependent and -independent mechanisms. In some embodiments, lipid moieties are modified on the CPP(s) for intracellular use.

[0100] In some embodiments, the foreign polypeptide sequence comprises a non-native cysteine ​​residue, e.g., a cysteine ​​residue not present in the native PNMA2 or endo-Gag polypeptide. The foreign cysteine ​​residue can be used to conjugate to a binding partner, e.g., via a suitable chemical reaction, such as maleimide chemistry. In some embodiments, the foreign polypeptide sequence comprises a non-native cysteine ​​residue and lacks one or more native cysteine ​​residues, e.g., one or more native cysteine ​​residues are deleted or replaced with a non-cysteine ​​residue. The foreign polypeptide sequence comprising a non-native cysteine ​​residue or a non-native cysteine ​​residue can be fused directly or via a linker to the C-terminus, N-terminus, or interior of the PNMA2 or endo Gag polypeptide, e.g., within or between domains of the PNMA2 or endo-Gag polypeptide disclosed herein.

[0101] In some cases, the foreign polypeptide sequence is directly, indirectly through a linker, or chemically fused to one or more of the cargo binding domain, NCD, UPD, CCD, KRs, PolyE, or VCS (if present). In some cases, the foreign polypeptide sequence is directly, indirectly through a linker, or chemically fused to the C-terminus of the PNMA2 polypeptide or endo-Gag polypeptide. In some cases, the foreign polypeptide sequence is directly, indirectly through a linker, or chemically fused to the N-terminus of the PNMA2 polypeptide or endo-Gag polypeptide.

[0102] In some embodiments, the exogenous polypeptide sequence may be inserted adjacent to a domain of the PNMA2 or endo-Gag polypeptide. For example, in some embodiments, the exogenous polypeptide sequence may be inserted adjacent (e.g., N- or C-terminally) to one or more of the NCD, CCD, UPD, KRs, PolyE, or VCS domains.

[0103] In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: a foreign polypeptide sequence, and PolyE. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: PolyE and a foreign polypeptide sequence.

[0104] In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: a foreign polypeptide sequence, and a KRs. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: a KRs and a foreign polypeptide sequence.

[0105] In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: a foreign polypeptide sequence, a KRs, and PolyE. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: a KRs, a foreign polypeptide sequence, and PolyE. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: a KRs, PolyE, and a foreign polypeptide sequence. In some cases, each of the domains is fused directly or indirectly to each of the two adjacent domains.

[0106] In some cases, the PNMA2 or endo-Gag polypeptide comprises the following domains from N-terminus to C-terminus: a foreign polypeptide sequence, an NCD, a UPD, a CCD, a KRs, a PolyE, and a VCS. In some cases, the PNMA2 or endo-Gag polypeptide comprises the following domains from N-terminus to C-terminus: an NCD, a foreign polypeptide sequence, an UPD, a CCD, a KRs, a PolyE, and a VCS. In some cases, the PNMA2 or endo-Gag polypeptide comprises the following domains from N-terminus to C-terminus: an NCD, a UPD, a foreign polypeptide sequence, a CCD, a KRs, a PolyE, and a VCS. In some cases, the PNMA2 or endo-Gag polypeptide comprises the following domains from N-terminus to C-terminus: an NCD, a UPD, a foreign polypeptide sequence, a CCD, a KRs, a PolyE, and a VCS. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: NCD, UPD, CCD, KRs, a foreign polypeptide sequence, PolyE, and a VCS. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: NCD, UPD, CCD, KRs, PolyE, a foreign polypeptide sequence, and a VCS. In some cases, the PNMA2 or endo-Gag polypeptide comprises, from N-terminus to C-terminus, the following domains: NCD, UPD, CCD, KRs, PolyE, a VCS, and a foreign polypeptide sequence. In some cases, each of the domains is fused directly or indirectly to each of the two adjacent domains.

[0107] In certain embodiments, domains of the PNMA2 or endo-Gag polypeptides are replaced, partially replaced, or serve as insertion sites for exogenous polypeptide sequences. For example, in some embodiments, one or more of the NCD, CCD, UPD, KRs, PolyE, or VCS domains can be replaced, partially replaced, or serve as insertion sites for exogenous polypeptide sequences.

[0108] In some cases, the PNMA2 is a human PNMA2 polypeptide. In some cases, the PNMA2 is a non-human PNMA2 polypeptide. In further cases, the PNMA2 polypeptide comprises one or more domains of a human PNMA2 polypeptide, at least one of the domains being involved in capsid formation. In further cases, the PNMA2 polypeptide comprises one or more domains of a non-human PNMA2 polypeptide, at least one of the domains being involved in capsid formation.

[0109] In some cases, the endo-Gag (e.g., a PNMA family member) is a human endo-Gag polypeptide. In some cases, the endo-Gag (e.g., a PNMA family member) is a non-human endo-Gag polypeptide. In further cases, the endo-Gag polypeptide comprises one or more domains of a human endo-Gag polypeptide, at least one of the domains being involved in capsid formation. In further cases, the endo-Gag polypeptide comprises one or more domains of a non-human endo-Gag polypeptide, at least one of the domains being involved in capsid formation.

[0110] In some cases, the engineered PNMA2 or endo-Gag polypeptide comprises a fragment of a PNMA2 or endo-Gag polypeptide derived from a first species and at least an additional fragment derived from a PNMA2 or endo-Gag polypeptide from a second species.

[0111] In some embodiments, exemplary mammalian PNMA2 or endo-Gag proteins for expression as recombinant or engineered PNMA2 or endo-Gag polypeptides are from the species Homo sapiens. Additional exemplary species of primate PNMA2 or endo-Gag proteins for expression as recombinant or engineered PNMA2 or endo-Gag polypeptides include gorilla, orangutan, pan paniscus, southern pigtailed macaque, chlorocebus sabaeus, papio anubis, rhinopithecus roxellana, macaca fascicularis, nomascus leucogenys, callithrix jacchus, aotus nancymaae, cebus capucinus imitator, saimiri boliviensis boliviensis, otolemur garnettii, macaca mulatta, and macaca fascicularis.

[0112] A list of exemplary species of rodent PNMA2 or endo-Gag proteins for expression as recombinant or engineered PNMA2 or endo-Gag polypeptides includes, but is not limited to, Fukomys damarensis, Microcebus murinus, Heterocephalus glaber, Propithecus coquereli, Marmota marmota marmota, Galeopterus variegatus, Cavia porcellus, Dipodomys ordii, Octodon degus, Castor canadensis nannospalax galili, Carlito syrichta, Chinchilla lanigera, Mus musculus, Ictidomys tridecemlineatus, Rattus norvegicus, Microtus ochrogaster, Otlemur garnettii, Meriones unguiculatus, Cricetulus griseus, Rattus norvegicus, Neotoma lepida, Jaculus jaculus, Mustela putorius furo, Mesocricetus auratus, tupaia chinensis, cricetulus griseus, chrysochloris asiatica, elephantulus edwardii, erinaceus europaeus, ochotona princeps, sorex araneus, monodelphis domestica, echinops telfairi, and condylura cristata.

[0113] In some embodiments, a nucleic acid or amino acid sequence of the disclosure (e.g., encoding a PNMA2 polypeptide or an endo-Gag polypeptide, or a fragment or domain thereof) has at least 70% homology, at least 71% homology, at least 72% homology, at least 73% homology, at least 74% homology, at least 75% homology, at least 76% homology, at least 77% homology, at least 78% homology, at least 79% homology, at least 80% homology, at least 81% homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, at least 99% homology, at least 100% homology, at least 101% homology, at least 102% homology, at least 103% homology, at least 104% homology, at least 105% homology, at least 106% homology, at least 107% homology, at least 108% homology, at least 109% homology, at least 110% homology, at least 111% homology, at least 112% homology, at least 113% homology, at least 114% homology, at least 115% homology, at least 116% homology, at least 117% homology, at least 118% homology, at least 119% The nucleic acid has 86% homology, at least 87% homology, at least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, at least 99% homology, at least 99.1% homology, at least 99.2% homology, at least 99.3% homology, at least 99.4% homology, at least 99.5% homology, at least 99.6% homology, at least 99.7% homology, at least 99.8% homology, at least 99.9% or at least 99.99% homology.

[0114] Various methods and software programs are used to determine the homology (e.g., identity or similarity) between two or more sequences. For example, the degree of sequence identity or similarity between two sequences can be determined by comparing the two sequences using a computer program commonly used for this purpose, such as a global or local alignment algorithm. Non-limiting examples include NCBI BLAST, BLASTp, BLASTn, BLASTx, tBLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, Needle(EMBOSS), Stretcher(EMBOSS), GGEARCH2SEQ, Water(EMBOSS), Matcher(EMBOSS), LALIGN, SSEARCH2SEQ, or another suitable method or algorithm, for example, using default parameters. The Needleman and Wunsch global alignment algorithm can be used to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps.

[0115] In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO:7.

[0116] In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to at least 100 contiguous amino acids of SEQ ID NO:7. In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to at least 200 contiguous amino acids of SEQ ID NO:7. In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to at least 250 contiguous amino acids of SEQ ID NO:7.In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to at least 300 contiguous amino acids of SEQ ID NO:7.

[0117] In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:7.

[0118] In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises one or more amino acid substitutions, deletions, or insertions from a polypeptide having the amino acid sequence of SEQ ID NO: 7. In some embodiments, an endo-Gag or PNMA2 polypeptide comprises from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions, or insertions compared to SEQ ID NO:7.

[0119] In some embodiments, the endo-Gag or PNMA2 polypeptide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least or at least 50 amino acid substitutions, deletions, or insertions compared to SEQ ID NO:7.

[0120] In some embodiments, the endo-Gag or PNMA2 polypeptide comprises up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, or up to 50 amino acid substitutions, deletions, or insertions compared to SEQ ID NO:7.

[0121] In some embodiments, the endo-Gag polypeptide or PNMA2 polypeptide is selected from the group consisting of 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-4, and including 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-15, 4-20, 4-30, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40, 10-15, 15-20, or 20-25 amino acid substitutions, deletions, or insertions.

[0122] In some embodiments, the endo-Gag or PNMA2 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, deletions, or insertions compared to SEQ ID NO: 7. The amino acid substitutions may be conservative or non-conservative. The one or more amino acid substitutions, deletions, or insertions may be at the N-terminus, C-terminus, or combinations thereof within the amino acid sequence. The amino acid substitutions, deletions, or insertions may be consecutive, non-consecutive, or combinations thereof.

[0123] In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 8. In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises, consists essentially of, or consists of an amino acid sequence that is SEQ ID NO:8.

[0124] In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to at least 100 contiguous amino acids of SEQ ID NO:8. In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to at least 200 contiguous amino acids of SEQ ID NO:8. In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to at least 250 contiguous amino acids of SEQ ID NO:8.In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to at least 300 contiguous amino acids of SEQ ID NO:8.

[0125] In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises one or more amino acid substitutions, deletions, or insertions from a polypeptide having the amino acid sequence of SEQ ID NO: 8. In some embodiments, an endo-Gag or PNMA2 polypeptide comprises from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions, or insertions compared to SEQ ID NO:8.

[0126] In some embodiments, the endo-Gag or PNMA2 polypeptide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least or at least 50 amino acid substitutions, deletions, or insertions compared to SEQ ID NO:8.

[0127] In some embodiments, the endo-Gag or PNMA2 polypeptide comprises up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, or up to 50 amino acid substitutions, deletions, or insertions compared to SEQ ID NO:8.

[0128] In some embodiments, the endo-Gag polypeptide or PNMA2 polypeptide is selected from the group consisting of 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-4, and including 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-15, 4-20, 4-30, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40, 10-15, 15-20, or 20-25 amino acid substitutions, deletions, or insertions.

[0129] In some embodiments, the endo-Gag or PNMA2 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, deletions, or insertions compared to SEQ ID NO:8. The amino acid substitutions may be conservative or non-conservative. The one or more amino acid substitutions, deletions, or insertions may be at the N-terminus, C-terminus, or combinations thereof within the amino acid sequence. The amino acid substitutions, deletions, or insertions may be consecutive, non-consecutive, or combinations thereof.

[0130] In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 1. In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises, consists essentially of, or consists of an amino acid sequence that is SEQ ID NO:1.

[0131] In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to at least 100 contiguous amino acids of SEQ ID NO:1. In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to at least 200 contiguous amino acids of SEQ ID NO:1. In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to at least 250 contiguous amino acids of SEQ ID NO:1.In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises, consists essentially of, or consists of an amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to at least 300 contiguous amino acids of SEQ ID NO:1.

[0132] In certain embodiments, an endo-Gag or PNMA2 polypeptide of the disclosure (e.g., a recombinant or engineered endo-Gag or PNMA2 polypeptide) comprises one or more amino acid substitutions, deletions, or insertions from a polypeptide having the amino acid sequence of SEQ ID NO: 1. In some embodiments, an endo-Gag or PNMA2 polypeptide comprises from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions, or insertions compared to SEQ ID NO:1.

[0133] In some embodiments, the endo-Gag or PNMA2 polypeptide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least or at least 50 amino acid substitutions, deletions, or insertions compared to SEQ ID NO:1.

[0134] In some embodiments, the endo-Gag or PNMA2 polypeptide comprises up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, or up to 50 amino acid substitutions, deletions, or insertions compared to SEQ ID NO:1.

[0135] In some embodiments, the endo-Gag polypeptide or PNMA2 polypeptide has a sequence similar to or similar to SEQ ID NO: 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20, 2-30, 2-40, 3-4, and including 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20, 3-30, 3-40, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-15, 4-20, 4-30, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40, 10-15, 15-20, or 20-25 amino acid substitutions, deletions, or insertions.

[0136] In some embodiments, the endo-Gag or PNMA2 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid substitutions, deletions, or insertions compared to SEQ ID NO:1. The amino acid substitutions may be conservative or non-conservative. The one or more amino acid substitutions, deletions, or insertions may be at the N-terminus, C-terminus, or combinations thereof within the amino acid sequence. The amino acid substitutions, deletions, or insertions may be consecutive, non-consecutive, or combinations thereof.

[0137] Linker In certain embodiments, a polypeptide or capsid of the disclosure includes a linker, eg, a peptide or non-peptide linker, that covalently or non-covalently links the two components.

[0138] A linker can connect a first component (e.g., a domain, a polypeptide, a cargo-binding domain, a cargo, a delivery component, or an exogenous polypeptide sequence) to a second component (e.g., a domain, a polypeptide, a cargo-binding domain, a cargo, a delivery component, or an exogenous polypeptide sequence).

[0139] The linker can link the cargo-binding domain to the cargo (e.g., covalently or non-covalently). The linker can link a first cargo-binding domain to a second cargo-binding domain. The linker can link a cargo-binding domain to a different domain. The linker can link a cargo-binding domain to a polypeptide (e.g., a PNMA2 polypeptide or an end-Gag polypeptide). The linker can link a cargo-binding domain to a foreign polypeptide sequence.

[0140] The linker can link the cargo to the cargo binding domain. The linker can link the first cargo to the second cargo. The linker can link the cargo to the domain. The linker can link the cargo to the polypeptide (e.g., the PNMA2 polypeptide or the end-Gag polypeptide). The linker can link the cargo to the exogenous polypeptide sequence.

[0141] The linker can link a foreign polypeptide sequence to a polypeptide (e.g., a PNMA2 polypeptide or an end-Gag polypeptide). The linker can link a first foreign polypeptide sequence to a second foreign polypeptide sequence. The linker can link a foreign polypeptide sequence to a domain. The linker can link a foreign polypeptide sequence to a cargo binding domain. The linker can link a foreign polypeptide sequence to a cargo.

[0142] The linker can link a first domain to a second domain (e.g., a cargo-binding domain). The linker can link a domain to a polypeptide (e.g., a PNMA2 polypeptide or an end-Gag polypeptide). The linker can link a domain to a cargo. The linker can link a domain to an exogenous polypeptide sequence.

[0143] A linker can link a first polypeptide (e.g., a PNMA2 polypeptide or an end-Gag polypeptide) to a second polypeptide. A linker can link a polypeptide (e.g., a PNMA2 polypeptide or an end-Gag polypeptide) to a domain. A linker can link a polypeptide (e.g., a PNMA2 polypeptide or an end-Gag polypeptide) to a cargo binding domain. A linker can link a polypeptide (e.g., a PNMA2 polypeptide or an end-Gag polypeptide) to a cargo. A linker can link a polypeptide (e.g., a PNMA2 polypeptide or an end-Gag polypeptide) to an exogenous polypeptide sequence.

[0144] A linker can link any two components of a polypeptide, capsid, complex, or composition disclosed herein. For example, a linker can link a PNMA2 polypeptide to a cargo, an endo-Gag polypeptide to a cargo, a PNMA2 polypeptide to a cargo binding domain, an endo-Gag polypeptide to a cargo binding domain, a PNMA2 polypeptide to a foreign polypeptide sequence, an endo-Gag polypeptide to a foreign polypeptide sequence, a PNMA2 polypeptide to a delivery component, an endo-Gag polypeptide to a delivery component, or any other two domains disclosed herein. A linker can link two domains in a PNMA polypeptide, two domains in a cargo to a domain in a cargo binding domain, two domains in a cargo binding domain, two domains in a foreign polypeptide sequence, two domains in a delivery component, etc. A polypeptide, capsid, complex, or composition can include multiple linkers. For example, a cargo, cargo binding domain, or foreign polypeptide sequence comprising an antigen-binding fragment can include two variable regions linked by a linker, such as an scFv. A polypeptide or domain comprising one or more linkers can be linked to another polypeptide or domain (e.g., an exogenous polypeptide sequence that is a cargo, cargo-binding domain, or scFv) via another linker that is the same or different, can comprise a linker between the VH and VL domains, and can be linked to a PNMA2 or endo-Gag polypeptide by a second linker.

[0145] In some embodiments, the linker is a peptide linker. In some cases, the linker is a rigid linker. In other cases, the linker is a flexible linker. In some cases, the linker is a non-cleavable linker. In other cases, the linker is a cleavable linker. In further cases, the linker comprises a linear structure or a non-linear structure (e.g., a cyclic structure).

[0146] Flexible peptide linkers may have sequences that include stretches of glycine and serine residues. The small size of glycine and serine residues provides flexibility and allows mobility of the tethered functional domains. The incorporation of serine or threonine may maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thereby reducing unfavorable interactions between the linker and the protein moiety. Flexible linkers may also include additional amino acids such as threonine and alanine to maintain flexibility, and polar amino acids such as lysine and glutamine to improve solubility. Rigid peptide linkers may have, for example, an α-helical structure. Rigid α-helical linkers may act as spacers between certain protein domains. The peptide linker can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues in length. In some cases, the linker sequence can be, for example, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, or at least about 50 amino acids in length. In some cases, the linker sequence can be, for example, up to about 2, up to about 3, up to about 4, up to about 5, up to about 6, up to about 7, up to about 8, up to about 9, up to about 10, up to about 15, up to about 20, up to about 30, up to about 40, up to about 50, up to about 60, up to about 70, up to about 80, or up to about 100 amino acids long. In some cases, the linker is 5-20 amino acids long. In some cases, the linker is 10-20 amino acids long. In some cases, the linker is 4-8 amino acids long.

[0147] The linker may be a non-cleavable linker. The non-cleavable linker of the present disclosure may include a stable chemical linker. Examples of non-cleavable linkers that can be used in the proteins of the present disclosure to link domains and / or polypeptides may include thioether linkers, alkyl linkers, polymer linkers. The linker may be an SMCC linker or a PEG linker. In some embodiments, the linker may be a PEG linker. The non-cleavable linker may also include a non-proteolytically cleavable peptide linker. A non-proteolytically cleavable peptide may be inactive against proteases present in a particular sample, tissue, or organism. For example, the peptide may be inactive or substantially inactive against all or most human protease cleavage sequences, thereby including a high degree of stability in humans and human samples. Such peptides may also include secondary structures that render the protease cleavage site inactive or inaccessible to proteases. A non-cleavable linker of the disclosure may comprise a cleavage half-life in the presence of a human protease in a pH 7 buffer at 25° C. of at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 1 day, at least 2 days, at least 3 days, at least 1 week, at least 2 weeks, or at least 1 month.

[0148] In certain embodiments, the non-cleavable linker comprises a short peptide of various lengths. Exemplary non-cleavable linkers include (EAAAK)n (SEQ ID NO:9), or (EAAAR)n (SEQ ID NO:10), where n is 1-5, and up to 30 residues of glutamic acid-proline or lysine-proline repeats. In some embodiments, the non-cleavable linker comprises (GS)n, (SG)n, (GGGGS)n (SEQ ID NO:11), or (GGGS)n (SEQ ID NO:12), where n is 1-10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; KESGSVSSEQLAQFRSLD (SEQ ID NO:13); or EGKSSGSGSESKST (SEQ ID NO:14). In some embodiments, the non-cleavable linker comprises a polyGly / Ala polymer.

[0149] In certain embodiments, the linker is a cleavable linker, for example, an extracellularly cleavable linker or an intracellularly cleavable linker.In some cases, the linker is designed to cleave under the presence of a specific condition or in a specific environment (for example, under physiological conditions).For example, the design of the linker for cleavage under a specific condition, such as a specific enzyme, allows targeting of cellular uptake to a specific location.

[0150] In some embodiments, the linker is a pH-sensitive linker. In one example, the linker is cleaved under basic pH conditions. In other cases, the linker is cleaved under acidic pH conditions.

[0151] In some embodiments, the linker is cleaved in vivo by an endogenous enzyme (e.g., a protease), such as a serine protease, including, but not limited to, thrombin, metalloprotease, furin, cathepsin B, necrotic enzyme (e.g., calpain), etc. For example, a cleavable linker can be used to covalently link a cargo to a cargo-binding domain, and the linker can be cleaved in vivo to release the cargo. Exemplary cleavable linkers include, but are not limited to, GGAANLVRGG (SEQ ID NO: 15); SGRIGFLRTA (SEQ ID NO: 16); SGRSA (SEQ ID NO: 17); GFLG (SEQ ID NO: 18); ALAL (SEQ ID NO: 19); FK; PIC(Et)FF (SEQ ID NO: 20), where C(Et) represents S-ethylcysteine; PR(S / T)(L / I)(S / T) (SEQ ID NO: 21); DEVD (SEQ ID NO: 22); GWEHDG (SEQ ID NO: 23); RPLALWRS (SEQ ID NO: 24); or a combination thereof.

[0152] In certain embodiments, the cleavable linker comprises a 2A linker that can be co-translationally or post-translationally processed into separate polypeptides. The inclusion of a 2A linker can increase the likelihood that the appropriate ratio of components is produced (e.g., 1:1, 1:2, 1:3, 1:4, or 1:5 ratio of two components). In some cases, the inclusion of a 2A linker can increase the likelihood that two components (e.g., capsid subunit and cargo) are produced at equal or nearly equal levels.

[0153] The linker may be a non-peptide linker. The linker may be a chemical linker. The linker may be a chemical bond, e.g., a covalent bond or a non-covalent bond. The linker of the present disclosure may include a chemical linker. For example, two first components (e.g., a domain, a polypeptide, a cargo-binding domain, a cargo, or a foreign polypeptide sequence) can be linked to a second component (e.g., a domain, a polypeptide, a cargo-binding domain, a cargo, or a foreign polypeptide sequence) by a chemical linker. Each chemical linker of the present disclosure may be an alkylene, an alkenylene, an alkynylene, a heteroalkylene, a cycloalkylene, a heterocycloalkylene, an arylene, or a heteroarylene, any of which may be optionally substituted. In some embodiments, the chemical linker of the present disclosure may be an ester, an ether, an amide, a thioether, or a polyethylene glycol (PEG). In some embodiments, the linker can reverse the order of the amino acid sequences in the compound where the two amino acid sequences are linked, for example, so that the amino acid sequences linked by the linkage are head to head, rather than head to tail. Non-limiting examples of such linkers include diesters of dicarboxylic acids, such as oxalyl diester, malonyl diester, succinyl diester, glutaryl diester, adipyl diester, p-methyl diester, fumaryl diester, maleyl diester, phthalyl diester, isophthalyl diester, and terephthalyl diester. Non-limiting examples of such linkers include diamides of dicarboxylic acids, such as oxalyl diamide, malonyl diamide, succinyl diamide, glutaryl diamide, adipyl diamide, p-methyl diamide, fumaryl diamide, maleyl diamide, phthalyl diamide, isophthalyl diamide, and terephthalyl diamide.Non-limiting examples of such linkers include, but are not limited to, diamides of diamino linkers, such as ethylenediamine, 1,2-di(methylamino)ethane, 1,3-diaminopropane, 1,3-di(methylamino)propane, 1,4-di(methylamino)butane, 1,5-di(methylamino)pentane, 1,6-di(methylamino)hexane, and piperidine.

[0154] Non-limiting examples of optional substituents include hydroxyl groups, sulfhydryl groups, halogens, amino groups, nitro groups, nitroso groups, cyano groups, azide groups, sulfoxide groups, sulfone groups, sulfonamide groups, carboxyl groups, carboxaldehyde groups, imine groups, alkyl groups, haloalkyl groups, alkenyl groups, haloalkenyl groups, alkynyl groups, haloalkynyl groups, alkoxy groups, aryl groups, aryloxy groups, aralkyl groups, arylalkoxy groups, heterocyclyl groups, acyl groups, acyloxy groups, carbamate groups, amide groups, ureido groups, epoxy groups, and ester groups.

[0155] In some embodiments, the non-peptide linker or chemical linker is a cleavable linker, such as a self-immolative linker.

[0156] In some embodiments, the cleavable linker comprises a chemical trigger that controls cleavage (and, e.g., release of the cargo). Chemical triggers that may be used in the linkers of the present disclosure may be a dipeptide trigger, a cathepsin cleavable trigger, an acid cleavable trigger, a GSH cleavable trigger, an Fe(II) cleavable trigger, an enzyme cleavable trigger, a photoresponsive-cleavable trigger, a bioorthogonal cleavable trigger, a glycosidase cleavable trigger, a phosphatase cleavable trigger, a sulfatase cleavable trigger, a hydrazone trigger, a carbonate trigger, a silyl ether trigger, a disulfide trigger, a 1,2,4-trioxolane trigger, a dipeptide trigger, a triglycyl (CX) trigger, a cBu-Cit trigger, a β-glucuronide trigger, a β-galactoside trigger, a pyrophosphate trigger, an aryl sulfate trigger, a heptamethine cyanine fluorophore trigger, an O-nitrobenzyl trigger, a PC4AP trigger, or a dsProc trigger.

[0157] In some embodiments, the linker comprises a maleimide bond, a bis(vinylsulfonyl)piperazine bond, an N-methyl-N-phenylvinylsulfonamide bond, a Pt(II)-based bond, a carbamate bond, a carbonate bond, a quaternary ammonium bond.

[0158] In some embodiments, the linker comprises a bond generated by carbonyl condensation, Staudinger reaction, modified Staudinger ligation, traceless Staudinger ligation, inverse electron demand Diels-Alter cycloaddition, copper catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), Heusgen cycloaddition, click chemistry, and the like.

[0159] In some embodiments, the linker comprises a bond generated between a cysteine ​​and a cysteine ​​reactive group, such as maleimide or iodoacetamide. For example, cysteine-based conjugation may be used to link two components, such as a cargo-binding domain and a cargo. One or more reactive cysteine ​​residue(s) may be introduced at a selected location, for example, via insertion or substitution at the C-terminus of the PNMA2 or endo-Gag polypeptide, the N-terminus of the PNMA2 or endo-Gag polypeptide, or within the PNMA2 or endo-Gag polypeptide (e.g., within or between domains disclosed herein). The introduced cysteine ​​residue(s) may be linked to another portion of the polypeptide via a linker or spacer as disclosed herein. The cysteine ​​residues may be introduced so as not to interfere with the structure or function of the PNMA or endo-Gag polypeptide, for example, its ability to assemble into a capsid state or form, its ability to disassemble into a non-capsid state or form, and / or its ability to reassemble into a capsid state or form. To reduce or eliminate reactivity or conjugation at undesirable sites, cysteine ​​residues can optionally be deleted or substituted elsewhere with non-reactive or less reactive residues.

[0160] In some embodiments, the linker comprises a covalent attachment of two components. In some embodiments, the linker comprises a non-covalent attachment of two components. Two components present in a polypeptide, complex, or capsid can be non-covalently linked, for example, by ionic bonds, hydrogen bonds, interactions mediated by oligomerization or dimerization domains, etc.

[0161] Capsid In some embodiments, capsids are disclosed herein. In some cases, the capsid comprises an endo-Gag polypeptide, e.g., a PNMA family polypeptide, such as a PNMA2 polypeptide. Further exemplary endo-Gag polypeptides include PNMA1, PNMA2, PNMA3, PNMA4 (MOAP1), PNMA5, PNMA6A, PNMA6B / 6D, PNMA6E, PNMA6F, PNMA7A, PNMA7B, PNMA8A, PNMA8B, PNMA8C, CCDC8, Arc, BOP, LDOC1, PEG10, RTL3, RTL6, RTL8A, RTL8B, and ZNF18. In some cases, the polypeptide is a functional fragment that can form, for example, a subunit of a capsid. The PNMA or end-Gag polypeptides of the present disclosure can self-assemble to form a capsid, also called a virus-like particle (VLP).

[0162] In some embodiments, the capsid comprises a PNMA2-based capsid. In some cases, the PNMA2-based capsid comprises a plurality of recombinant PNMA2 polypeptides of the present disclosure. In some cases, the PNMA2-based capsid comprises a plurality of engineered PNMA2 polypeptides of the present disclosure. In some embodiments, the PNMA2 polypeptides are recombinant and engineered.

[0163] In some embodiments, the capsid comprises an endo-Gag-based capsid. In some cases, the endo-Gag capsid comprises a plurality of recombinant endo-Gag polypeptides of the disclosure. In some cases, the endo-Gag capsid comprises a plurality of engineered endo-Gag polypeptides of the disclosure. In some embodiments, the endo-Gag polypeptides are recombinant and engineered.

[0164] In certain embodiments, assembly of PNMA2 and / or endo-Gag-based capsids occurs ex vivo or in vitro, in some cases, PNMA2 and / or endo-Gag-based capsids are assembled in vivo.

[0165] In some embodiments, the capsid comprises a disulfide bond. The PNAM2 polypeptide or endo-Gag polypeptide that assembles to form the capsid can comprise one or more cysteine ​​residues that form one or more disulfide bonds. Disulfide bonds can contribute, for example, to the assembly, reassembly, and / or stability of the capsids disclosed herein. In some embodiments, disulfide bonds can be reduced at a target site, such as an intracellular (e.g., cytoplasmic) environment, to facilitate capsid disassembly and cargo delivery.

[0166] In some embodiments, a first PNAM2 or endo-Gag polypeptide subunit of a capsid forms an intermolecular disulfide bond with a second PNMA2 or endo-Gag polypeptide. In some embodiments, a PNAM2 or endo-Gag polypeptide subunit of a capsid forms an intramolecular disulfide bond. In some embodiments, a PNAM2 or endo-Gag polypeptide subunit of a capsid forms an intramolecular disulfide bond and an intermolecular disulfide bond. In some embodiments, the disulfide bond contributes to the formation of dimers, trimers, tetramers, and / or other higher order multimers of PNMA2 or endo-Gag polypeptides.

[0167] In some embodiments, the PNMA2 or endo-Gag polypeptide comprises at least one, at least two, at least three, or at least four cysteines that form disulfide bonds, e.g., when assembled to form a capsid. In some embodiments, the PNMA2 or endo-Gag polypeptide comprises one cysteine ​​that forms a disulfide bond. In some embodiments, the PNMA2 or endo-Gag polypeptide comprises two cysteines that form disulfide bonds. In some embodiments, the PNMA2 or endo-Gag polypeptide comprises three cysteines that form disulfide bonds. In some embodiments, the PNMA2 or endo-Gag polypeptide comprises four cysteines that form disulfide bonds.

[0168] In some embodiments, a PNMA2 or endo-Gag polypeptide comprises a cysteine ​​residue, e.g., a disulfide-forming cysteine ​​residue, at a position corresponding to any one or more of positions 10, 136, 233, and 310 of SEQ ID NO:1. In some embodiments, a PNMA2 or endo-Gag polypeptide comprises a cysteine ​​residue (e.g., a disulfide-forming cysteine ​​residue) at a position corresponding to residue 10 of SEQ ID NO:1. In some embodiments, a PNMA2 or endo-Gag polypeptide comprises a cysteine ​​residue (e.g., a disulfide-forming cysteine ​​residue) at a position corresponding to residue 136 of SEQ ID NO:1. In some embodiments, a PNMA2 or endo-Gag polypeptide comprises a cysteine ​​residue (e.g., a disulfide-forming cysteine ​​residue) at a position corresponding to residue 233 of SEQ ID NO:1. In some embodiments, a PNMA2 or endo-Gag polypeptide comprises a cysteine ​​residue (e.g., a disulfide-forming cysteine ​​residue) at a position corresponding to residue 310 of SEQ ID NO:1.

[0169] In some embodiments, the capsids have an average diameter of at least 1 nm or more. In some cases, the capsids have an average diameter of at least 2 nm, at least 3 nm, at least 4 nm, at least 5 nm, at least 10 nm, at least 11 nm, at least 12 nm, at least 13 nm, at least 14 nm, at least 15 nm, at least 16 nm, at least 17 nm, at least 18 nm, at least 19 nm, at least 20 nm, at least 25 nm, at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 300 nm, at least 400 nm, at least 500 nm, at least 600 nm, or more. In some cases, the capsids have an average diameter of at least 5 nm or more. In some cases, the capsids have an average diameter of at least 7 nm or more. In some cases, the capsids have an average diameter of at least 10 nm or more. In some cases, the capsids have an average diameter of at least 12 nm or more. In some cases, the capsids have an average diameter of at least 15 nm or more. In some cases, the capsids have an average diameter of at least 17 nm or more. In some cases, the capsids have an average diameter of at least 20 nm or more. In some cases, the capsids have an average diameter of at least 30 nm or more. In some cases, the capsids have an average diameter of at least 40 nm or more. In some cases, the capsids have an average diameter of at least 50 nm or more. In some cases, the capsids have an average diameter of at least 80 nm or more. In some cases, the capsids have an average diameter of at least 100 nm or more. In some cases, the capsids have an average diameter of at least 200 nm or more. In some cases, the capsids have an average diameter of at least 300 nm or more.In some cases, the capsids have an average diameter of at least 400 nm or more. In some cases, the capsids have an average diameter of at least 500 nm or more. In some cases, the capsids have an average diameter of at least 600 nm or more.

[0170] In some embodiments, the capsid has an average diameter of at most 100 nm or less. In some cases, the capsid has an average diameter of at most 1 nm, at most 5 nm, at most 10 nm, at most 20 nm, at most 30 nm, at most 40 nm, at most 50 nm, at most 60 nm, at most 70 nm, at most 80 nm, at most 90 nm, at most 95 nm, at most 100 nm, at most 105 nm, at most 110 nm, at most 115 nm, at most 120 nm, at most 125 nm, at most 130 nm, at most 140 nm, at most 150 nm, at most 200 nm, at most 300 nm, at most 400 nm, at most 500 nm, at most 600 nm or less. In some cases, the capsid has an average diameter of at most 50 nm or less. In some cases, the capsid has an average diameter of at most 80 nm or less.

[0171] In some cases, the capsids have an average diameter of up to 90 nm or less. In some cases, the capsids have an average diameter of up to 95 nm or less. In some cases, the capsids have an average diameter of up to 100 nm or less. In some cases, the capsids have an average diameter of up to 110 nm or less. In some cases, the capsids have an average diameter of up to 120 nm or less. In some cases, the capsids have an average diameter of up to 150 nm or less. In some cases, the capsids have an average diameter of up to 200 nm or less. In some cases, the capsids have an average diameter of up to 300 nm or less. In some cases, the capsids have an average diameter of up to 400 nm or less. In some cases, the capsids have an average diameter of up to 500 nm or less. In some cases, the capsids have an average diameter of up to 600 nm or less.

[0172] In some embodiments, the capsids have an average diameter of about 1 nm, about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 120 nm, about 150 nm, about 200 nm, about 300 nm, about 400 nm, about 500 nm, or about 600 nm. In some cases, the capsids have an average diameter of about 20 nm. In some cases, the capsids have an average diameter of about 30 nm. In some cases, the capsids have an average diameter of about 40 nm. In some cases, the capsids have an average diameter of about 50 nm. In some cases, the capsids have an average diameter of about 60 nm. In some cases, the capsids have an average diameter of about 70 nm. In some cases, the capsids have an average diameter of about 80 nm. In some cases, the capsids have an average diameter of about 100 nm. In some cases, the capsids have an average diameter of about 200 nm.

[0173] In some embodiments, the capsids have an average diameter of about 1 nm to about 600 nm. In some cases, the capsid may be between about 5 nm and about 500 nm, between about 5 nm and about 400 nm, between about 5 nm and about 300 nm, between about 5 nm and about 200 nm, between about 5 nm and about 100 nm, between about 5 nm and about 50 nm, between about 5 nm and about 30 nm, between about 10 nm and about 500 nm, between about 10 nm and about 400 nm, between about 10 nm and about 300 nm, between about 10 nm and about 200 nm, between about 10 nm and about 150 nm, between about 10 nm and about 120 nm, between about 10 nm and about 100 nm, between about 10 nm and about 90 nm, between about 10 nm and about 50 nm, between about 10 nm and about 30 nm, between about 15 nm and about 500 nm, between about 15 nm and about 400 nm, between about 15 nm and about 300 nm, between about 15 nm and about 200 nm, between about 15 nm and about 150 nm, between about 1 The average diameter is from 5 nm to about 120 nm, from about 15 nm to about 100 nm, from about 15 nm to about 90 nm, from about 15 nm to about 50 nm, from about 20 nm to about 500 nm, from about 20 nm to about 400 nm, from about 20 nm to about 300 nm, from about 20 nm to about 200 nm, from about 20 nm to about 150 nm, from about 20 nm to about 120 nm, from about 20 nm to about 100 nm, from about 20 nm to about 90 nm, from about 20 nm to about 50 nm, from about 30 nm to about 500 nm, from about 30 nm to about 400 nm, from about 30 nm to about 300 nm, from about 30 nm to about 200 nm, from about 30 nm to about 100 nm, from about 30 nm to about 50 nm, from about 50 nm to about 300 nm, from about 50 nm to about 200 nm, or from about 50 nm to about 100 nm.

[0174] The capsids in the compositions of the present disclosure can be, for example, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% pure. Purity can be determined, for example, by SDS-PAGE.

[0175] The capsids in the compositions of the present disclosure may exhibit, for example, a particle uniformity of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.Particle uniformity can be measured, for example, by multi-angle dynamic light scattering (MADLS) using the particle diameter size ranges disclosed herein.

[0176] In some cases, the PNMA2 and / or endo-Gag based capsids are stable at room temperature. In some cases, the PNMA2 and / or endo-Gag based capsids are empty. In other cases, the PNMA2 and / or endo-Gag based capsids are loaded (e.g., loaded with a heterologous cargo as disclosed herein). In some embodiments, the heterologous cargo is present within at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the plurality of capsids. In some embodiments, the heterologous cargo is present within at least 50% of the plurality of capsids. In some embodiments, the heterologous cargo is external to at least 1%, at least 3%, at least 5%, at least 7%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the plurality of capsids.

[0177] In some embodiments, at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the heterologous cargo is within the multiple capsids. In some embodiments, up to 1%, up to 3%, up to 5%, up to 7%, up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, up to 91%, up to 92%, up to 93%, up to 94%, up to 95%, up to 96%, up to 97%, up to 98%, up to 99%, or up to 99.5% of the heterologous cargo is external to the plurality of capsids. In some embodiments, at least 1%, at least 3%, at least 5%, at least 7%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of the heterologous cargo is external to the multiple capsids.

[0178] In some cases, the PNMA2 and / or endo-Gag based capsid is stable at a temperature of about 2° C. to about 37° C. In some cases, the PNMA2 and / or endo-Gag based capsid is stable at a temperature of about 2° C. to about 40° C., 2° C. to about 37° C., 2° C. to about 25° C., 2° C. to about 20° C., 4° C. to about 40° C., 4° C. to about 37° C., 4° C. to about 25° C., 4° C. to about 20° C., 2° C. to about 8° C., about 2° C. to about 4° C., about 20° C. to about 37° C., about 25° C. to about 37° C., about 20° C. to about 30° C., about 25° C. to about 30° C., or about 30° C. to about 37° C. In some cases, the PNMA2 and / or endo-Gag based capsid is empty. In other cases, PNMA2 and / or endo-Gag based capsids are loaded (e.g., loaded with heterologous cargo and / or therapeutic agents, such as DNA or RNA).

[0179] In some cases, the PNMA2 and / or endo-Gag based capsids are stable for at least about 1 day, at least about 2 days, at least about 4 days, at least about 5 days, at least about 7 days, at least about 14 days, at least about 28 days, at least about 30 days, at least about 60 days, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 3 years, at least about 5 years, or more. In some cases, the PNMA2 and / or endo-Gag based capsids exhibit low or minimal degradation, e.g., less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5%, based on the total number of degraded PNMA2 and / or endo-Gag based capsids. In some cases, the PNMA2 and / or endo-Gag based capsids are empty. In other cases, the PNMA2 and / or endo-Gag based capsids are loaded (eg, loaded with a therapeutic agent, such as DNA or RNA).

[0180] In some embodiments, the capsid comprises a first endogenous retroviral capsid polypeptide and a second endogenous retroviral capsid polypeptide, wherein the amino acid sequence of the first endogenous retroviral capsid polypeptide is not identical to the amino acid sequence of the second endogenous retroviral capsid polypeptide.

[0181] In some embodiments, the first endogenous retroviral capsid polypeptide is an endo-Gag polypeptide or comprises the amino acid sequence of an endo-Gag polypeptide. In some embodiments, the first endogenous retroviral capsid polypeptide is a native endo-Gag polypeptide or comprises the amino acid sequence of a native endo-Gag polypeptide. In some embodiments, the first endogenous retroviral capsid polypeptide is an engineered endo-Gag polypeptide or comprises the amino acid sequence of an engineered endo-Gag polypeptide. In some embodiments, the first endogenous retroviral capsid polypeptide is an engineered endo-Gag polypeptide that comprises an amino acid deletion compared to the corresponding native endo-Gag polypeptide. In some embodiments, the first endogenous retroviral capsid polypeptide is an engineered endo-Gag polypeptide that comprises an amino acid substitution compared to the corresponding native endo-Gag polypeptide. In some embodiments, the first endogenous retroviral capsid polypeptide is an engineered endo-Gag polypeptide that comprises an amino acid insertion compared to the corresponding native endo-Gag polypeptide. In some embodiments, the first endogenous retroviral capsid polypeptide is an engineered endo Gag polypeptide that includes a non-native cysteine ​​that is not present in the corresponding native endo Gag polypeptide.

[0182] In some embodiments, the first endogenous retroviral capsid polypeptide is a PNMA2 polypeptide or comprises the amino acid sequence of a PNMA2 polypeptide. In some embodiments, the first endogenous retroviral capsid polypeptide is a native PNMA2 polypeptide or comprises the amino acid sequence of a native PNMA2 polypeptide. In some embodiments, the first endogenous retroviral capsid polypeptide is an engineered PNMA2 polypeptide or comprises the amino acid sequence of an engineered PNMA2 polypeptide. In some embodiments, the first endogenous retroviral capsid polypeptide is an engineered PNMA2 polypeptide that comprises an amino acid deletion compared to the corresponding native PNMA2 polypeptide. In some embodiments, the first endogenous retroviral capsid polypeptide is an engineered PNMA2 polypeptide that comprises an amino acid substitution compared to the corresponding native PNMA2 polypeptide. In some embodiments, the first endogenous retroviral capsid polypeptide is an engineered PNMA2 polypeptide that comprises an amino acid insertion compared to the corresponding native PNMA2 polypeptide. In some embodiments, the first endogenous retroviral capsid polypeptide is an engineered PNMA2 polypeptide that includes a non-native cysteine ​​that is not present in the corresponding native PNMA2 polypeptide.

[0183] In some embodiments, the first endogenous retroviral capsid polypeptide is not a PNMA2 polypeptide or does not include the amino acid sequence of a PNMA2 polypeptide. In some embodiments, the first endogenous retroviral capsid polypeptide is not a native PNMA2 polypeptide or does not include the amino acid sequence of a native PNMA2 polypeptide. In some embodiments, the first endogenous retroviral capsid polypeptide is not an engineered PNMA2 polypeptide or does not include the amino acid sequence of an engineered PNMA2 polypeptide.

[0184] In some embodiments, the first endogenous retrovirus capsid polypeptide comprises a foreign polypeptide sequence disclosed herein. In some embodiments, the first endogenous retrovirus capsid polypeptide comprises a cargo binding domain (e.g., an RNA, DNA, or protein binding domain) disclosed herein. In some embodiments, the first endogenous retrovirus capsid polypeptide comprises a domain that binds to a cell surface molecule. In some embodiments, the first endogenous retrovirus capsid polypeptide comprises an antibody or an antigen-binding fragment thereof.

[0185] In some embodiments, the second endogenous retroviral capsid polypeptide is an endo-Gag polypeptide or comprises the amino acid sequence of an endo-Gag polypeptide. In some embodiments, the second endogenous retroviral capsid polypeptide is a native endo-Gag polypeptide or comprises the amino acid sequence of a native endo-Gag polypeptide. In some embodiments, the second endogenous retroviral capsid polypeptide is an engineered endo-Gag polypeptide or comprises the amino acid sequence of an engineered endo-Gag polypeptide. In some embodiments, the second endogenous retroviral capsid polypeptide is an engineered endo-Gag polypeptide that comprises an amino acid deletion compared to the corresponding native endo-Gag polypeptide. In some embodiments, the second endogenous retroviral capsid polypeptide is an engineered endo-Gag polypeptide that comprises an amino acid substitution compared to the corresponding native endo-Gag polypeptide. In some embodiments, the second endogenous retroviral capsid polypeptide is an engineered endo-Gag polypeptide that comprises an amino acid insertion compared to the corresponding native endo-Gag polypeptide. In some embodiments, the second endogenous retroviral capsid polypeptide is an engineered endo Gag polypeptide that includes a non-native cysteine ​​that is not present in the corresponding native endo Gag polypeptide.

[0186] In some embodiments, the second endogenous retroviral capsid polypeptide is a PNMA2 polypeptide or comprises the amino acid sequence of a PNMA2 polypeptide. In some embodiments, the second endogenous retroviral capsid polypeptide is a native PNMA2 polypeptide or comprises the amino acid sequence of a native PNMA2 polypeptide. In some embodiments, the second endogenous retroviral capsid polypeptide is an engineered PNMA2 polypeptide or comprises the amino acid sequence of an engineered PNMA2 polypeptide. In some embodiments, the second endogenous retroviral capsid polypeptide is an engineered PNMA2 polypeptide that comprises an amino acid deletion compared to the corresponding native PNMA2 polypeptide. In some embodiments, the second endogenous retroviral capsid polypeptide is an engineered PNMA2 polypeptide that comprises an amino acid substitution compared to the corresponding native PNMA2 polypeptide. In some embodiments, the second endogenous retroviral capsid polypeptide is an engineered PNMA2 polypeptide that comprises an amino acid insertion compared to the corresponding native PNMA2 polypeptide. In some embodiments, the second endogenous retroviral capsid polypeptide is an engineered PNMA2 polypeptide that includes a non-native cysteine ​​that is not present in the corresponding native PNMA2 polypeptide.

[0187] In some embodiments, the second endogenous retrovirus capsid polypeptide comprises a foreign polypeptide sequence disclosed herein. In some embodiments, the second endogenous retrovirus capsid polypeptide comprises a cargo binding domain (e.g., an RNA, DNA, or protein binding domain) disclosed herein. In some embodiments, the second endogenous retrovirus capsid polypeptide comprises a domain that binds to a cell surface molecule. In some embodiments, the second endogenous retrovirus capsid polypeptide comprises an antibody or an antigen-binding fragment thereof.

[0188] In some embodiments, the first endogenous retroviral capsid polypeptide and the second endogenous retroviral capsid polypeptide are present in the capsid at a ratio of about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, 100:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, or 1:50. In some cases, the ratio is a comparison in molar concentrations. In some cases, the ratio is a comparison in number of encapsidating subunits. In some cases, the ratio is based on the mass (e.g., ng) of encapsidating subunits present.

[0189] In some embodiments, the PNMA2-based capsid or endo-Gag-based capsid comprises a plurality of recombinant or engineered PNMA2 polypeptides and a plurality of non-PNMA2 proteins. Exemplary species of non-PNMA2 proteins include, but are not limited to, PNMA1, PNMA3, PNMA4 (MOAP1), PNMA5, PNMA6A, PNMA6B / 6D, PNMA6E, PNMA6F, PNMA7A, PNMA7B, PNMA8A, PNMA8B, PNMA8C, CCDC8, Arc, BOP, LDOC1, PEG10, RTL3, RTL6, RTL8A, RTL8B, ZNF18, Copia, ASPRV1, a protein or combination of proteins selected from the SCAN domain family, and a protein or combination of proteins selected from the retrotransposon Gag-like family.

[0190] In some cases, the ratio of the plurality of recombinant or engineered PNMA2 polypeptides to the plurality of non-PNMA2 proteins is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, 100:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:20, or 1:50. In some cases, the ratio is a comparison in molar concentrations. In some cases, the ratio is a comparison in number of encapsidating subunits.

[0191] cargo In some embodiments, the capsid of the present disclosure comprises a cargo. In some embodiments, the cargo is a heterologous cargo that is not native to the PNMA2 polypeptide. In some embodiments, the cargo is a therapeutic agent. In some embodiments, the cargo is a nucleic acid molecule, a small molecule, a protein, a peptide, an antibody or a binding fragment thereof, a peptidomimetic, or a nucleotide mimetic. In some cases, the cargo is a therapeutic cargo, for example, including one or more drugs. In some cases, the cargo comprises a diagnostic tool or a component thereof for profiling, for example, one or more markers (such as markers associated with one or more disease phenotypes). In additional cases, the cargo comprises an imaging tool or a component thereof.

[0192] In some cases, the cargo is a nucleic acid molecule. Examples of nucleic acid molecules include DNA, RNA, and mixtures of DNA and RNA. In some embodiments, the nucleic acid molecule comprises a hybrid of DNA and RNA.

[0193] In some cases, the nucleic acid molecule is a DNA polymer. In some cases, the DNA is a single-stranded DNA polymer. In other cases, the DNA is a double-stranded DNA polymer. In additional cases, the DNA is a hybrid of single-stranded and double-stranded DNA polymers.

[0194] In some embodiments, the nucleic acid molecule is an RNA polymer, such as a single-stranded RNA polymer, a double-stranded RNA polymer, or a hybrid of a single-stranded and double-stranded RNA polymer. In some cases, the RNA comprises and / or encodes an antisense oligoribonucleotide, siRNA, mRNA, tRNA, rRNA, snRNA, shRNA, microRNA, or non-coding RNA.

[0195] In some embodiments, the nucleic acid molecule is an antisense oligonucleotide, optionally including DNA, RNA, or a hybrid of DNA and RNA. In some cases, the nucleic acid molecule comprises and / or encodes an mRNA molecule. In some embodiments, the nucleic acid molecule comprises and / or encodes an RNAi molecule. In some cases, the RNAi molecule is a microRNA (miRNA) molecule. In other cases, the RNAi molecule is an siRNA molecule. The miRNA and / or siRNA are optionally double-stranded or hairpin, and are further optionally encapsulated as precursor molecules.

[0196] In some embodiments, the nucleic acid molecule is for use in nucleic acid-based therapy. In some cases, the nucleic acid molecule is for regulating gene expression (e.g., regulating translation or degradation of mRNA), regulating RNA splicing, or RNA interference. In some cases, the nucleic acid molecule comprises and / or codes for antisense oligonucleotides, microRNA molecules, siRNA molecules, mRNA molecules for use in regulating gene expression, regulating RNA splicing, or RNA interference.

[0197] In some cases, the nucleic acid molecule is used for gene editing.Exemplary gene editing systems include, but are not limited to, CRISPR-Cas system, zinc finger nuclease (ZFN) system, and transcription activator-like effector nuclease (TALEN) system.In some cases, the nucleic acid molecule comprises and / or codes for the components involved in CRISPR-Cas system, ZFN system, or TALEN system.

[0198] In some cases, the nucleic acid molecule is for use in antigen production for therapeutic and / or prophylactic vaccine production, for example, the nucleic acid molecule encodes an antigen that is expressed to elicit a desired immune response (e.g., a proinflammatory immune response, an anti-inflammatory immune response, a tolerogenic immune response, a B cell response, an antibody response, a T cell response, a CD4+ T cell response, a CD8+ T cell response, a Th1 immune response, a Th2 immune response, a Th17 immune response, a Treg immune response, an M1 macrophage response, an M2 macrophage response, or a combination thereof).

[0199] In some cases, the nucleic acid molecule comprises a nucleic acid enzyme. The nucleic acid enzyme is an RNA molecule (e.g., a ribozyme) or a DNA molecule (e.g., a deoxyribozyme) that has catalytic activity. In some cases, the nucleic acid molecule is a ribozyme. In other cases, the nucleic acid molecule is a deoxyribozyme. In some cases, the nucleic acid molecule is an MNAzyme that functions as a biosensor and / or molecular switch (see, e.g., Mokany, et al., (2010) MNAzymes, a versatile new class of nucleic acid enzymes that can function as biosensors and molecular switches, JACS 132(2):1051-1059).

[0200] In some cases, exemplary targets of the nucleic acid molecule include, but are not limited to, UL123 (human cytomegalovirus), APOB, AR (androgen receptor) gene, KRAS, PCSK9, CFTR, and SMN (e.g., SMN2).

[0201] In some embodiments, the nucleic acid molecule is at least 5 nucleotides in length. In some cases, the nucleic acid molecule is at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, or at least 9000 or more nucleotides in length. In some cases, the nucleic acid molecule is at least 10 nucleotides or more in length. In some cases, the nucleic acid molecule is at least 15 nucleotides or more in length. In some cases, the nucleic acid molecule is at least 20 nucleotides or more in length. In some cases, the nucleic acid molecule is at least 30 nucleotides or more in length. In some cases, the nucleic acid molecule is at least 40 nucleotides or more in length. In some cases, the nucleic acid molecule is at least 50 nucleotides or more in length. In some cases, the nucleic acid molecule is at least 100 nucleotides or more in length. In some cases, the nucleic acid molecule is at least 200 nucleotides or more in length. In some cases, the nucleic acid molecule is at least 300 nucleotides or more in length. In some cases, the nucleic acid molecule is at least 500 nucleotides or more in length. In some cases, the nucleic acid molecule is at least 1000 nucleotides or more in length.In some cases, the nucleic acid molecule is at least 2000 nucleotides or more in length. In some cases, the nucleic acid molecule is at least 3000 nucleotides or more in length. In some cases, the nucleic acid molecule is at least 4000 nucleotides or more in length. In some cases, the nucleic acid molecule is at least 5000 nucleotides or more in length. In some cases, the nucleic acid molecule is at least 8000 nucleotides or more in length.

[0202] In some embodiments, the nucleic acid molecule is up to 10,000 nucleotides or less in length. In some cases, the nucleic acid molecule is up to 15, up to 20, up to 25, up to 30, up to 35, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 150, up to 200, up to 250, up to 300, up to 400, up to 500, up to 1000, up to 1500, up to 2000, up to 3000, up to 4000, up to 5000, up to 6000, up to 7000, up to 8000, up to 9000, or up to 10,000 or less in length. In some cases, the nucleic acid molecule is up to 15 nucleotides or less in length. In some cases, the nucleic acid molecule is up to 20 nucleotides or less in length. In some cases, the nucleic acid molecule is up to 25 nucleotides or less in length. In some cases, the nucleic acid molecule is up to 30 nucleotides or less in length. In some cases, the nucleic acid molecule is up to 40 nucleotides or less in length. In some cases, the nucleic acid molecule is up to 50 nucleotides or less in length. In some cases, the nucleic acid molecule is up to 100 nucleotides or less in length. In some cases, the nucleic acid molecule is up to 200 nucleotides or less in length. In some cases, the nucleic acid molecule is up to 300 nucleotides or less in length. In some cases, the nucleic acid molecule is up to 500 nucleotides or less in length. In some cases, the nucleic acid molecule is up to 1000 nucleotides or less in length. In some cases, the nucleic acid molecule is up to 2000 nucleotides or less in length. In some cases, the nucleic acid molecule is up to 3000 nucleotides or less in length. In some cases, the nucleic acid molecule is up to 4000 nucleotides or less in length. In some cases, the nucleic acid molecule is up to 5000 nucleotides or less in length. In some cases, the nucleic acid molecule is up to 8000 nucleotides or less in length. In some cases, the nucleic acid molecule is up to 9000 nucleotides or less in length.

[0203] In some embodiments, the nucleic acid molecule is about 100 nucleotides in length. In some cases, the nucleic acid molecule is about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 400, about 500, about 1000, about 1500, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, or about 10000 nucleotides in length. In some cases, the nucleic acid molecule is about 10 nucleotides in length. In some cases, the nucleic acid molecule is about 15 nucleotides in length. In some cases, the nucleic acid molecule is about 20 nucleotides in length. In some cases, the nucleic acid molecule is about 25 nucleotides in length. In some cases, the nucleic acid molecule is about 30 nucleotides in length. In some cases, the nucleic acid molecule is about 40 nucleotides in length. In some cases, the nucleic acid molecule is about 50 nucleotides in length. In some cases, the nucleic acid molecule is about 100 nucleotides in length. In some cases, the nucleic acid molecule is about 150 nucleotides in length. In some cases, the nucleic acid molecule is about 200 nucleotides in length. In some cases, the nucleic acid molecule is about 300 nucleotides in length. In some cases, the nucleic acid molecule is about 500 nucleotides in length. In some cases, the nucleic acid molecule is about 1000 nucleotides in length. In some cases, the nucleic acid molecule is about 2000 nucleotides in length. In some cases, the nucleic acid molecule is about 3000 nucleotides in length. In some cases, the nucleic acid molecule is about 4000 nucleotides in length. In some cases, the nucleic acid molecule is about 5000 nucleotides in length. In some cases, the nucleic acid molecule is about 8000 nucleotides in length. In some cases, the nucleic acid molecule is about 9000 nucleotides in length.

[0204] In some embodiments, the nucleic acid molecule is about 5 to about 10,000 nucleotides in length. In some cases, the nucleic acid molecule is about 5 to about 9000 nucleotides in length, about 5 to about 8000 nucleotides in length, about 5 to about 7000 nucleotides in length, about 5 to about 6000 nucleotides in length, about 5 to about 5000 nucleotides in length, about 5 to about 4000 nucleotides in length, about 5 to about 3000 nucleotides in length, about 5 to about 2000 nucleotides in length, about 5 to about 1000 nucleotides in length, about 5 to about 500 nucleotides in length, about 5 to about 100 nucleotides in length, about 5 to about 50 nucleotides in length, about 5 to about 40 nucleotides in length, about 5 to about 30 nucleotides in length, about 5 to about 25 nucleotides in length, about 5 to about 20 nucleotides in length, or about 10 to about 10,000 nucleotides in length. In some cases, the nucleic acid molecule is from about 10 to about 9000 nucleotides in length, from about 10 to about 8000 nucleotides in length, from about 10 to about 7000 nucleotides in length, from about 10 to about 6000 nucleotides in length, from about 10 to about 5000 nucleotides in length, from about 10 to about 4000 nucleotides in length, from about 10 to about 3000 nucleotides in length, from about 10 to about 2000 nucleotides in length, from about 10 to about 1000 nucleotides in length, from about 10 to about 500 nucleotides in length, from about 10 to about 100 nucleotides in length, from about 10 to about 50 nucleotides in length, from about 10 to about 40 nucleotides in length, from about 10 to about 30 nucleotides in length, from about 10 to about 25 nucleotides in length, from about 10 to about 20 nucleotides in length, or from about 50 to about 10,000 nucleotides in length. In some cases, the nucleic acid molecule is about 50 to about 9000 nucleotides in length, about 50 to about 8000 nucleotides in length, about 50 to about 7000 nucleotides in length, about 50 to about 6000 nucleotides in length, about 50 to about 5000 nucleotides in length, about 50 to about 4000 nucleotides in length, about 50 to about 3000 nucleotides in length, about 50 to about 2000 nucleotides in length, about 50 to about 1000 nucleotides in length, about 50 to about 500 nucleotides in length, about 50 to about 100 nucleotides in length, about 50 to about 50 nucleotides in length, about 50 to about 40 nucleotides in length, about 50 to about 30 nucleotides in length, about 50 to about 25 nucleotides in length, about 50 to about 20 nucleotides in length, or about 100 to about 10,000 nucleotides in length.In some cases, the nucleic acid molecule is about 100 to about 9000 nucleotides in length, about 100 to about 8000 nucleotides in length, about 100 to about 7000 nucleotides in length, about 100 to about 6000 nucleotides in length, about 100 to about 5000 nucleotides in length, about 100 to about 4000 nucleotides in length, about 100 to about 3000 nucleotides in length, about 100 to about 2000 nucleotides in length, about 100 to about 1000 nucleotides in length, about 100 to about 500 nucleotides in length, about 100 to about 100 nucleotides in length, about 100 to about 50 nucleotides in length, about 100 to about 40 nucleotides in length, about 100 to about 30 nucleotides in length, about 100 to about 25 nucleotides in length, about 100 to about 20 nucleotides in length, or about 500 to about 10,000 nucleotides in length. In some cases, the nucleic acid molecule is about 500 to about 9000 nucleotides in length, about 500 to about 8000 nucleotides in length, about 500 to about 7000 nucleotides in length, about 500 to about 6000 nucleotides in length, about 500 to about 5000 nucleotides in length, about 500 to about 4000 nucleotides in length, about 500 to about 3000 nucleotides in length, about 500 to about 2000 nucleotides in length, about 500 to about 1000 nucleotides in length, about 500 to about 500 nucleotides in length, about 500 to about 100 nucleotides in length, about 500 to about 50 nucleotides in length, about 500 to about 40 nucleotides in length, about 500 to about 30 nucleotides in length, about 500 to about 25 nucleotides in length, or about 500 to about 20 nucleotides in length.

[0205] In some embodiments, the nucleic acid molecule comprises natural, synthetic, or artificial nucleotide analogs or bases. In some cases, the nucleic acid molecule comprises DNA, RNA, and / or a combination of nucleotide analogs. In some cases, the synthetic or artificial nucleotide analogs or bases comprise modifications to one or more of the ribose moiety, the phosphate moiety, the nucleoside moiety, or a combination thereof.

[0206] In some embodiments, the nucleotide analog or artificial nucleotide base described above comprises a nucleic acid having a modification at the 2' hydroxyl group of the ribose moiety. In some cases, the modification includes H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, where R is an alkyl moiety. Exemplary alkyl moieties include, but are not limited to, halogen, sulfur, thiol, thioether, thioester, amine (primary, secondary, or tertiary), amide, ether, ester, alcohol, and oxygen. In some cases, the alkyl moiety further comprises a modification. In some cases, the modification includes an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso group, a nitrile group, a heterocyclic (e.g., imidazole, hydrazino, or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur-containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some cases, the alkyl moiety further comprises a heterosubstitution. In some cases, a carbon of the heterocyclic group is replaced with nitrogen, oxygen, or sulfur. In some cases, heterocyclic substitutions include, but are not limited to, morpholino, imidazole, and pyrrolidino.

[0207] In some cases, the modification at the 2' hydroxyl group is a 2'-O-methyl modification or a 2'-O-methoxyethyl (2'-O-MOE) modification. In some cases, the 2'-O-methyl modification adds a methyl group to the 2' hydroxyl group of the ribose moiety, and the 2'O-methoxyethyl modification adds a methoxyethyl group to the 2' hydroxyl group of the ribose moiety.

[0208] In some cases, the modification at the 2' hydroxyl group is a 2'-O-aminopropyl modification in which an extended amine group containing a propyl linker attaches the amine group to the 2' oxygen. In some cases, this modification neutralizes the overall negative charge from the phosphate of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar, thereby improving cellular uptake properties due to its zwitterionic properties.

[0209] In some cases, the modification at the 2' hydroxyl group is a locked or bridged ribose modification (e.g., locked nucleic acid or LNA) in which an oxygen molecule attached to the 2' carbon is linked to the 4' carbon by a methylene group, thus forming a 2'-C,4'-C-oxymethylene linked bicyclic ribonucleotide monomer.

[0210] In some embodiments, additional modifications at the 2' hydroxyl group include 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA).

[0211] In some embodiments, nucleotide analogs include modified bases, including, but not limited to, N1-methylpseudouridine, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides with modifications at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides (e.g., 7-deaza-adenosine, 6- azouridine, 6-azocytidine, or 6-azothymidine), 5-methyl-2-thiouridine, other thio bases (2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, queosine, archaeosine, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines (e.g., N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridin-4-one, or pyridin-2-one), phenyl and modified phenyl groups, such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonyl alkylated nucleotides. Modified nucleotides also include nucleotides modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moiety is, or is based on, mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbocycles, in some cases. The term nucleotide also includes universal bases.Exemplary universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularine.

[0212] In some embodiments, the nucleotide analogue further comprises morpholino, peptide nucleic acid (PNA), methyl phosphonate nucleotide, thiol phosphonate nucleotide, 2'-fluoro N3-P5'-phosphoramidite, or 1',5'-anhydrohexitol nucleic acid (HNA). Morpholino or phosphorodiamidate morpholino oligos (PMOs) include synthetic molecules whose structure mimics that of natural nucleic acids, but deviates from the normal sugar and phosphate structures. In some cases, the five-membered ribose ring is replaced with a six-membered morpholino ring containing four carbons, one nitrogen, and one oxygen. In some cases, the ribose monomers are linked by phosphorodiamidate groups instead of phosphate groups. In such cases, the backbone changes eliminate all positive and negative charges, allowing the morpholino neutral molecule to cross cell membranes without the aid of a cell delivery agent, as used by charged oligonucleotides.

[0213] In some embodiments, peptide nucleic acids (PNAs) do not contain sugar rings or phosphate linkages, and the bases are attached and appropriately spaced with oligoglycine-like molecules so that the backbone charge is eliminated.

[0214] In some embodiments, one or more modifications are optionally made in the internucleotide linkage.In some cases, modified internucleotide linkages include, but are not limited to, phosphorothioates, phosphorodithioates, methyl phosphonates, 5'-alkylene phosphonates, 5'-methyl phosphonates, 3'-alkylene phosphonates, trifluoroborate salts, boranophosphate esters and 3'-5' or 2'-5' selenophosphates, phosphotriesters, thionoalkyl phosphotriesters, phosphonate hydrogen linkages, alkyl phosphonates, alkyl phosphonothioates, aryl phosphonothioates, phosphoroselenoates, phosphorodiselenoates, phosphinates, phosphoramidates, 3'-alkyl phosphoramidates, aminoalkyl phosphoramidates, thionophosphos. Foramidates; phosphoropiperazidates; phosphoroanilothioates; phosphoroanilidates; ketones; sulfones; sulfonamides; carbonates; carbamates; methylenehydrazo; methylenedimethylhydrazo; formacetals; thioformacetals; oximes; methyleneimino; methylenemethylimino; thioamidates; linkages with riboacetyl groups; aminoethylglycines; silyl or siloxane linkages; alkyl or cycloalkyl linkages which are saturated or unsaturated and / or substituted and / or containing heteroatoms, e.g., with or without heteroatoms of 1 to 10 carbons; linkages with morpholino structures, amides, or polyamides (wherein the bases are attached directly or indirectly to the aza nitrogens of the backbone); and combinations thereof.

[0215] In some embodiments, the one or more modifications include a modified phosphate backbone, where the modification produces a neutral or uncharged backbone. In some cases, the phosphate backbone is modified by alkylation to produce an uncharged or neutral phosphate backbone. As used herein, alkylation includes methylation, ethylation, and propylation. In some cases, an alkyl group as used herein in the context of alkylation refers to a linear or branched saturated hydrocarbon group containing 1 to 6 carbon atoms. In some cases, exemplary alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, and 2-ethylbutyl groups. In some cases, the modified phosphate is a phosphate group as described in U.S. Pat. No. 9,481,905.

[0216] In some embodiments, the additional modified phosphate backbone comprises a methylphosphonate, an ethylphosphonate, a methylthiophosphonate, or a methoxyphosphonate. In some embodiments, the modified phosphate is a methylphosphonate. In some embodiments, the modified phosphate is an ethylphosphonate. In some embodiments, the modified phosphate is a methylthiophosphonate. In some embodiments, the modified phosphate is a methoxyphosphonate.

[0217] In some embodiments, the one or more modifications further include modifications of the ribose moiety, the phosphate backbone and the nucleoside, or modifications of the nucleotide analogue at the 3' or 5' end. For example, the 3' end is optionally comprised of a 3' cationic group or by inverting the nucleoside at the 3' end with a 3'-3' linkage. In another alternative, the 3' end is optionally conjugated with an amino alkyl group, such as a 3'C5-amino alkyl dT. In a further alternative, the 3' end is optionally conjugated with an abasic site, such as a non-purine or non-pyrimidine site. In some cases, the 5' end is conjugated with an amino alkyl group, such as a 5'-O-alkylamino substituent. In some cases, the 5' end is conjugated with an abasic site, such as a non-purine or non-pyrimidine site.

[0218] In some embodiments, exemplary nucleic acid cargoes include, but are not limited to, Fomivirsen, Mipomersen, AZD5312 (AstraZeneca), Nusinersen, and SB010 (Sterna Biologicals).

[0219] Gene editing In some embodiments, the cargo comprises or encodes a gene editing system or a component thereof. Non-limiting examples of gene editing tools and techniques include CRISPR, TALEN, zinc finger nucleases (ZFNs), meganucleases, Mega-TALs, and transposon-based systems.

[0220] In some embodiments, the cargo comprises or encodes: a CRISPR-associated polypeptide (Cas), a zinc finger nuclease (ZFN), a zinc finger associated gene regulatory polypeptide, a transcription activator-like effector nuclease (TALEN), a transcription activator-like effector associated gene regulatory polypeptide, a meganuclease, a natural master transcription factor, an epigenetic modification enzyme, a recombinase, a flippase, a transposase, an RNA binding protein (RBP), an Argonaute protein, any derivative thereof, any variant thereof, or any fragment thereof.

[0221] In some embodiments, cargo comprises or codes for CRISPR system or its components.CRISPR system can be used to promote the insertion of recombinant nucleic acid that codes for regulatable membrane protein or its components into cell genome.For example, CRISPR system can introduce double-strand break at target site in genome or random site in genome.

[0222] In some embodiments, the cargo comprises or encodes a Cas protein. Non-limiting examples of Cas proteins that can be used in the CRISPR system include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Cs m3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, c2c1, c2c3, Cas9HiFi, its homologues, and its modified forms. Unmodified CRISPR enzymes, such as Cas9, can have DNA cleavage activity. CRISPR enzymes can direct the cleavage of one or both strands in a target sequence, such as within the target sequence and / or within the complement of the target sequence. For example, the CRISPR enzyme can direct the cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence. The Cas protein can be a high-fidelity Cas protein. Alternatives to S. pyogenes Cas9 can include the Cpf1 family of RNA-guided endonucleases that exhibit cleavage activity in mammalian cells.

[0223] In some embodiments, the cargo comprises or encodes, for example, a guide RNA (gRNA) that forms a complex with a Cas protein.

[0224] In some embodiments, the cargo comprises or encodes a nucleotide sequence to be inserted into the genome. In some embodiments, the cargo comprises or encodes a repair template, for example for homology-directed repair.

[0225] In some embodiments, the cargo comprises or encodes a dual nickase CRISPR system or its components. The dual nickase approach can be used to introduce double-strand breaks. Cas protein can be mutated at specific amino acids in either nuclease domain, which eliminates the activity of one nuclease domain, generating a nickase Cas protein that can generate single-strand breaks. Two different guide RNAs that target the nickase and the opposite strand can be utilized to generate DSBs in the target site (often referred to as "double nick" or "dual nickase" CRISPR system).

[0226] In some embodiments, the cargo comprises or encodes a transposon-based system or a component thereof. A transposon-based system can be utilized to insert a recombinant nucleic acid encoding a regulatable membrane protein or a component thereof of the present disclosure into a genome. A transposon can comprise a recombinant nucleic acid that can be inserted into a DNA sequence. Class I transposons can be transcribed into an RNA intermediate and then reverse transcribed and inserted into a DNA sequence. Class II transposons can comprise a DNA sequence that is excised from one DNA sequence and / or inserted into another DNA sequence. A class II transposon system can comprise (i) a transposon vector that comprises a sequence (e.g., including a transgene) flanked by inverted terminal repeats, and (ii) a source of transposase enzyme.

[0227] In some embodiments, the cargo comprises or encodes a TALEN system or a component thereof. TALEN may refer to an engineered transcription activator-like effector nuclease, which generally comprises a central domain of DNA-binding tandem repeats and a cleavage domain. TALENs may be produced by fusing a TAL effector DNA-binding domain to a DNA cleavage domain. In some cases, the DNA-binding tandem repeat comprises 33-35 amino acids in length and contains two hypervariable amino acid residues at positions 12 and 13 that can recognize at least one specific DNA base pair. Transcription activator-like effector (TALE) proteins can be fused to a nuclease, such as a wild-type or mutant Fok1 endonuclease, or the catalytic domain of Fok1.

[0228] In some embodiments, the cargo comprises or encodes a zinc finger nuclease (ZFN), or a variant, fragment, or derivative thereof. ZFN can refer to a fusion between a cleavage domain, such as the cleavage domain of Fok1, and at least one zinc finger motif (e.g., at least two, at least three, at least four, or at least five zinc finger motifs) capable of binding to polynucleotides, such as DNA and RNA.

[0229] In some embodiments, the cargo comprises or encodes a meganuclease. Meganucleases generally refer to rare-cutting or homing endonucleases that may have high sequence specificity. Meganucleases can recognize DNA target sites at least 12 base pairs in length, for example, in the range of 12-40 base pairs in length, 12-50 base pairs in length, or 12-60 base pairs in length. Meganucleases can be modular DNA-binding nucleases, such as any fusion protein that includes at least one catalytic domain of an endonuclease and at least one DNA-binding domain or protein that specifies a nucleic acid target sequence. The DNA-binding domain can include at least one motif that recognizes single-stranded or double-stranded DNA. Nuclease-active meganucleases can generate double-stranded breaks. Meganucleases can be monomeric or dimeric. In some embodiments, meganucleases are naturally occurring (found in nature) or wild-type, while in other cases, meganucleases are non-natural, artificial, engineered, synthetic, rationally designed, or man-made. In some embodiments, meganucleases of the present disclosure include I-CreI meganuclease, I-CeuI meganuclease, I-Msol meganuclease, I-SceI meganuclease, variants thereof, derivatives thereof, and fragments thereof.

[0230] low molecule In some embodiments, the cargo is a small molecule. In some cases, the small molecule is an inhibitor (e.g., a pan-inhibitor or a selective inhibitor). In other cases, the small molecule is an activator. In further cases, the small molecule is an agonist, antagonist, partial agonist, mixed agonist / antagonist, or competitive antagonist.

[0231] In some embodiments, the small molecule is a drug that falls into the following classes: analgesics, anxiolytics, antiarrhythmics, antimicrobials, antibiotics, anticoagulants and thrombolytics, anticonvulsants, antidepressants, antidiarrheals, antiemetics, antifungals, antihistamines, antihypertensives, anti-inflammatory drugs, antineoplastics, antipsychotics, antipyretics, antivirals, barbiturates, beta blockers, bronchodilators, cold remedies, corticosteroids, cough suppressants, cytotoxic drugs, decongestants, diuretics, expectorants, hormones, hypoglycemic drugs, immunosuppressants, laxatives, muscle relaxants, sex hormones, hypnotics, or tranquilizers.

[0232] In some embodiments, the small molecule is an inhibitor, for example an inhibitor of a kinase pathway, such as a tyrosine kinase pathway or a serine / threonine kinase pathway. In some cases, the small molecule is a dual protein kinase inhibitor. In some cases, the small molecule is a lipid kinase inhibitor.

[0233] In some cases, the small molecule is a neuraminidase inhibitor.

[0234] In some cases, the small molecule is a carbonic anhydrase inhibitor.

[0235] In some embodiments, exemplary targets of small molecules include, but are not limited to, vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), vascular endothelial growth factor receptor 3 (VEGFR3), fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), fibroblast growth factor receptor 4 (FGFR4), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 6 (CDK6), receptor tyrosine kinase, phosphoinositide 3-kinase (PI3K) isoforms (e.g., PI3Kδ, also known as p110δ), Janus kinase 1 (JAK1), Janus kinase 3 (JAK3), receptors of the platelet-derived growth factor receptor family (PDFG-R), and carbonic anhydrase (e.g., carbonic anhydrase I).

[0236] In some embodiments, the small molecule targets a viral protein, such as a viral envelope protein. In some embodiments, the small molecule reduces viral adsorption to a host cell. In some embodiments, the small molecule reduces viral entry into a host cell. In some embodiments, the small molecule reduces viral replication in a host or host cell. In some embodiments, the small molecule reduces viral assembly.

[0237] In some embodiments, exemplary small molecule cargoes include, but are not limited to, lenvatinib, palbociclib, regorafenib, idelalisib, tofacitinib, nintedanib, zanamivir, ethoxzolamide, and artemisinin.

[0238] protein In some embodiments, the cargo is a protein. In some cases, the protein is a full-length protein. In other cases, the protein is a fragment, e.g., a functional fragment. In some cases, the protein is a naturally occurring protein. In additional cases, the protein is a de novo engineered protein. In further cases, the protein is a fusion protein. In further cases, the protein is a recombinant protein. Examples of proteins include, but are not limited to, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic drugs.

[0239] In some cases, the protein is for use in enzyme replacement therapy.

[0240] In some cases, the protein is for use in antigen production for therapeutic and / or prophylactic vaccine production. For example, the protein comprises an antigen that elicits a desired immune response (e.g., a proinflammatory immune response, an anti-inflammatory immune response, a tolerogenic immune response, a B cell response, an antibody response, a T cell response, a CD4+ T cell response, a CD8+ T cell response, a Th1 immune response, a Th2 immune response, a Th17 immune response, a Treg immune response, an M1 macrophage response, an M2 macrophage response, or a combination thereof).

[0241] In some cases, exemplary protein cargoes include, but are not limited to, romiplostim, liraglutide, human growth hormone (rHGH), human insulin (BHI), follicle-stimulating hormone (FSH), factor VIII, erythropoietin (EPO), granulocyte-colony stimulating factor (G-CSF), alpha-galactosidase A, alpha-L-iduronidase, N-acetylgalactosamine-4-sulfatase, dornase alpha, tissue plasminogen activator (TPA), glucocerebrosidase, interferon-beta-1a, insulin-like growth factor 1 (IGF-1), or rasburicase.

[0242] peptide In some embodiments, the cargo is a peptide. In some cases, the peptide is a naturally occurring peptide. In other cases, the peptide is an engineered or recombinant peptide. In some cases, the peptide targets G protein-coupled receptors, ion channels, microorganisms, antibacterial targets, catalytic receptors or other Ig family receptors, intracellular targets, membrane-anchored targets, or extracellular targets.

[0243] In some cases, the peptide comprises at least 2 amino acids. In some cases, the peptide comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids. In some cases, the peptide comprises at least 10 amino acids. In some cases, the peptide comprises at least 15 amino acids. In some cases, the peptide comprises at least 20 amino acids. In some cases, the peptide comprises at least 30 amino acids. In some cases, the peptide comprises at least 40 amino acids. In some cases, the peptide comprises at least 50 amino acids. In some cases, the peptide comprises at least 60 amino acids. In some cases, the peptide comprises at least 70 amino acids. In some cases, the peptide comprises at least 80 amino acids. In some cases, the peptide comprises at least 90 amino acids. In some cases, the peptide comprises at least 100 amino acids.

[0244] In some cases, the peptide comprises up to 3 amino acids. In some cases, the peptide comprises up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 15, up to 20, up to 25, up to 30, up to 35, up to 40, up to 45, up to 50, up to 60, up to 70, up to 80, up to 90, or up to 100 amino acids. In some cases, the peptide comprises up to 10 amino acids. In some cases, the peptide comprises up to 15 amino acids. In some cases, the peptide comprises up to 20 amino acids. In some cases, the peptide comprises up to 30 amino acids. In some cases, the peptide comprises up to 40 amino acids. In some cases, the peptide comprises up to 50 amino acids. In some cases, the peptide comprises up to 60 amino acids. In some cases, the peptide comprises up to 70 amino acids. In some cases, the peptide comprises up to 80 amino acids. In some cases, the peptide comprises up to 90 amino acids. In some cases, the peptide comprises up to 100 amino acids.

[0245] In some cases, the peptide comprises about 1 to about 10 kDa. In some cases, the peptide comprises about 1 to about 9 kDa, about 1 to about 6 kDa, about 1 to about 5 kDa, about 1 to about 4 kDa, about 1 to about 3 kDa, about 2 to about 8 kDa, about 2 to about 6 kDa, about 2 to about 4 kDa, about 1.2 to about 2.8 kDa, about 1.5 to about 2.5 kDa, or about 1.5 to about 2 kDa.

[0246] In some embodiments, the peptide is a cyclic peptide. In some cases, the cyclic peptide is a macrocyclic peptide. In other cases, the cyclic peptide is a constrained peptide. The cyclic peptide is assembled by various linkages, such as head to tail, tail to side chain, side chain to tail, and side chain to side chain. In some cases, the cyclic peptide (e.g., macrocyclic or constrained peptide) has a molecular weight of about 500 Daltons to about 2000 Daltons. In other cases, the cyclic peptide (e.g., macrocyclic or constrained peptide) ranges from about 10 amino acids to about 100 amino acids, about 10 amino acids to about 70 amino acids, or about 10 amino acids to about 50 amino acids.

[0247] In some cases, the peptides are for use in producing antigens for the production of therapeutic and / or prophylactic vaccines. For example, the peptides include antigens that elicit a desired immune response (e.g., a proinflammatory immune response, an anti-inflammatory immune response, a tolerogenic immune response, a B cell response, an antibody response, a T cell response, a CD4+ T cell response, a CD8+ T cell response, a Th1 immune response, a Th2 immune response, a Th17 immune response, a Treg immune response, an M1 macrophage response, an M2 macrophage response, or a combination thereof).

[0248] In some embodiments, the peptide comprises natural amino acids, unnatural amino acids, or a combination thereof. In some cases, an amino acid residue refers to a molecule that contains both an amino group and a carboxyl group. Suitable amino acids include, but are not limited to, both D and L isomers of naturally occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic pathways. The term amino acid as used herein includes, but is not limited to, α-amino acids, natural amino acids, unnatural amino acids, and amino acid analogs.

[0249] In some cases, an α-amino acid refers to a molecule that contains both an amino group and a carboxyl group bonded to a carbon called the α-carbon.

[0250] In some cases, a β-amino acid refers to a molecule that contains both an amino group and a carboxyl group in the β configuration.

[0251] In some embodiments, the amino acid analogs are racemic mixtures. In some cases, the D isomers of the amino acid analogs are used. In some cases, the L isomers of the amino acid analogs are used. In some cases, the amino acid analogs contain chiral centers in the R or S configuration.

[0252] In some embodiments, exemplary peptide cargoes include, but are not limited to, peginestatide, insulin, adrenocorticotropic hormone (ACTH), calcitonin, oxytocin, vasopressin, octreolide, and leuprorelin.

[0253] In some embodiments, exemplary peptide cargoes include, but are not limited to, telavancin, dalbavancin, oritavancin, anidulafungin, lanreotide, pasireotide, romidepsin, linaclotide, and peginesatide.

[0254] antibody In some embodiments, the cargo is an antibody or a binding fragment thereof. In some cases, the antibody or binding fragment thereof comprises a humanized antibody or a binding fragment thereof, a murine antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a bispecific antibody or a binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv, (scFv)2, a diabody, a minibody, a nanobody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a camelid antibody or a binding fragment thereof, or a chemically modified derivative thereof.

[0255] In some cases, the antibody or binding fragment thereof recognizes a cell surface protein. In some cases, the cell surface protein is an antigen expressed by a cancerous cell. In some cases, the cell surface protein is a neoepitope. In some cases, the cell surface protein comprises one or more mutations compared to the wild-type protein.Exemplary cancer antigens that may be bound by the exogenous polypeptide sequence include, but are not limited to, the following: alpha-fetoprotein, ASLG659, B7-H3, BAFF-R, brevican, CA125 (MUC16), CA15-3, CA19-9, carcinoembryonic antigen (CEA), CA242, CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor), CTLA-4, CXCR5, E16 (LAT1, SLC7A5), FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase anchor protein 1a), SPAP1B, SPAP1C), epidermal growth factor, ETBR, Fc receptor-like protein 1 (FCRH 1), GEDA, HLA-DOB (β subunit of MHC class II molecule (Ia antigen), human chorionic gonadotropin, ICOS, IL-2 receptor, IL20Rα, immunoglobulin superfamily receptor translocation associated 2 (IRTA2), L6, LewisY, LewisX, MAGE-1, MAGE-2, MAGE-3, MAGE4, ​​MART1, mesothelin, MDP, MPF (SMR, MSLN), MCP1 (CCL2), macrophage inhibitory factor (MIF), MPG, MSG783, mucin, MUC1-KLH, Napi3b (SLC34A2), nectin-4, Neu oncogene product, NCA, placental alkaline phosphatase, prostate-specific membrane antigen (PMSA), prostatic acid phosphatase, PSCA hlg, anti-transferrin receptor, p97, purinergic receptor P2X ligand-gated ion channel 5 (P2X5), LY64 (lymphocyte antigen 64 (RP105), gp100, P21, six-transmembrane epithelial antigen of the prostate (STEAP1)), STEAP2, Sema5b, tumor-associated glycoprotein 72 (TAG-72), TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4), etc.

[0256] In some cases, the cell surface protein comprises a cluster of differentiation (CD) cell surface marker. Exemplary CD cell surface markers include, but are not limited to, CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L (L-selectin), CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD71 , CD79 (e.g., CD79a, CD79b), CD90, CD95 (Fas), CD103, CD104, CD125 (IL5RA), CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), CD221, CD274, CD279 (PD-1), CD319 (SLAMF7), and CD326 (EpCAM).

[0257] In some embodiments, exemplary antibodies or binding fragments thereof include, but are not limited to, zalutumumab (HuMax-EFGr, Genmab), abagovomab (Menarini), avituzumab (Merck), adecatumumab (MT201), alacizumab pegol, alemtuzumab (Campath®, MabCampath, or Campath-1H; Leukosite), AlloMune (BioTransplant), amatuximab (Morphotek, Inc.), anti-VEGF (Genetech), anatumomab mafenatox, apolizumab (hu1D10), asculinbacumab (Pfizer Inc.), atezolizumab (MPDL3280A; Genentech / Roche), B43.13 (OvaRex, AltaRex), Corporation), basiliximab (Simulect®, Novartis), belimumab (Benlysta®, GlaxoSmithKline), bevacizumab (Avastin®, Genentech), blinatumomab (Blincyto, AMG103; Amgen), BEC2 (ImGlone Systems Inc.), carlumab (Janssen Biotech), catumaxomab (Removab, Trion Pharma), CEAcide (Immunomedics), cetuximab (Erbitux®, ImClone), sitatuzumab vogatox (VB6-845), cixutumumab (IMC-A12, ImClone Systems Inc.), conatumumab (AMG 655, Amgen), dacetuzumab (SGN-40, huS2C6; Seattle Genetics, Inc.), daratumumab (Darzalex®, Janssen Biotech), detumomab, drozitumab (Genentech), durvalumab (MedImmune), dusigitumab (MedImmune), edrecolomab (MAb17-1A, Panorex, Glaxo Wellcome), elotuzumab (Empliciti™, Bristol-Myers Squibb), emibetuzumab (Eli Lilly), enavatuzumab (Facet Biotech Corp.), enfortumab vedotin (Seattle Genetics, Inc.), enoblitutumab (MGA271, MacroGenics, Inc.), ensituxumab (Neogenix Oncology, Inc.), epratuzumab (LymphoCide, Immunomedics, Inc.), ertumaxomab (Rexomun®, Trion Pharma), etaracizumab (Abegrin, MedImmune), farletuzumab (MORAb-003, Morphotek, Inc.), FBTA05 (Lymphomun, Trion Pharma), ficlatuzumab (AVEO Pharmaceuticals), figitumumab (CP-751871, Pfizer), flanvotumab (ImClone Systems), fresolimumab (GC1008, Aanofi-Aventis), futuximab, glaximab, ganitumab (Amgen), girentuximab (Rencarex®, Wilex AG), IMAB362 (Claudiximab, Ganymed Pharmaceuticals AG), imalumab (Baxalta), IMC-1C11 (ImClone Systems), IMC-C225 (Imclone Systems Inc.), imgatuzumab (Genentech / Roche), intetumumab (Centocor, Inc.), ipilimumab (Yervoy®, Bristol-Myers Squibb), iratumumab (Medarex, Inc.), isatuximab (SAR650984, Sanofi-Aventis), labetuzumab (CEA-CIDE, Immunomedics), lexatumumab (ETR2-ST01, Cambridge Antibody Technology), lintuzumab (SGN-33, Seattle Genetics), lucatumumab (Novartis), rumiliximab, mapatumumab (HGS-ETR1, Human Genome Sciences), matuzumab (EMD 72000, Merck), milatuzumab (hLL1, Immunomedics, Inc.), mitumomab (BEC-2, ImClone Systems), narunatumab (ImClone Systems), necitumumab (Portrazza™, Eli Lilly), nesvacumab (Regeneron Pharmaceuticals), nimotuzumab (h-R3, BIOMAb EGFR, TheraCIM, Theraloc, or CIMAher; Biotech Pharmaceutical Co.), nivolumab (Opdivo®, Bristol-Myers Squibb), obinutuzumab (Gazyva or Gazyvaro, Hoffmann-La Roche), ocaratu-zumab (AME-133v, LY2469298; Mentrik Biotech, LLC), ofatumumab (Arzerra®, Genmab), onartuzumab (Genentech), ontuxizumab (Morphotek, Inc.), oregovomab (OvaRex®, AltaRex Corp.), otlertuzumab (Emergent BioSolutions), panitumumab (ABX-EGF, Amgen), pancomab (Glycotope GMBH), palsatuzumab (Genentech), patritumab, pembrolizumab (Keytruda®, Merck), pemtumomab (Theragyn, Antisoma), pertuzumab (Perjeta, Genentech), pidilizumab (CT-011, Medivation), popolatuzumab vedotin (Genentech / Roche), pritumumab, racotumomab (Vaxira®, Recombio), ramucirumab (Cyramza®, ImClone Systems Inc.), rituximab (Rituxan®, Genentech), lobatumab (Schering-Plough), serivantumab (Sanofi / Merrimack Pharmaceuticals, Inc.), sibrotuzumab, siltuximab (Sylvant™, Janssen Biotech), Smart MI95 (Protein Design Labs, Inc.), Smart ID10 (Protein Design Labs, Inc.), tabalumab (LY2127399, Eli Lilly), taplitumomab paptox (taplitumomab paptox), tenatumomab, teprotumumab (Roche), tetulomab, TGN1412 (CD28-SuperMAB or TAB08), tigatuzumab (CD-1008, Daiichi Sankyo), tositumomab, trastuzumab (Herceptin®), tremelimumab (CP-672,206; Pfizer), tucotuzumab celmoleukin (EMD Pharmaceuticals), ublituximab, urelumab (BMS-663513, Bristol-Myers Squibb), vorociximab (M200, Biogen Idec), and zatuximab.

[0258] In some cases, the antibody or binding fragment thereof is an antibody drug conjugate (ADC). In some cases, the payload of the ADC includes, but is not limited to, for example, an auristatin derivative, a maytansine, a maytansinoid, a taxane, a calicheamicin, a cemadotin, a duocarmycin, a pyrrolobenzodiazepine (PDB), or a tubulysin. In some cases, the payload includes monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). In some cases, the payload includes DM2 (mertansine) or DM4. In some cases, the payload includes a pyrrolobenzodiazepine dimer.

[0259] Extra Cargo In some embodiments, the cargo is a peptidomimetic. Peptidomimetics are small protein-like polymers designed to mimic peptides. In some cases, peptidomimetics include D-peptides. In other cases, peptidomimetics include L-peptides. Exemplary peptidomimetics include peptoids and beta-peptides.

[0260] In some embodiments, the cargo is a nucleotide mimic.

[0261] Vectors and Expression Systems The PNMA2 and endo-Gag polypeptides of the present disclosure can be encoded by a nucleic acid, for example, for expression in a host cell or for use in a cell-free expression system. In certain embodiments, the PNMA2, endo-Gag, engineered PNMA2 and engineered endo-Gag polypeptides described herein are encoded by a vector, such as a plasmid vector. In some embodiments, the vector includes any suitable vector derived from either eukaryotic or prokaryotic sources. In some cases, the vector is obtained from a bacterial (e.g., E. coli), insect, yeast (e.g., Pichia pastoris), algae, or mammalian source.

[0262] Exemplary bacterial vectors include pACYC177, pASK75, pBAD vector series, pBADM vector series, pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, pQE vector series, pRSETA, pRSETB, pRSETC, pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or pTAC-MAT-2.

[0263] Examples of insect vectors include pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, a FLAG vector such as pPolh-FLAG1 or pPolh-MAT 2, or a MAT vector such as pPolh-MAT1, or pPolh-MAT2.

[0264] Optionally, the yeast vector includes a Gateway® pDEST™ 14 vector, a Gateway® pDEST™ 15 vector, a Gateway® pDEST™ 17 vector, a Gateway® pDEST™ 24 vector, a Gateway® pYES-DEST52 vector, a pBAD-DEST49 Gateway® Destination vector, vector, pAO815 Pichia vector, pFLD1 Pichia yeast vector, pGAPZA, B, &C Pichia yeast vector, pPIC3.5K Pichia vector, pPIC6A, B, &C Pichia vector, pPIC9K Pichia vector, pTEF1 / Zeo, pYES2 yeast vector, pYES2 / CT yeast vector, pYES2 / NT A, B, &C yeast vector, or pYES3 / CT yeast vector.

[0265] Exemplary algal vectors include the pChlamy-4 vector or the MCS vector.

[0266] Examples of mammalian vectors include transient expression vectors or stable expression vectors.Mammalian transient expression vectors include p3xFLAG-CMV8, pFLAG-Myc-CMV19, pFLAG-Myc-CMV23, pFLAG-CMV2, pFLAG-CMV6a, b, c, pFLAG-CMV5.1, pFLAG-CMV5a, b, c, p3xFLAG-CMV7.1, pFLAG-CMV20, p3xFLAG-Myc-CMV24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV3 or pBICEP-CMV4. Mammalian stable expression vectors include pFLAG-CMV3, p3xFLAG-CMV9, p3xFLAG-CMV13, pFLAG-Myc-CMV21, p3xFLAG-Myc-CMV25, pFLAG-CMV4, p3xFLAG-CMV10, p3xFLAG-CMV14, pFLAG-Myc-CMV22, p3xFLAG-Myc-CMV26, pBICEP-CMV1, or pBICEP-CMV2.

[0267] In certain embodiments, the PNMA2 polypeptides, endo-Gag polypeptides, engineered PNMA2 polypeptides and engineered endo-Gag polypeptides described herein, and / or heterologous cargo are expressed using a cell-free expression system. In some cases, the cell-free system is a mixture of cytoplasmic and / or nuclear components from a cell, or isolated transcription and / or translation machinery, used to express RNA and / or proteins in vitro. In some cases, the cell-free system utilizes either prokaryotic or eukaryotic components, or transcription and / or translation machinery derived therefrom. In some embodiments, nucleic acid synthesis is obtained in a cell-free system based, for example, on Drosophila cells, Xenopus eggs, or HeLa cells (ATCC® CCL-2™). Exemplary cell-free systems include, but are not limited to, the E. coli S30 extract system, the E. coli T7 S30 system, or PURExpress®.

[0268] host cell In certain embodiments, the PNMA2 polypeptides, endo-Gag polypeptides, engineered PNMA2 polypeptides and engineered endo-Gag polypeptides described herein, and / or heterologous cargo are expressed using a host cell. In some embodiments, the host cell includes any suitable cell, such as a naturally occurring cell or a genetically modified cell. In some cases, the host cell is a production host cell. In some cases, the host cell is a eukaryotic cell. In other cases, the host cell is a prokaryotic cell. In some cases, the eukaryotic cell includes a fungus (e.g., a yeast cell), an animal cell, or a plant cell. In some cases, the prokaryotic cell is a bacterial cell. Examples of bacterial cells include gram-positive or gram-negative bacteria. In some embodiments, the gram-negative bacteria is an anaerobic, bacillus, or both.

[0269] In some cases, the gram-positive bacteria include Actinobacteria, Firmicutes, or Tenericutes bacteria. In some cases, the gram-negative bacteria include Aquificae, Deinococcus-Thermus, Fibrobacteres-Chlorobi / Bacteroidetes (FCB group), Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes-Verrucomicrobia / Chlamydiae (PVC group), Proteobacteria, Spirochaetes, or Synergistetes. In some embodiments, the bacteria is Acidobacteria, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Dictyoglomi, Thermodesulfobacteria, or Thermotogae. In some embodiments, the bacterial cell is Escherichia coli, Clostridium botulinum, or Coli bacilli.

[0270] Exemplary prokaryotic host cells include, but are not limited to, BL21, Mach1™, DH10B™, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F', INVαF, TOP10 / P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™.

[0271] In some cases, animal cells include cells derived from vertebrates or invertebrates. In some cases, animal cells include cells from marine invertebrates, fish, insects, amphibians, reptiles, mammals, or humans. In some cases, fungal cells include yeast cells, such as brewer's yeast, baker's yeast, or wine yeast.

[0272] Fungi include ascomycota such as yeasts, molds, filamentous fungi, basidiomycota, or zygomycota. In some cases, yeasts include Ascomycota or Basidiomycota. In some cases, Ascomycota include Saccharomycotina (true yeast, such as Saccharomyces cerevisiae (baker's yeast)) or Taphrinomycota (e.g., Schizosaccharomycetes (fission yeast)). In some cases, Basidiomycota include Agaricota (e.g., Tremella) or Urobacterium (e.g., Microbotryomycetes).

[0273] Exemplary yeasts or filamentous fungi include, for example, the genera Saccharomyces, Schizosaccharomyces, Candida, Pichia, Hansenula, Kluyveromyces, Zygosaccharomyces, Yarrowia, Trichosporon, Rhodosporidi, Aspergillus, Fusarium, or Trichoderma. Exemplary yeasts or filamentous fungi include, for example, the following species: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Candida boidini, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanathii, Candida lusitaniae, Rhodotorula mucilaginosa, Pichia methanolica, Pichia angusta, Pichia pastoris, Pichia anomala, Hansenula polymorpha, Kluyveromyces lactis, Zygosaccharomyces rouxii, Yarrowia lipolytica, Trichosporon pullulans, Rhodosporidium toru-Aspergillus niger, Aspergillus nidulans, Aspergillus awamori, Aspergillus oryzae, Trichoderma reesei, Yarrowia lipolytica, Brettanomyces bruxellensis, Candida stellata, Schizosaccharomyces pombe, Torulaspora delbrueckii, Zygosaccharomyces bailii, Cryptococcus neoformans, Cryptococcus gattii, or Saccharomyces boulardii.

[0274] Exemplary yeast host cells include, but are not limited to, Pichia pastoris yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33, and Saccharomyces cerevisiae yeast strains such as INVSc1.

[0275] In some cases, the additional animal cells include cells obtained from mollusks, arthropods, annelids, or sponges. In some cases, the additional animal cells are mammalian cells, such as from humans, primates, apes, horses, cows, pigs, dogs, cats, or rodents. In some cases, the rodents include mice, rats, hamsters, gerbils, hamsters, chinchillas, fancy rats, or guinea pigs.

[0276] Exemplary mammalian host cells include, but are not limited to, 293A cell line, 293FT cell line, 293F cells, 293H cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, Expi293F™ cells, Flp-In™ T-REx™ 293 cell line, Flp-In™-293 cell line, Flp-In™-3T3 cell line, Flp-In™-BHK cell line, Flp-In™-CHO cell line, Flp-In™-CV-1 cell line, Flp-In™-Jurkat cell line. , FreeStyle™ 293-F cells, FreeStyle™ CHO-S cells, GripTite™ 293MSR cell line, GS-CHO cell line, HepaRG™ cells, T-REx™ Jurkat cell line, Per.C6 cells, T-REx™-293 cell line, T-REx™-CHO cell line, and T-REx™-HeLa cell line.

[0277] In some cases, the mammalian host cell is a primary cell. In some cases, the mammalian host cell is a stable cell line, or a cell line that has integrated the desired genetic material into its own genome and has the ability to express the product of the genetic material after many generations of cell division. In some cases, the mammalian host cell is a transient cell line, or a cell line that has not integrated the desired genetic material into its own genome and has the ability to express the product of the genetic material after many generations of cell division.

[0278] Exemplary insect host cells include, but are not limited to, Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expresSF+® cells. Exemplary insect cell lines include, but are not limited to, strains derived from Chlamydomonas reinhardtii 137c or Synechococcus elongatus PPC 7942. In some cases, plant cells include cells from algae.

[0279] method Disclosed herein, in certain embodiments, are methods of preparing capsids, eg, capsids that encapsulate heterologous cargo.

[0280] The PNMA2 and endo-Gag polypeptides of the disclosure exhibit favorable properties for capsid assembly, disassembly, and / or reassembly.

[0281] The presence of PNMA2 and endo-Gag polypeptides in capsid or non-capsid form can be determined, for example, using size exclusion chromatography, multi-angle dynamic light scattering (MADLS), transmission electron microscopy (TEM), or a combination thereof. The efficiency of capsid assembly, disassembly, and reassembly can be calculated, for example, by comparing the amount of protein present in assembled capsids to the total amount of protein, or the amount of protein present in particles smaller and / or larger in size than capsids. In some embodiments, when calculating reassembly, the amount of protein loss during disassembly and reassembly can be measured and taken into account in the calculation.

[0282] In some embodiments, measuring capsid assembly efficiency involves quantifying the amount of purified protein present in a capsid state (e.g., as capsid particles) and quantifying the amount of purified protein present in a non-capsid state (e.g., unassembled and / or partially assembled). For example, size exclusion chromatography can be used to separate monomers and oligomers from assembled capsids and quantify the amount of protein present in a capsid state and the amount of protein present in an unassembled or partially assembled non-capsid state.

[0283] In some embodiments, measuring the capsid disassembly efficiency includes quantifying the amount of encapsidating protein remaining in solution after disassembly from a capsid state to a non-capsid state (e.g., oligomers, capsomers, and / or monomers). In some embodiments, the disassembly efficiency can be determined by quantifying the percent of total protein that is the monomer peak as measured by MADLS or SEC. In some embodiments, the capsids are treated with a disassembly buffer and optionally centrifuged to obtain a precipitate (e.g., aggregates) and / or to remove the remaining capsids from the solution. The loss of capsid structure is confirmed by electron microscopy (e.g., TEM) and / or dynamic light scattering (e.g., MADLS). The efficiency of disassembly can be determined by MADLS (e.g., via the capsid peak and the monomer peak) or size exclusion chromatography (SEC). The amount of protein remaining in solution can be quantified. The amount of monomer in solution can be quantified. The disassembly efficiency can be calculated. For example, recovery of 90 ng of protein in solution from 100 ng of starting capsid material indicates a disassembly efficiency of 90%.

[0284] In some embodiments, measuring the capsid reassembly efficiency comprises quantifying the amount of disassembled protein in solution that reassembles into a capsid state. In some embodiments, the amount of input protein in a non-capsid state in solution is quantified (e.g., after treatment with a disassembly buffer, optionally centrifugation to remove precipitates and / or capsids, and confirmation of the absence of capsid structures by TEM and / or MADLS). In some embodiments, the disassembled protein is treated with a reassembly buffer as disclosed herein, e.g., high salt, no chaotropic agent, and / or no reducing agent. Aggregates can be removed by centrifugation, e.g., at +4°C for 5 minutes at 15,000xg. The efficiency of reassembly can be determined by comparing the amount of endo-Gag polypeptides in the monomer and capsid peaks assayed by MADLS or SEC. Capsid formation can be confirmed by TEM and / or MADLS. In some embodiments, size exclusion chromatography can be performed to quantify the amount of protein in the capsid state, which can be compared to the amount of input protein. The reassembly efficiency is calculated. For example, starting with 90 ng of protein in a non-capsid state in solution, 81 ng of protein detected in a capsid state by size exclusion chromatography indicates a reassembly efficiency of 90%. In some embodiments, the reassembly efficiency is determined by comparing the amount of endo-Gag polypeptides in reassembled capsids without further purification to the amount of endo-Gag monomer polypeptides input. In some embodiments, the reassembly efficiency is determined by comparing the amount of endo-Gag polypeptides in reassembled capsids with further purification (e.g., by SEC or IEC) to the amount of endo-Gag monomer polypeptides input.

[0285] In some embodiments, MADLS is used to determine particle concentration and verify that it is within the expected range for the amount of protein in solution. For example, for PNMA2, which has a molecular weight of 41509.37, the expected number of particles can be calculated from the amount of PNMA2 polypeptide, based on the assumption that there are 60 PNMA2 monomers per capsid: 1 g capsid = 2.42 x 10 17 Capsid.

[0286] In some embodiments, the observed concentration of capsids disclosed herein is within about ±5%, within about ±10%, within about ±20%, within about ±30%, within about ±40%, within about ±50%, within about ±60%, within about ±70%, within about ±80%, within about ±90%, within about ±2-fold, within about ±5-fold, or within about ±10-fold of the predicted particle concentration.

[0287] In some embodiments, if capsid reassembly is less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 85%, less than 86%, less than 87%, less than 88%, less than 89%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 99.5% efficient, further purification is performed (e.g., by SEC or ion exchange chromatography).

[0288] In some embodiments, the PNMA2 or endo-Gag polypeptides of the disclosure exhibit favorable assembly properties: In some embodiments, when isolated, the PNMA2 or endo-Gag polypeptides of the disclosure assemble to form capsids with an efficiency of at least about 0.1%, at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99%. In some embodiments, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of the isolated PNMA2 or endo-Gag polypeptides assemble to form capsids.

[0289] In some embodiments, the PNMA2 or endo-Gag polypeptides of the present disclosure exhibit favorable disassembly properties. In some embodiments, after incubation in a disassembly buffer, the PNMA2 or endo-Gag capsid disassembles (e.g., into monomers or smaller subunits of capsids) with an efficiency of at least about 0.1%, at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99%. In some embodiments, at least about 0.1%, at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of the PNMA2 or endo-Gag polypeptide present in the capsid is disassembled into a non-capsid state.

[0290] In some embodiments, the PNMA2 or endo-Gag polypeptides of the disclosure exhibit favorable reassembly properties: in some embodiments, after disassembly, when incubated in a suitable reassembly buffer, the PNMA2 or endo-Gag polypeptides of the disclosure reassemble to form capsids with an efficiency of at least about 0.1%, at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99%. In some embodiments, the PNMA2 or endo-Gag polypeptides are at least about 0.1%, at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, or less than about 5%, e.g., as a percentage of PNMA2 or endo-Gag protein present after disassembly, as a percentage of PNMA2 or endo-Gag protein originally present in the capsid, or as a percentage of PNMA2 or endo-Gag protein present upon initial protein isolation. In any case, about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% reassemble to form capsids.

[0291] In some embodiments, the assembly buffer comprises a physiological buffer. In some embodiments, the assembly buffer is or comprises a balanced salt solution. In some embodiments, the assembly buffer is or comprises a saline solution, e.g., buffered saline. In some embodiments, the assembly buffer is or comprises PBS. In some embodiments, the assembly buffer is or comprises HBSS. In some embodiments, the assembly buffer comprises or is Ringer's solution. In some embodiments, the assembly buffer comprises sodium phosphate.

[0292] In some embodiments, the PNMA2 or endo-Gag polypeptides of the disclosure exhibit favorable assembly properties in buffers containing low salt levels or physiological levels of salt.

[0293] In some embodiments, the assembly buffer comprises less than 2000 mOsm / kg, less than 1000 mOsm / kg, less than 900 mOsm / kg, less than 800 mOsm / kg, less than 700 mOsm / kg, less than 600 mOsm / kg, less than 500 mOsm / kg, less than 400 mOsm / kg, less than 300 mOsm / kg, or less than 200 mOsm / kg of salt. In some embodiments, the assembly buffer is not a balanced salt solution. In some embodiments, the assembly buffer comprises more than 200 mOsm / kg of salt.

[0294] In some embodiments, the assembly buffer comprises glycerol. One example of an assembly buffer is 75 mM NaCl, 50 mM Tris pH 8.0, 10% glycerol. An additional example of an assembly buffer is phosphate buffered saline (PBS).

[0295] In some embodiments, capsid assembly is performed at a temperature of at least -20°C, at least -10°C, at least 0°C, at least 2°C, at least 4°C, at least 6°C, at least 8°C, at least 10°C, at least 12°C, at least 14°C, at least 16°C, at least 18°C, at least 20°C, at least 22°C, at least 25°C, at least 30°C, or at least 37°C. In some embodiments, capsid assembly is performed at a temperature of up to 2°C, up to 4°C, up to 6°C, up to 8°C, up to 10°C, up to 12°C, up to 14°C, up to 16°C, up to 18°C, up to 20°C, up to 22°C, up to 25°C, up to 30°C, up to 37°C, up to 40°C, or up to 45°C. In some embodiments, capsid assembly is performed at a temperature of about -20°C, about -10°C, about 0°C, about 2°C, about 4°C, about 6°C, about 8°C, about 10°C, about 12°C, about 14°C, about 16°C, about 18°C, about 20°C, about 22°C, about 25°C, about 30°C, or about 37°C. In some embodiments, capsid assembly is performed at a temperature of about 2°C to about 40°C, 2°C to about 37°C, 2°C to about 25°C, 2°C to about 20°C, 4°C to about 40°C, 4°C to about 37°C, 4°C to about 25°C, 4°C to about 20°C, 2°C to about 8°C, about 2°C to about 4°C, about 20°C to about 37°C, about 25°C to about 37°C, about 20°C to about 30°C, about 25°C to about 30°C, or about 30°C to about 37°C.

[0296] In some embodiments, capsid assembly comprises incubating in the assembly buffer for at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 16 hours, at least about 20 hours, at least about 24 hours, or more.

[0297] In some embodiments, the assembly buffer comprises a pH of about 3, about 4, about 5, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9, about 10, or about 11. In some embodiments, the assembly buffer comprises a pH of at least about 3, at least about 4, at least about 5, at least about 6, at least about 6.1, at least about 6.2, at least about 6.3, at least about 6.4, at least about 6.5, at least about 6.6, at least about 6.7, at least about 6.8, at least about 6.9, at least about 7, at least about 7.1, at least about 7.2, at least about 7.3, at least about 7.4, at least about 7.5, at least about 7.6, at least about 7.7, at least about 7.8, at least about 7.9, or at least about 8. In some embodiments, the assembly buffer comprises a pH of up to about 7, up to about 7.5, up to about 7.6, up to about 7.7, up to about 7.8, up to about 7.9, up to about 8, up to about 8.1, up to about 8.2, up to about 8.3, up to about 8.4, up to about 8.5, up to about 8.6, up to about 8.7, up to about 8.8, up to about 8.9, up to about 9, up to about 10, or up to about 11. In some embodiments, the assembly buffer comprises a pH of about 5-9, 5-8, 5-7.8, 5-7.6, 5-7.5, 5-7.4, 6-8, 6-7.8, 6-7.6, 6-7.5, 6-7.4, 7-8, 7-7.8, 7-7.6, or 7-7.5.

[0298] The disassembly buffer may include one or more reducing agents, such as reduced glutathione (GSH), beta-mercaptoethanol (β-ME), dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or combinations thereof. The disassembly buffer may include one or more solubilizing agents, detergents, such as non-denaturing detergents, such as CHAPS, nP-40, n-OG, DDM, Triton X-100, LDAO, Tween 20, or combinations thereof. The disassembly buffer may include one or more non-detergent sulfobetaines, such as NDSB-195, NDSB-201, NDSB-211, NDSB-221, NDSB-256, NDSB-256-4T, or combinations thereof. The disassembly buffer may include one or more chaotropic agents. The disassembly buffer can include n-butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, urea, MgCl2, CaCl2, NaI, or combinations thereof.

[0299] The disassembly buffer can include Tris. The disassembly buffer can include NaCl. The disassembly buffer can include NaP. The disassembly buffer can include glycerol. In some embodiments, the disassembly buffer includes MgCl2. The disassembly buffer can include urea. The disassembly buffer can include DTT. The disassembly buffer can include urea and DTT. A specific example of a disassembly buffer is 10% CHAPS, 20 mM TCEP, 100 mM Tris pH 8.0. A further example of a disassembly buffer is 10 mM TCEP, 10 mM MgCl2, 50 mM Tris pH 8.0. A further example of a disassembly buffer is a buffer containing 12 mM GSH. Additional examples of disassembly buffers include: (i) 5.33 M urea, 66.67 mM DTT, 500 mM NaCl, 20 mM NaP pH 7.4, 10% glycerol; (ii) 4 M urea, 50 mM DTT, 500 mM NaCl, 20 mM NaP pH 7.4, 10% glycerol; (iii) 4-6 M urea, 30-50 mM DTT, 500 mM NaCl, 10% glycerol.

[0300] The disassembly buffer can include components disclosed herein at a concentration of at least 1 pm, at least 10 pM, at least 100 pM, at least 1 nM, at least 10 nM, at least 100 nM, at least 1 μM, at least 10 μM, at least 100 μM, at least 1 mM, at least 10 mM, at least 100 mM, at least 1 M, or at least 5 M. The disassembly buffer can include components disclosed herein at a concentration of up to 1 pm, up to 10 pM, up to 100 pM, up to 1 nM, up to 10 nM, up to 100 nM, up to 1 μM, up to 10 μM, up to 100 μM, up to 1 mM, up to 10 mM, up to 100 mM, up to 1 M, or up to 5 M.

[0301] In some embodiments, capsid disassembly occurs at a temperature of at least -20°C, at least -10°C, at least 0°C, at least 2°C, at least 4°C, at least 6°C, at least 8°C, at least 10°C, at least 12°C, at least 14°C, at least 16°C, at least 18°C, at least 20°C, at least 22°C, at least 25°C, at least 30°C, or at least 37°C. In some embodiments, capsid disassembly occurs at a temperature of up to 2°C, up to 4°C, up to 6°C, up to 8°C, up to 10°C, up to 12°C, up to 14°C, up to 16°C, up to 18°C, up to 20°C, up to 22°C, up to 25°C, up to 30°C, up to 37°C, up to 40°C, or up to 45°C. In some embodiments, capsid disassembly is performed at a temperature of about -20°C, about -10°C, about 0°C, about 2°C, about 4°C, about 6°C, about 8°C, about 10°C, about 12°C, about 14°C, about 16°C, about 18°C, about 20°C, about 22°C, about 25°C, about 30°C, or about 37°C. In some embodiments, capsid disassembly is performed at a temperature of about 2°C to about 40°C, 2°C to about 37°C, 2°C to about 25°C, 2°C to about 20°C, 4°C to about 40°C, 4°C to about 37°C, 4°C to about 25°C, 4°C to about 20°C, 2°C to about 8°C, about 2°C to about 4°C, about 20°C to about 37°C, about 25°C to about 37°C, about 20°C to about 30°C, about 25°C to about 30°C, or about 30°C to about 37°C.

[0302] In some embodiments, capsid disassembly comprises incubating in the disassembly buffer for at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 16 hours, at least about 20 hours, at least about 24 hours, or more.

[0303] In some embodiments, the disassembly buffer comprises a pH of about 3, about 4, about 5, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9, about 10, or about 11. In some embodiments, the disassembly buffer comprises a pH of at least about 3, at least about 4, at least about 5, at least about 6, at least about 6.1, at least about 6.2, at least about 6.3, at least about 6.4, at least about 6.5, at least about 6.6, at least about 6.7, at least about 6.8, at least about 6.9, at least about 7, at least about 7.1, at least about 7.2, at least about 7.3, at least about 7.4, at least about 7.5, at least about 7.6, at least about 7.7, at least about 7.8, at least about 7.9, or at least about 8. In some embodiments, the disassembly buffer comprises a pH of up to about 7, up to about 7.5, up to about 7.6, up to about 7.7, up to about 7.8, up to about 7.9, up to about 8, up to about 8.1, up to about 8.2, up to about 8.3, up to about 8.4, up to about 8.5, up to about 8.6, up to about 8.7, up to about 8.8, up to about 8.9, up to about 9, up to about 10, or up to about 11. In some embodiments, the disassembly buffer comprises a pH of about 5-9, about 5-8, about 5-7.8, about 5-7.6, about 5-7.5, about 5-7.4, about 6-8, about 6-7.8, about 6-7.6, about 6-7.5, about 6-7.4, about 7-8, about 7-7.8, about 7-7.6, or about 7-7.5.

[0304] In some embodiments, the reassembly buffer comprises one or more monovalent salts, such as NaCl, KCl, LiCl, NaBr, NaF, or combinations thereof. In some embodiments, the reassembly buffer comprises one or more osmolytes, such as glycerol, ethylene glycol, mannitol, glycine, glycine betaine, trehalose, sorbitol, or combinations thereof.

[0305] In some embodiments, the reassembly buffer is free of or substantially devoid of chaotropic agents, hi some embodiments, the reassembly buffer is free of or substantially devoid of reducing agents.

[0306] The reassembly buffer can include components disclosed herein at a concentration of at least 1 pm, at least 10 pM, at least 100 pM, at least 1 nM, at least 10 nM, at least 100 nM, at least 1 μM, at least 10 μM, at least 100 μM, at least 1 mM, at least 10 mM, at least 100 mM, at least 1 M, or at least 5 M. The reassembly buffer can include components disclosed herein at a concentration of up to 1 pm, up to 10 pM, up to 100 pM, up to 1 nM, up to 10 nM, up to 100 nM, up to 1 μM, up to 10 μM, up to 100 μM, up to 1 mM, up to 10 mM, up to 100 mM, up to 1 M, or up to 5 M.

[0307] In some embodiments, the reassembly buffer comprises a physiological buffer. In some embodiments, the reassembly buffer comprises a balanced salt solution. In some embodiments, the reassembly buffer is or comprises a saline solution, such as buffered saline. In some embodiments, the reassembly buffer is or comprises PBS. In some embodiments, the reassembly buffer comprises sodium phosphate. In some embodiments, the reassembly buffer is or comprises HBSS. In some embodiments, the reassembly buffer comprises or is Ringer's solution.

[0308] In some embodiments, the PNMA2 or endo-Gag polypeptides of the disclosure exhibit favorable reassembly properties in buffers containing low salt levels or physiological levels of salt.

[0309] In some embodiments, the reassembly buffer comprises less than 2000 mOsm / kg, less than 1000 mOsm / kg, less than 900 mOsm / kg, less than 800 mOsm / kg, less than 700 mOsm / kg, less than 600 mOsm / kg, less than 500 mOsm / kg, less than 400 mOsm / kg, less than 300 mOsm / kg, or less than 200 mOsm / kg salt. In some embodiments, the reassembly buffer is not a balanced salt solution.

[0310] In some embodiments, the reassembly buffer comprises high salt. In some embodiments, the reassembly buffer comprises greater than 200 mOsm / kg of salt. In some embodiments, the reassembly buffer comprises at least 8000, at least 7000, at least 6000, at least 5000, at least 4000, at least 3000, at least 2000, at least 1000, at least 900, at least 800, at least 700, at least 600, at least 500, at least 400, at least 300, or at least 200 mOsm / kg of salt.

[0311] In some embodiments, the reassembly buffer comprises glycerol. One example of a reassembly buffer is 75 mM NaCl, 50 mM Tris pH 8.0, 10% glycerol. An additional example of a reassembly buffer is PBS. A further example of a reassembly buffer is 20 mM Na2HPO4 / NaH2PO4, pH 7.4, 0.5 M NaCl, 10% glycerol, 40 mM imidazole, 1 mM DTT.

[0312] In some embodiments, capsid reassembly is performed at a temperature of at least -20°C, at least -10°C, at least 0°C, at least 2°C, at least 4°C, at least 6°C, at least 8°C, at least 10°C, at least 12°C, at least 14°C, at least 16°C, at least 18°C, at least 20°C, at least 22°C, at least 25°C, at least 30°C, or at least 37°C. In some embodiments, capsid reassembly is performed at a temperature of up to 2°C, up to 4°C, up to 6°C, up to 8°C, up to 10°C, up to 12°C, up to 14°C, up to 16°C, up to 18°C, up to 20°C, up to 22°C, up to 25°C, up to 30°C, up to 37°C, up to 40°C, or up to 45°C. In some embodiments, capsid reassembly is performed at a temperature of about -20°C, about -10°C, about 0°C, about 2°C, about 4°C, about 6°C, about 8°C, about 10°C, about 12°C, about 14°C, about 16°C, about 18°C, about 20°C, about 22°C, about 25°C, about 30°C, or about 37°C. In some embodiments, capsid reassembly is performed at a temperature of about 2°C to about 40°C, 2°C to about 37°C, 2°C to about 25°C, 2°C to about 20°C, 4°C to about 40°C, 4°C to about 37°C, 4°C to about 25°C, 4°C to about 20°C, 2°C to about 8°C, about 2°C to about 4°C, about 20°C to about 37°C, about 25°C to about 37°C, about 20°C to about 30°C, about 25°C to about 30°C, or about 30°C to about 37°C.

[0313] In some embodiments, the capsid reassembly comprises incubating in a reassembly buffer for at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 16 hours, at least about 20 hours, at least about 24 hours, or more.

[0314] In some embodiments, the reassembly buffer comprises a pH of about 3, about 4, about 5, about 6, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9, about 10, or about 11. In some embodiments, the reassembly buffer comprises a pH of at least about 3, at least about 4, at least about 5, at least about 6, at least about 6.1, at least about 6.2, at least about 6.3, at least about 6.4, at least about 6.5, at least about 6.6, at least about 6.7, at least about 6.8, at least about 6.9, at least about 7, at least about 7.1, at least about 7.2, at least about 7.3, at least about 7.4, at least about 7.5, at least about 7.6, at least about 7.7, at least about 7.8, at least about 7.9, or at least about 8. In some embodiments, the reassembly buffer comprises a pH of up to about 7, up to about 7.5, up to about 7.6, up to about 7.7, up to about 7.8, up to about 7.9, up to about 8, up to about 8.1, up to about 8.2, up to about 8.3, up to about 8.4, up to about 8.5, up to about 8.6, up to about 8.7, up to about 8.8, up to about 8.9, up to about 9, up to about 10, or up to about 11. In some embodiments, the reassembly buffer comprises a pH of about 5-9, 5-8, 5-7.8, 5-7.6, 5-7.5, 5-7.4, 6-8, 6-7.8, 6-7.6, 6-7.5, 6-7.4, 7-8, 7-7.8, 7-7.6, or 7-7.5.

[0315] In some embodiments, the method comprises incubating multiple PNMA2 or endo-Gag polypeptides, engineered PNMA2 or endo-Gag polypeptides, and / or recombinant PNMA2 or endo-Gag polypeptides with a heterologous cargo in solution for a period of time sufficient to generate loaded PNMA2-based capsids or endo-Gag-based capsids.

[0316] In some cases, the time sufficient to generate loaded PNMA2-based capsids or endo-Gag-based capsids is at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 16 hours, at least about 20 hours, at least about 24 hours, or more. In some embodiments, the time sufficient to generate loaded PNMA2-based capsids or endo-Gag-based capsids is up to about 5 minutes, up to about 10 minutes, up to about 20 minutes, up to about 30 minutes, up to about 1 hour, up to about 2 hours, up to about 4 hours, up to about 6 hours, up to about 8 hours, up to about 10 hours, up to about 12 hours, up to about 16 hours, up to about 20 hours, up to about 24 hours, or up to about 48 hours.

[0317] In some cases, the PNMA2-based capsid or endo-Gag-based capsid assembles, disassembles, or reassembles at a temperature between about 2° C. and about 37° C. In some cases, the PNMA2-based capsid or endo-Gag-based capsid is prepared at a temperature between about 2° C. and about 8° C., between about 2° C. and about 4° C., between about 20° C. and about 37° C., between about 25° C. and about 37° C., between about 20° C. and about 30° C., between about 25° C. and about 30° C., between about 30° C. and about 37° C., between about 2° C. and about 40° C., between 2° C. and about 25° C., between 2° C. and about 20° C., between 4° C. and about 40° C., between 4° C. and about 37° C., between 4° C. and about 25° C., or between 4° C. and about 20° C. In some cases, the PNMA2-based capsid or endo-Gag-based capsid assembles, disassembles, or reassembles at room temperature.

[0318] In some cases, the method includes mixing a solution containing the plurality of engineered and / or recombinant PNMA2 polypeptides with a plurality of non-PNMA2 encapsidation subunits prior to incubation with the cargo. In some cases, the plurality of non-PNMA2 encapsidation subunits are mixed with the plurality of engineered and / or recombinant PNMA2 polypeptides in a ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In other cases, the plurality of non-PNMA2 encapsidation subunits are mixed with the plurality of engineered and / or recombinant PNMA2 polypeptides in a ratio of 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0319] In some cases, the PNMA2-based capsid or endo-Gag-based capsid is further formulated for systemic administration. In some cases, the PNMA2-based capsid or endo-Gag-based capsid is further formulated for local administration. In some cases, the PNMA2-based capsid or endo-Gag-based capsid is further formulated for parenteral (e.g., intra-arterial, intra-articular, intradermal, intralesional, intramuscular, intraocular, intraosseous injection, intraperitoneal, intrathecal, intravenous, intravitreal, or subcutaneous) administration. In some cases, the PNMA2-based capsid or endo-Gag-based capsid is further formulated for local administration. In some cases, the PNMA2-based capsid or endo-Gag-based capsid is further formulated for oral administration. In some cases, the PNMA2-based capsid or endo-Gag-based capsid is further formulated for sublingual administration. In some cases, the PNMA2-based capsid or the endo-Gag-based capsid is further formulated for administration by aerosol.

[0320] Also described herein, in certain embodiments, is the use of PNMA2-based capsids or endo-Gag-based capsids for delivery of cargo to a site of interest.

[0321] In some cases, the methods of the disclosure include contacting a cell with a PNMA2-based capsid or an endo-Gag-based capsid. The contacting can be in vitro, ex vivo, or in vivo. In some embodiments, the contacting is performed in vivo at a site of interest.

[0322] In some cases, the method includes contacting a cell with a PNMA2-based capsid or an endo-Gag-based capsid at a site of interest for a time sufficient to promote cellular uptake of the capsid. The time sufficient to promote cellular uptake of the capsid can be at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, at least about 12 hours, at least about 16 hours, at least about 20 hours, at least about 24 hours, or more. In some embodiments, the time sufficient to promote cellular uptake of the capsid is up to about 5 minutes, up to about 10 minutes, up to about 20 minutes, up to about 30 minutes, up to about 1 hour, up to about 2 hours, up to about 4 hours, up to about 6 hours, up to about 8 hours, up to about 10 hours, up to about 12 hours, up to about 16 hours, up to about 20 hours, up to about 24 hours, or up to about 48 hours.

[0323] In some embodiments, the method includes contacting the cell with the PNMA2-based capsid or endo-Gag-based capsid at a concentration sufficient to induce a desired effect on the cell, which may be at least about 0.001 pg / mL, at least about 0.01 pg / mL, at least about 0.1 pg / mL, at least about 1 pg / mL, at least about 10 pg / mL, at least about 100 pg / mL, at least about 1 ng / mL, at least about 10 ng / mL, at least about 100 ng / mL, at least about 1 μg / mL, at least about 10 μg / mL, at least about 100 μg / mL, at least about 1 mg / mL, at least about 10 mg / mL, or at least about 100 mg / mL.

[0324] In some cases, the cell is a muscle cell, a skin cell, a blood cell, or an immune cell (eg, a T cell or a B cell).

[0325] In some cases, the cell is a tumor cell, such as a solid tumor cell or a cell derived from a hematological malignancy. In some cases, the solid tumor cell is a cell derived from bladder cancer, breast cancer, brain cancer, colon cancer, kidney cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, or thyroid cancer. In some cases, the cell derived from a hematological malignancy is a B-cell malignancy or a T-cell malignancy. In some cases, the cell is derived from leukemia, lymphoma, myeloma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, mantle cell lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, peripheral T-cell lymphoma, multiple myeloma, plasmacytoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), or chronic myeloid leukemia (CML).

[0326] In some embodiments, the cell is a somatic cell. In some cases, the cell is a blood cell, a skin cell, a connective tissue cell, a bone cell, a muscle cell, or a cell from an organ.

[0327] In some embodiments, the cell is an epithelial cell, a connective tissue cell, a muscle cell, or a neuron.

[0328] In some cases, the cell is an endodermal cell, a mesodermal cell, or an ectodermal cell. In some cases, the endoderm includes cells of the respiratory system, the intestine, the liver, the gallbladder, the pancreas, the islets of Langerhans, the thyroid gland, or the hindgut. In some cases, the mesoderm includes osteochondroma progenitor cells, muscle cells, cells of the digestive system, renal stem cells, cells of the reproductive system, cells of the circulatory system (such as endothelial cells). Examples of cells from the ectoderm include epithelial cells, cells of the anterior pituitary gland, cells of the peripheral nervous system, cells of the neuroendocrine system, cells of the eye, cells of the central nervous system, cells of the ependyma, or cells of the pineal gland. In some cases, cells derived from the central and peripheral nervous systems include neurons, Schwann cells, satellite glial cells, oligodendrocytes, or astrocytes. In some cases, the neuron further comprises an interneuron, a pyramidal neuron, a GABAergic neuron, a dopaminergic neuron, a serotonergic neuron, a glutamatergic neuron, a motor neuron from the spinal cord, or an inhibitory spinal neuron.

[0329] In some embodiments, the cells are stem or progenitor cells. In some cases, the cells are mesenchymal stem or progenitor cells. In other cases, the cells are hematopoietic stem or progenitor cells.

[0330] In some cases, the target protein is overexpressed or depleted in the cell. In some cases, the target protein is overexpressed in the cell. In further cases, the target protein is depleted in the cell.

[0331] In some cases, the target gene in the cell has one or more mutations. In some cases, the cell has an impaired splicing machinery.

[0332] In some cases, the PNMA2-based capsid is administered systemically to a subject in need thereof. In other cases, the PNMA2-based capsid or endo-Gag-based capsid is administered locally to a subject in need thereof. In some embodiments, the PNMA2-based capsid or endo-Gag-based capsid is administered parenterally, orally, topically, sublingually, or by aerosol to a subject in need thereof. In some cases, the PNMA2-based capsid or endo-Gag-based capsid is administered parenterally to a subject in need thereof. In other cases, the PNMA2-based capsid or endo-Gag-based capsid is administered orally to a subject in need thereof. In further cases, the PNMA2-based capsid or endo-Gag-based capsid is administered locally, sublingually, or by aerosol to a subject in need thereof.

[0333] In some embodiments, the delivery component is combined with the PNMA2-based capsid or the endo-Gag-based capsid for targeted delivery to a site of interest. In some cases, the delivery component includes a carrier, e.g., an extracellular vesicle such as a micelle, liposome, or microvesicle; or a viral envelope.

[0334] In some cases, the delivery moiety functions as a primary delivery vehicle for a PNMA2-based capsid or an endo-Gag-based capsid that does not contain its own delivery moiety, in such cases the delivery moiety directs the PNMA2-based capsid or the endo-Gag-based capsid to a desired target site and optionally facilitates intracellular uptake.

[0335] In other cases, the delivery component improves the targeting specificity and / or sensitivity of the PNMA2-based capsid. In such cases, the delivery component improves the specificity and / or affinity of the PNMA2-based capsid or endo-Gag-based capsid for the target site. In some embodiments, the delivery component improves the specificity and / or affinity by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or more. In further cases, the delivery component improves the specificity and / or affinity by about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 200-fold, 500-fold, or more. In further cases, the delivery component increases specificity and / or affinity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more.

[0336] In some embodiments, the capsid has low or reduced off-target effect. Off-target effect can be, for example, the effect that occurs when cargo is delivered to unintended tissue or cell type, or the effect that occurs when cargo binds to unintended interaction partner. In some cases, the off-target effect is less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5%. In some cases, the capsid does not have off-target effect.

[0337] In some cases, the delivery component increases specificity and / or affinity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more. In further cases, the delivery component increases specificity and / or affinity by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500% or more. Additionally, the delivery component optionally reduces off-target effects by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold or more. Furthermore, the delivery component optionally reduces off-target effects by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more.

[0338] In additional cases, the delivery component functions as a first vehicle to transport the PNMA2-based capsid to a general target area (e.g., the tumor microenvironment), and the PNMA2-based or endo-Gag-based capsid includes a second delivery component (e.g., an exogenous polypeptide sequence disclosed herein) that directs the PNMA2-based capsid or endo-Gag-based capsid to a more specific target site and optionally facilitates cellular uptake. In some embodiments, the delivery component (e.g., the delivery component that functions as a first vehicle) minimizes off-target effects by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or more. In some embodiments, the delivery components minimize off-target effects by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 500%, or more.

[0339] In further cases, the delivery component functions as a first vehicle to transport the PNMA2-based capsid to a desired target site, and the PNMA2-based or endo-Gag-based capsid includes a second delivery component, e.g., an exogenous polypeptide sequence disclosed herein, that facilitates intracellular uptake.

[0340] In some embodiments, the delivery component comprises an extracellular vesicle. In some cases, the extracellular vesicle comprises a microvesicle, a liposome, or a micelle. In some cases, the extracellular vesicle has an average diameter of about 10 nm to about 2000 nm, about 10 nm to about 1000 nm, about 10 nm to about 800 nm, about 20 nm to about 600 nm, about 30 nm to about 500 nm, about 50 nm to about 200 nm, or about 80 nm to about 100 nm.

[0341] In some embodiments, the delivery component comprises a microvesicle. Microvesicles, also known as circulating microvesicles or microparticles, are membrane-bound vesicles that contain phospholipids. In some cases, the microvesicles have an average diameter of about 50 nm to about 1000 nm, about 100 nm to about 800 nm, about 200 nm to about 500 nm, or about 50 nm to about 400 nm.

[0342] In some cases, microvesicles are derived from cell membrane inversion, exocytosis, shedding, blebbing, or budding. In some cases, microvesicles are produced from differentiated cells. In other cases, microvesicles are produced from undifferentiated cells, such as by blast cells, progenitor cells, or stem cells.

[0343] In some embodiments, the delivery component comprises a liposome. In some cases, the liposome comprises multiple lipopeptides displayed on the surface of the liposome for targeted delivery to a site or region of interest. In some cases, the liposome fuses with a target cell, thereby discharging the contents of the liposome into the target cell. In some cases, the liposome is endocytosed by phagocytic cells. After endocytosis, liposomal lipids are degraded in the lysosome, releasing the encapsulated agent.

[0344] Exemplary liposomes suitable for incorporation include, but are not limited to, multilamellar vesicles (MLV), oligolamellar vesicles (OLV), unilamellar vesicles (UV), small unilamellar vesicles (SUV), medium unilamellar vesicles (MUV), large unilamellar vesicles (LUV), giant unilamellar vesicles (GUV), multilamellar vesicles (MVV), single or oligolamellar vesicles produced by reverse phase evaporation (REV), multilamellar vesicles produced by reverse phase evaporation (MLV-REV), stable multilamellar vesicles (SPLV), freeze-thawed MLV (FATMLV), vesicles prepared by extrusion (VET), vesicles prepared by French press (FPV), vesicles prepared by fusion (FUV), dehydrated rehydrated vesicles (DRV), and bubblesomes (BSV). In some cases, the liposome comprises amphipol (A8-35). Techniques for preparing liposomes are described, for example, in COLLOIDAL DRUG DELIVERY SYSTEMS, vol. 66 (J. Kreuter ed., Marcel Dekker, Inc. (1994)), which is incorporated herein by reference for such disclosure.

[0345] Depending on the method of preparation, liposomes can be unilamellar or multilamellar and vary in size, ranging from about 20 nm to over about 1000 nm in diameter.

[0346] In some cases, liposome provided herein also comprises carrier lipid.In some embodiments, carrier lipid is phospholipid.Carrier lipid that can form liposome includes, but is not limited to, dipalmitoyl phosphatidylcholine (DPPC), phosphatidylcholine (PC; lecithin), phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE) or phosphatidylserine (PS). Other suitable phospholipids further include distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylglycerol (DSPG), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidic acid (DPPA), dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dipalmitoylphosphatidylserine (DPPS), dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), and the like, or combinations thereof. In some embodiments, the liposome further comprises a sterol (e.g., cholesterol) that regulates liposome formation. The carrier lipid is optionally any non-phosphorylated polar lipid. In some embodiments, the liposome comprises an electrically neutral lipid.

[0347] In some embodiments, the liposome or delivery component comprises cationic lipid. Cationic lipid has a head group with a permanent positive charge. Non-limiting examples of cationic lipid include 1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium bromide (DDAB), and 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), as well as commercially available transfection reagents.

[0348] In some embodiments, the delivery component comprises a micelle, in some cases, the micelle has an average diameter of about 2 nm to about 250 nm, about 20 nm to about 200 nm, about 20 nm to about 100 nm, or about 50 to about 100 nm.

[0349] In some cases, the micelles are polymeric micelles characterized by a core-shell structure in which a hydrophobic core is surrounded by a hydrophilic shell. In some cases, the hydrophilic shell further comprises a hydrophilic polymer or copolymer and a pH-sensitive component.

[0350] Exemplary hydrophilic polymers or copolymers include, but are not limited to, poly(N-substituted acrylamides), poly(N-acryloylpyrrolidine), poly(N-acryloylpiperidine), poly(N-acryl-L-amino acid amides), poly(ethyloxazoline), methyl cellulose, hydroxypropyl acrylate, hydroxyalkyl cellulose derivatives and poly(vinyl alcohol), poly(N-isopropylacrylamide), poly(N-vinyl-2-pyrrolidone), polyethylene glycol derivatives, and combinations thereof.

[0351] The delivery component may include a pH-sensitive moiety, which may include, but is not limited to, alkyl acrylates, such as methacrylate, ethyl acrylate, propyl acrylate, and butyl acrylate, or amino acids, such as glutamic acid.

[0352] In some cases, the hydrophobic moiety constitutes the core of the micelle and includes, for example, a single alkyl chain, such as octadecyl acrylate, or a double-chain alkyl compound, such as phosphatidylethanolamine or dioctadecylamine. In some cases, the hydrophobic moiety is optionally a water-insoluble polymer, such as poly(lactic acid) or poly(e-caprolactone).

[0353] Polymeric micelles that exhibit pH-sensitive properties are also contemplated, for example, formed by using pH-sensitive polymers such as, but not limited to, copolymers from methacrylic acid, methacrylic acid esters and acrylic acid esters, polyvinyl acetate phthalate, hydroxypropylmethylcellulose phthalate, cellulose acetate phthalate, or cellulose acetate trimellitate.

[0354] The delivery component can include a cationic moiety, such as a cationic lipid, a cationic peptide, or a cationic polymer. The cationic moiety (e.g., lipid, peptide, or moiety) can associate with the capsid (e.g., its negative spike portion) through electrostatic interactions. The presence of cationic lipid, polymer, or peptide on the capsid can enhance binding to negatively charged cell surface, thereby facilitating increased uptake of the capsid.

[0355] In some embodiments, the delivery component comprises a cationic peptide, such as a cationic cell-penetrating peptide disclosed herein.

[0356] In some embodiments, the delivery component comprises a cationic polymer. Non-limiting examples of cationic polymers include cationic peptides and their derivatives (e.g., polylysine, polyornithine), linear or branched synthetic polymers (e.g., polybrene, polyethyleneimine), polysaccharide-based delivery molecules (e.g., cyclodextrin, chitosan), natural polymers (e.g., histones, collagen), and activated and non-activated dendrimers. Cationic reagents can attach to cell membranes through electrostatic interactions and proceed to uptake into cells via endocytosis.

[0357] In some embodiments, the delivery component comprises a viral envelope or a component thereof. For example, the viral envelope comprises glycoproteins, phospholipids, and additional proteins obtained from the host, any of which may be included in the delivery component. In some cases, the viral envelope or a component thereof is permissive to a wide range of target cells. In other cases, the viral envelope or a component thereof is not permissive and is specific to the target cell of interest. In some cases, the viral envelope or a component thereof comprises a cell-specific binding protein, and optionally a fusion molecule that helps fuse the cargo to the target cell. In some cases, the viral envelope or a component thereof comprises an endogenous viral envelope. In other cases, the viral envelope is a modified envelope that comprises one or more foreign proteins.

[0358] In some cases, viral envelope or its components are derived from DNA virus.Examples of enveloped DNA virus include Herpesviridae, Poxviridae and Hepadnaviridae.In other cases, viral envelope or its components are derived from RNA virus.Exemplary enveloped RNA virus include Bunyaviridae, Coronaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae and Togaviridae.In further cases, viral envelope or its components are derived from Retroviridae.

[0359] In some embodiments, the viral envelope or a component thereof is derived from an oncolytic virus, such as an oncolytic DNA virus from the family of Herpesviridae (e.g., HSV1) or Poxviridae (e.g., Vaccinia virus and Myxoma virus), or an oncolytic RNA virus from the family of Rhabdoviridae (e.g., VSV) or Paramyxoviridae (e.g., MV and NDV).

[0360] In some cases, the delivery component, viral envelope, or components thereof further comprise a foreign or engineered protein that binds to an antigen or cell surface molecule. Exemplary antigens and cell surface molecules for targeting include, but are not limited to, P-glycoprotein, Her2 / Neu, erythropoietin (EPO), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGF-R), cadherin, carcinoembryonic antigen (CEA), CD4, CD8, CD19, CD20, CD33, CD34, CD45, CD117 (c-kit), CD133, HLA-A, HLA-B, HLA-C, chemokine receptor 5 (CCRS), stem cells. Cell proliferation-associated human nuclear antigen defined by the markers ABCG2 transporter, ovarian cancer antigen CA125, immunoglobulins, integrins, prostate-specific antigen (PSA), prostate stem cell antigen (PSCA), dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), thyroglobulin, granulocyte-macrophage colony-stimulating factor (GM-CSF), myogenic differentiation factor 1 (MyoD-1), Leu-7 (CD57), LeuM-1, monoclonal antibody Ki-67 (Ki-67), viral envelope protein, HIV gp120, or transferrin receptor.

[0361] In some embodiments, the PNMA2-based capsid or the endo-Gag-based capsid is for in vitro use.

[0362] In some cases, the PNMA2-based capsid or the endo-Gag-based capsid is for ex vivo use.

[0363] In some cases, the PNMA2-based capsid or the endo-Gag-based capsid is for use in vivo.

[0364] Kits and Products In certain embodiments, kits and products are disclosed herein for use in one or more of the methods described herein. Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers, such as vials, tubes, and each container(s) includes one of the separate elements used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the container is formed from a variety of materials, such as glass or plastic.

[0365] For example, the container(s) can contain a recombinant or engineered PNMA2 or endo-Gag polypeptide as described above. Such kits optionally include an identifying description or label or instructions for its use in the methods described herein. For example, kits typically include a label describing the contents and / or instructions for use, and a package insert with instructions for use. A set of instructions is also typically included.

[0366] Specific Terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. It is to be understood that the detailed description is merely exemplary and explanatory, and does not limit any of the claimed subject matter. In this application, the use of the singular includes the plural unless expressly stated otherwise. It should be noted that as used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless otherwise stated. Furthermore, the use of the term "including" as well as other forms such as "include", "includes" and "included" are not limiting.

[0367] Conversely, various features of the invention that may be described for brevity in the context of a single embodiment may also be provided separately or in any suitable combination. Conversely, although the invention may be described for clarity herein in the context of separate embodiments, the invention may also be practiced in a single embodiment.

[0368] References herein to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments, but not necessarily in all embodiments.

[0369] As used herein, ranges and amounts may be expressed as "about" a particular value or range. "About" includes the exact amount. Thus, "about 5 μL" also means "about 5 μL" and "5 μL." In general, the term "about" includes amounts that are expected to be within experimental error.

[0370] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0371] As used herein, the terms "individual(s)," "subject(s)," and "patient(s)" refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human. None of the terms require, and are not limited to, situations characterized by the supervision (e.g., continuous or intermittent) of a medical professional (e.g., a physician, registered nurse, nurse practitioner, physician assistant, healthcare professional, or hospice professional).

[0372] Embodiment Embodiment 1. A capsid comprising a recombinant PNMA2 polypeptide and a heterologous cargo.

[0373] Embodiment 2. The capsid of embodiment 1, wherein the heterologous cargo is a nucleic acid.

[0374] Embodiment 3. The capsid of embodiment 1 or 2, wherein the heterologous cargo is DNA.

[0375] Embodiment 4. The capsid of embodiment 1 or 2, wherein the heterologous cargo is RNA.

[0376] Embodiment 5. The capsid of any one of embodiments 1 to 4, wherein the heterologous cargo is a therapeutic agent.

[0377] Embodiment 6. The capsid of embodiment 1, wherein the heterologous cargo is a protein, peptide, antibody or binding fragment thereof, peptidomimetic, nucleotide mimetic, drug, diagnostic tool, imaging tool, or small molecule.

[0378] Embodiment 7. The capsid of any one of embodiments 1 to 6, wherein the recombinant PNMA2 polypeptide is a mammalian PNMA2.

[0379] Embodiment 8. A capsid according to any one of embodiments 1 to 6, wherein the recombinant PNMA2 polypeptide is human PNMA2.

[0380] Embodiment 9. A capsid described in any one of embodiments 1 to 8, wherein the recombinant PNMA2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:1.

[0381] Embodiment 10. The capsid of any one of embodiments 1 to 8, wherein the PNMA2 polypeptide comprises an amino acid sequence that comprises at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:1.

[0382] Embodiment 11. The capsid of any one of embodiments 1 to 8, wherein the PNMA2 polypeptide comprises an amino acid sequence that is SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:1.

[0383] Embodiment 12. A capsid described in any one of embodiments 1 to 10, wherein the recombinant PNMA2 polypeptide comprises a sequence modification relative to SEQ ID NO:1.

[0384] Embodiment 13. A capsid according to any one of embodiments 1 to 12, wherein the capsid comprises a disulfide bond.

[0385] Embodiment 14. A capsid according to any one of embodiments 1 to 12, comprising a disulfide bond involving a cysteine ​​at position 10, 136, 233, or 310 of the recombinant PNMA2 polypeptide relative to SEQ ID NO:1.

[0386] Embodiment 15. A capsid according to any one of embodiments 1 to 12, comprising a disulfide bond involving a cysteine ​​at position 136 of the recombinant PNMA2 polypeptide relative to SEQ ID NO:1.

[0387] Embodiment 16. A capsid according to any one of embodiments 1 to 12, comprising a disulfide bond involving a cysteine ​​at position 310 of the recombinant PNMA2 polypeptide relative to SEQ ID NO:1.

[0388] Embodiment 17. A composition comprising a plurality of capsids of any one of embodiments 1-16, wherein the capsids are at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% pure as measured by SDS-PAGE.

[0389] Embodiment 18. A composition comprising a plurality of capsids according to any one of embodiments 1 to 16, wherein the capsids comprise a particle uniformity of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% as measured by multi-angle dynamic light scattering.

[0390] Embodiment 19. An engineered PNMA2 polypeptide comprising sequence modifications relative to SEQ ID NO:1.

[0391] Embodiment 20 The engineered PNMA2 polypeptide of embodiment 19, wherein the sequence modification comprises an amino acid deletion.

[0392] Embodiment 21 The engineered PNMA2 polypeptide of embodiment 19 or 20, wherein the sequence modification comprises an amino acid insertion.

[0393] Embodiment 22. The engineered PNMA2 polypeptide of any one of embodiments 19 to 21, wherein the sequence modification comprises an amino acid substitution.

[0394] Embodiment 23. The engineered PNMA2 polypeptide of any one of embodiments 19 to 22, wherein the sequence modification comprises a cargo binding domain.

[0395] Embodiment 24. The engineered PNMA2 polypeptide of any one of embodiments 19 to 23, wherein the sequence modification comprises a nucleic acid binding domain.

[0396] Embodiment 25. The engineered PNMA2 polypeptide of any one of embodiments 19 to 24, wherein the sequence modification comprises a DNA binding domain.

[0397] Embodiment 26 The engineered PNMA2 polypeptide of any one of embodiments 19 to 25, wherein the sequence modification comprises an RNA binding domain.

[0398] Embodiment 27. The engineered PNMA2 polypeptide of any one of embodiments 19 to 26, wherein the sequence modification comprises a zinc finger domain.

[0399] Embodiment 28. The engineered PNMA2 polypeptide of any one of embodiments 19 to 27, wherein the sequence modification comprises a subcellular localization signal.

[0400] Embodiment 29. The engineered PNMA2 polypeptide of any one of embodiments 19 to 28, wherein the sequence modification comprises a nuclear localization signal (NLS).

[0401] Embodiment 30. The engineered PNMA2 polypeptide of any one of embodiments 19 to 29, wherein the sequence modification comprises an arginine-rich domain.

[0402] Embodiment 31. The engineered PNMA2 polypeptide of any one of embodiments 19 to 30, wherein the sequence modification is at the N-terminus of SEQ ID NO:1.

[0403] Embodiment 32. The engineered PNMA2 polypeptide of any one of embodiments 19 to 30, wherein the sequence modification is at the C-terminus of SEQ ID NO:1.

[0404] Embodiment 33. The engineered PNMA2 polypeptide of any one of embodiments 19 to 30, wherein the sequence modification is within SEQ ID NO:1.

[0405] Embodiment 34. A nucleic acid encoding a PNMA2 polypeptide according to any one of embodiments 19 to 33.

[0406] Embodiment 35. A vector comprising the nucleic acid of embodiment 34.

[0407] Embodiment 36. A cell comprising the nucleic acid of embodiment 34.

[0408] Embodiment 37. A method for delivering a heterologous cargo to a cell, comprising contacting said cell with a capsid according to any one of embodiments 1 to 18.

[0409] Embodiment 38 The method of embodiment 37, wherein the cell is a eukaryotic cell.

[0410] Embodiment 39 The method of embodiment 37 or 38, wherein the cell is a vertebrate cell.

[0411] Embodiment 40. The method of any one of embodiments 37 to 39, wherein the cell is a mammalian cell.

[0412] Embodiment 41. The method of any one of embodiments 37 to 40, wherein the cell is a human cell.

[0413] Embodiment 42. The method of any one of embodiments 37 to 41, wherein the cells are contacted with the capsid at a concentration of at least about 0.001 pg / mL, at least about 0.01 pg / mL, at least about 0.1 pg / mL, at least about 1 pg / mL, at least about 10 pg / mL, at least about 100 pg / mL, at least about 1 ng / mL, at least about 10 ng / mL, at least about 100 ng / mL, at least about 1 μg / mL, at least about 10 μg / mL, at least about 100 μg / mL, at least about 1 mg / mL, at least about 10 mg / mL, or at least about 100 mg / mL.

[0414] Embodiment 43. The method of any one of embodiments 37 to 42, wherein the contacting is performed in vivo.

[0415] Embodiment 44. The method of any one of embodiments 37 to 43, wherein the contacting is performed in vitro or ex vivo.

[0416] Embodiment 45. The method of any one of embodiments 37 to 44, wherein the heterologous cargo comprises a nucleic acid and the cell expresses a gene encoded by the nucleic acid after delivery.

[0417] Embodiment 46. The method of any one of embodiments 37 to 44, wherein the heterologous cargo is a protein, peptide, antibody or binding fragment thereof, peptidomimetic, nucleotide mimetic, drug, diagnostic tool, imaging tool, or small molecule.

[0418] Embodiment 47. An engineered endogenous retroviral capsid polypeptide comprising a cysteine ​​residue, wherein when isolated, said engineered endogenous retroviral capsid polypeptide is capable of assembling to form a capsid with an efficiency of at least 5%.

[0419] Embodiment 48. The engineered endogenous retroviral capsid polypeptide of embodiment 47, wherein upon isolation, the engineered endogenous retroviral capsid polypeptide is capable of assembling to form a capsid with an efficiency of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

[0420] Embodiment 49. The engineered endogenous retroviral capsid polypeptide of embodiment 47 or 48, which is an endo-Gag polypeptide.

[0421] Embodiment 50. An engineered endogenous retroviral capsid polypeptide according to any one of embodiments 47 to 49, which is an engineered PNMA protein.

[0422] Embodiment 51. The engineered endogenous retroviral capsid polypeptide of any one of embodiments 47 to 50, which is an engineered PNMA2 protein.

[0423] Embodiment 52. The engineered endogenous retroviral capsid polypeptide of any one of embodiments 47-51, wherein the assembling is performed in a buffer containing less than 2000 mOsm / kg, less than 1000 mOsm / kg, less than 900 mOsm / kg, less than 800 mOsm / kg, less than 700 mOsm / kg, less than 600 mOsm / kg, less than 500 mOsm / kg, less than 400 mOsm / kg, less than 300 mOsm / kg, or less than 200 mOsm / kg of salt.

[0424] Embodiment 53. A capsid comprising engineered endogenous retroviral capsid polypeptides that can be disassembled into a non-capsid state with at least 1% efficiency and reassembled into a capsid state with at least 1% efficiency.

[0425] Embodiment 54. The capsid of embodiment 53, which can be disassembled into a non-capsid state with an efficiency of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

[0426] Embodiment 55. The capsid of embodiment 53 or 54, wherein when in the disassembled non-capsid state, the capsid can be reassembled into the capsid state with an efficiency of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

[0427] Embodiment 56. A capsid according to any one of embodiments 53 to 55, wherein the engineered endogenous retroviral capsid polypeptide is an endo-Gag polypeptide.

[0428] Embodiment 57. A capsid according to any one of embodiments 53 to 56, wherein the engineered endogenous retroviral capsid polypeptide is an engineered PNMA protein.

[0429] Embodiment 58. A capsid described in any one of embodiments 53 to 57, wherein the engineered endogenous retroviral capsid polypeptide is an engineered PNMA2 protein.

[0430] Embodiment 59. The capsid of any one of embodiments 53 to 58, wherein the efficiency of reassembly is determined in a buffer containing less than 2000 mOsm / kg, less than 1000 mOsm / kg, less than 900 mOsm / kg, less than 800 mOsm / kg, less than 700 mOsm / kg, less than 600 mOsm / kg, less than 500 mOsm / kg, less than 400 mOsm / kg, less than 300 mOsm / kg, or less than 200 mOsm / kg of salt.

[0431] Embodiment 60. A method for producing a capsid, comprising: (a) expressing a PNMA2 polypeptide in a host cell or a cell-free expression system; and (b) isolating the PNMA2 polypeptide, wherein the isolated PNMA2 polypeptide assembles to form the capsid.

[0432] Embodiment 61 The method of any embodiment 60, wherein at least about 5% of the isolated PNMA2 polypeptides assemble to form the capsid.

[0433] Embodiment 62. The method of embodiment 60 or 61, wherein at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the isolated PNMA2 polypeptide assembles to form the capsid.

[0434] Embodiment 63. The method of any one of embodiments 60 to 62, wherein the percentage of the isolated PNMA2 polypeptide present in capsid form is measured by a multi-angle dynamic light scattering assay.

[0435] Embodiment 64. The method of any one of embodiments 60-63, producing assembled capsids that are at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% pure as measured by SDS-PAGE.

[0436] Embodiment 65. The method of any one of embodiments 60 to 64, producing assembled capsids having a particle uniformity of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% as measured by multi-angle dynamic light scattering.

[0437] Embodiment 66. The method of any one of embodiments 60 to 65, wherein the isolated PNMA2 polypeptide is assembled in a buffer containing less than 2000 mOsm / kg, less than 1000 mOsm / kg, less than 900 mOsm / kg, less than 800 mOsm / kg, less than 700 mOsm / kg, less than 600 mOsm / kg, less than 500 mOsm / kg, less than 400 mOsm / kg, less than 300 mOsm / kg, or less than 200 mOsm / kg of salt.

[0438] Embodiment 67. A method for producing a capsid described in any one of embodiments 1 to 18 or 37 to 59, comprising: (a) expressing a PNMA2 polypeptide in a host cell or a cell-free expression system; (b) isolating the PNMA2 polypeptide; (c) treating the isolated PNMA2 polypeptide with a disassembly buffer, thereby producing a disassembled form of the PNMA2 polypeptide; and (d) incubating the disassembled PNMA2 polypeptide in a reassembly buffer in the presence of the heterologous cargo; thereby producing the capsid comprising the heterologous cargo.

[0439] Embodiment 68. The method of embodiment 67, wherein the isolated PNMA2 polypeptide is present at least partially in capsid form prior to step (c).

[0440] Embodiment 69. The method of any one of embodiments 60 to 68, wherein the PNMA2 polypeptide is a recombinant PNMA2 polypeptide described in any one of the preceding embodiments.

[0441] Embodiment 70. The method of any one of embodiments 60 to 68, wherein the PNMA2 polypeptide is an engineered PNMA2 polypeptide described in any one of the preceding embodiments.

[0442] Embodiment 71. The method of any one of embodiments 67 to 70, wherein at least about 5% of the PNMA2 polypeptide present in capsid form prior to step (c) is disassembled after step (c).

[0443] Embodiment 72. The method of any one of embodiments 67 to 71, wherein at least about 5% of the PNMA2 polypeptide that is present in a disassembled form after step (c) is present in a capsid form after step (d).

[0444] Embodiment 73. The method of any one of embodiments 71 or 72, wherein the percentage of the PNMA2 polypeptide present in a capsid form or in a disassembled state is measured by a multi-angle dynamic light scattering assay.

[0445] Embodiment 74. The method of any one of embodiments 67 to 73, wherein the disassembly buffer comprises a reducing agent.

[0446] Embodiment 75. The method of embodiment 74, wherein the reducing agent is GSH or TCEP.

[0447] Embodiment 76 The method of any one of embodiments 67 to 73, wherein the disassembly buffer comprises a non-denaturing detergent.

[0448] Embodiment 77. The method of embodiment 76, wherein the non-denaturing detergent is CHAPS.

[0449] Embodiment 78. The method of any one of embodiments 67-77, wherein the reassembly buffer comprises less than 2000 mOsm / kg, less than 1000 mOsm / kg, less than 900 mOsm / kg, less than 800 mOsm / kg, less than 700 mOsm / kg, less than 600 mOsm / kg, less than 500 mOsm / kg, less than 400 mOsm / kg, less than 300 mOsm / kg, or less than 200 mOsm / kg of salt.

[0450] Embodiment 79. A method for loading PNMA capsids comprising: a) disassembling PNMA capsids into a composition comprising PNMA monomers; b) contacting a cargo with said composition comprising PNMA monomers; and c) reassembling said composition comprising PNMA monomers into PNMA capsids, thereby loading the cargo into the interior of the PNMA capsids.

[0451] Embodiment 80 The method of embodiment 79, wherein said disassembling comprises reducing disulfide bonds.

[0452] Embodiment 81. The method of any one of embodiments 67 to 80, wherein the reassembling comprises incubating the cargo with the composition comprising PNMA monomers in a physiological buffer.

[0453] Embodiment 82. The method of any one of embodiments 67 to 80, wherein the reassembling comprises incubating the cargo and the composition comprising PNMA monomers in a buffer containing about 270 to 330 mOsm / kg salt. EXAMPLES

[0454] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

[0455] Example 1 - Design and construction of a DNA vector encoding a recombinant PNMA protein To construct a recombinant DNA vector for PNMA2 expression, the full-length cDNA open reading frame, excluding the initial methionine, was inserted into a cloning vector and then transferred to an expression vector according to standard methods.

[0456] The cDNA encoding PNMA2 was inserted into an expression vector derived from pET-41-a(+) (EMD Millipore (Novagen) catalog number 70566). The entire cloning site of pET-41-a(+) was removed and replaced with DNA having the nucleotide sequence of SEQ ID NO:25, which encodes an N-terminal tag having the amino acid sequence of SEQ ID NO:26, including a 6xHis tag (SEQ ID NO:27), a 6 amino acid spacer (SEQ ID NO:28), and a TEV cleavage site (SEQ ID NO:29), followed in some cases by an additional SG linker. The PNMA2 open reading frame without the initiating methionine codon (codon-optimized, SEQ ID NO:30, encoding SEQ ID NO:7) was inserted after the TEV cleavage site by Gibson assembly. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, Smith HO(2009),Enzymatic assembly of DNA molecules up to several hundred kilobases,Nature Methods.6(5):343-345. Figure 2A provides a schematic of the PNMA2 construct.

[0457] After expression and Tobacco Etch Virus (TEV) cleavage, the resulting N-terminus of PNMA2 has one residual glycine from the TEV cleavage (SEQ ID NO:8). SEQ ID NOs:31 and 1 show the nucleotide and amino acid sequences, respectively, of PNMA2 including the N-terminal methionine.

[0458] [Table 2-1] [Table 2-2] [Table 2-3]

[0459] Example 2 - Expression and purification of His-tagged PNMA2 protein The expression vector construct containing the His-tagged PNMA2 open reading frame was transformed into the Rosetta2(DE3) E. coli strain (Millipore Sigma, Cat. No. 71400). PNMA2 expression was induced with 0.1 mM IPTG followed by incubation at 16°C for 16 hours. PNMA2 protein was purified as outlined in Figure 2B. Cell pellets were lysed by sonication in 20 mM sodium phosphate pH 7.4, 0.1 M NaCl, 40 mM imidazole, 1 mM DTT, 2 mM MgCl2, 1 mM ATP, and 10% glycerol. The lysate was treated with excess TURBO DNase (Thermo Fisher Scientific, Cat. No. AM2238), RNase Cocktail (Thermo Fisher Scientific, Cat. No. AM2286), and Benzonase Nuclease (Millipore Sigma, Cat. No. 71205) to eliminate nucleic acids. The lysate was cleared of cell debris by centrifugation and subsequent filtration. The 6xHis-tagged recombinant protein was loaded onto a HisTrap HP column (Cytiva, Cat. No. 17-5247-01), washed with HisTrap Buffer A (HTA) (20 mM sodium phosphate pH 7.4, 0.5 M NaCl, 40 mM imidazole, and 10% glycerol) and eluted with a linear gradient of HisTrap Buffer B (HTB) (20 mM sodium phosphate, pH 7.4, 0.5 M NaCl, 500 mM imidazole, and 10% glycerol) ("6xHis tag" disclosed as SEQ ID NO:27). The eluted protein peak was collected, diluted 5-fold with MonoQ dilution buffer (25 mM TrisHCl, pH 8.0, 10% glycerol) and loaded onto a MonoQ5 / 50GL anion exchange column (Cytiva, catalog no. 175166...

Claims

1. 1. A method of making a capsid, the method comprising: (a) expressing an endogenous retroviral capsid polypeptide in a host cell or a cell-free expression system; and (b) isolating the endogenous retroviral capsid polypeptide. Including, The method, wherein at least about 5% of said isolated endogenous retroviral capsid polypeptides assemble to form said capsid.

2. 2. The method of claim 1, wherein the percentage of the isolated endogenous retroviral capsids that assemble to form the capsid is determined by size exclusion chromatography.

3. The method of claim 1 or 2, wherein at least 20% of the isolated endogenous retroviral capsid polypeptides assemble to form the capsid.

4. 1. A method of making a capsid containing a cargo, the method comprising: (a) expressing an endogenous retroviral capsid polypeptide in a host cell or a cell-free expression system; (b) isolating capsids comprising said endogenous retroviral capsid polypeptide; (c) disassembling the capsid, thereby producing a disassembled endogenous retroviral capsid polypeptide; (d) contacting the disassembled endogenous retroviral capsid polypeptide with a cargo; (e) allowing the disassembled endogenous retroviral capsid polypeptides to reassemble, thereby generating the capsid containing the cargo. A method comprising:

5. 5. The method of claim 4, wherein the isolated polypeptide is present at least partially in an encapsidated form prior to step (c).

6. 6. The method of claim 4 or 5, wherein at least about 5% of the polypeptide present in encapsid form prior to step (c) is disassembled after step (c).

7. The method of any one of claims 4 to 6, wherein at least about 5% of the polypeptides present in disassembled form after step (c) are present in capsid form after step (d).

8. 1. A method for loading an endogenous retroviral capsid, the method comprising: a) disassembling endogenous retroviral capsids into a composition comprising endogenous retroviral capsid monomers; b) contacting the cargo with said composition comprising an endogenous retroviral capsid monomer; and c) allowing said composition comprising endogenous retroviral capsid monomers to reassemble into endogenous retroviral capsids. Including, Thereby, the cargo is loaded inside the endogenous retroviral capsid.

9. A method according to any one of claims 1 to 8, wherein the endogenous retroviral capsid polypeptide is a native endo-Gag polypeptide or an engineered endo-Gag polypeptide.

10. The method of any one of claims 1 to 9, wherein the method produces assembled capsids that (i) have a purity of at least about 50% as measured by SDS-PAGE, or (ii) have a particle uniformity of at least about 50% as measured by multi-angle dynamic light scattering.

11. 8. The method of any one of claims 4 to 7, wherein the disassembly occurs in a disassembly buffer comprising a reducing agent, such as reduced glutathione (GSH), beta mercaptoethanol (β-ME), dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).

12. The method of any one of claims 4 to 7 and 11, wherein the disassembly occurs in a disassembly buffer comprising a non-denaturing detergent, such as CHAPS.

13. The method of any one of claims 1 to 12, wherein the reassembly is performed in a reassembly buffer containing less than 500 mOsm / kg salt, for example, the reassembly is performed in a reassembly buffer containing about 270-330 mOsm / kg salt.

14. The method of any one of claims 4 to 7, wherein the disassembly is carried out in a disassembly buffer, the disassembly buffer comprising (i) one or more solubilizing agents, (ii) a non-detergent sulfobetaine, (iii) a chaotropic agent, (iv) n-butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, urea, MgCl2, CaCl2, NaI, or combinations thereof, or (v) DTT and urea.

15. A method according to any one of claims 4 to 7 and 11 to 14, wherein the dismantling step is carried out at -20°C to 45°C.

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