Compositions comprising modified finger ring virus capsid proteins and uses thereof

Abstract

The present invention relates generally to modified ring virus capsid proteins, ring vectors, ring VLPs, and compositions and uses thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 344,019, filed May 19, 2022, and U.S. Provisional Application No. 63 / 387,337, filed December 14, 2022. The contents of the foregoing applications are hereby incorporated by reference in their entirety. Background Art

[0003] There is an ongoing need to develop suitable delivery vehicles to deliver therapeutic agents to patients. Summary of the Invention

[0004] The present disclosure provides a ring vector (e.g., a synthetic ring vector) that can be used as a delivery vehicle, for example, for delivering genetic material, delivering effectors (e.g., loads), or delivering therapeutic agents or therapeutic effectors to eukaryotic cells (e.g., human cells or human tissues). In some embodiments, the ring vector (e.g., a particle, e.g., a viral particle, e.g., anellovirus particle) comprises a genetic element (e.g., a genetic element comprising a therapeutic DNA sequence) encapsulated in a protein shell (e.g., a protein shell comprising an anellovirus capsid protein, e.g., an anellovirus ORF1 molecule or a polypeptide encoded by an anellovirus ORF1 nucleic acid, e.g., as described herein), which is capable of introducing the genetic element into a cell (e.g., a mammalian cell, e.g., a human cell). In some embodiments, the ring vector is a particle comprising a protein shell comprising a polypeptide encoded by an anellovirus ORF1 nucleic acid (e.g., an ORF1 nucleic acid of a type B ring virus (Betatorquevirus), e.g., as described herein).

[0005] In some embodiments, the protein coat of the ring vector or ring VLP comprises a modified ring virus ORF1 molecule. In some embodiments, the ring virus ORF1 molecule is modified to delete at least a portion of a structural arginine-rich region (e.g., as described herein). In some embodiments, the ring virus ORF1 molecule is modified to delete at least a portion of a structural C-terminal domain (e.g., as described herein). In some embodiments, the ring virus ORF1 molecule is a chimeric ORF1 molecule comprising a fragment or domain (e.g., a structural arginine-rich region, a P1 domain, a P2 domain, a P1-1 domain, and / or a P1-2 domain, e.g., as described herein) from a different ring virus ORF1 protein (e.g., as described herein). In some embodiments, the ring virus ORF1 molecule is a chimeric ORF1 molecule comprising a fragment or domain from a protein other than an ring virus ORF1 protein (e.g., a protein from another virus, e.g., as described herein).

[0006] In some embodiments, the ring vector or ring VLP comprises a surface moiety as described herein on its outer surface (e.g., attached to a protein shell). In some embodiments, the protein shell comprises an ORF1 molecule attached to a surface moiety. In some embodiments, the protein shell comprises an ORF1 molecule comprising a click handle. In some embodiments, the protein shell comprises an ORF1 molecule fused to a polypeptide surface moiety. In some embodiments, the protein shell comprises a plurality of ORF1 molecules each attached to a surface moiety, for example, wherein the plurality of ORF1 molecules form a multimer (e.g., a dimer, trimer, or pentamer).

[0007] The genetic elements of the finger ring vectors of the present disclosure are typically circular and / or single-stranded DNA molecules (e.g., circular and single-stranded), and typically include a protein binding sequence that is bound to the protein coat surrounding it, or a polypeptide connected thereto, which may help to enclose the genetic elements within the protein coat, and / or enrich the genetic elements within the protein coat relative to other nucleic acids. In some cases, the genetic elements are circular or linear. In some cases, the genetic elements contain or encode effectors (e.g., nucleic acid effectors (e.g., non-coding RNA), or polypeptide effectors (e.g., proteins)), for example, effectors that can be expressed in cells. In some embodiments, the effector is a therapeutic agent or therapeutic effector, for example, as described herein. In some cases, the effector is, for example, an endogenous effector or an exogenous effector for wild-type anellovirus or target cell. In some embodiments, the effector is exogenous for wild-type anellovirus or target cell. In some embodiments, the finger ring vector can be delivered to the cell by contacting the cell and introducing the genetic elements encoding the effector into the cell so that the effector is produced or expressed by the cell. In some cases, the effector is an endogenous effector (e.g., an endogenous effector that is endogenous to the target cell, but, for example, is provided in greater amounts by the finger ring vector). In other cases, the effector is an exogenous effector. In some cases, the effector can modulate the function of the cell or modulate the activity or level of a target molecule in the cell. For example, the effector can reduce the level of a target protein in the cell. In another example, the finger ring vector can deliver and express effectors, such as exogenous proteins, in vivo. For example, the finger ring vector can be used to deliver genetic material to a target cell, tissue, or subject; to deliver effectors to a target cell, tissue, or subject; or to treat diseases and disorders, for example, by delivering effectors that can act as therapeutic agents to desired cells, tissues, or subjects. In some cases, the finger ring vector is prepared by in vitro assembly. The in vitro assembly of the finger ring vector typically involves the formation of a protein shell surrounding the genetic element, which occurs outside the host cell (e.g., in a cell-free suspension, lysate, or supernatant). In vitro assembly can, in some cases, utilize components produced in the host cell, but generally does not require a host cell for particle assembly.

[0008] This disclosure provides a ring-shaped VLP (e.g., a synthetic ring-shaped VLP) that can be used as a delivery medium, for example, to deliver genetic material, effector (e.g., load), or therapeutic agent or therapeutic effector to eukaryotic cells (e.g., human cells or human tissues). The ring-shaped VLP typically includes a surface portion as described herein on its outer surface (e.g., attached to a protein shell). In some embodiments, the surface portion contains an effector. In some embodiments, the surface portion contains a targeting agent (e.g., an agent that targets the ring-shaped VLP to target cells or tissues). In some embodiments, the ring-shaped VLP (e.g., a particle, such as a viral particle, such as a ringvirus particle) includes a protein shell (e.g., a protein shell containing a ringvirus capsid protein, such as a ringvirus ORF1 molecule or polypeptide encoded by a ringvirus ORF1 nucleic acid, such as as described herein). In some embodiments, the ring-shaped VLP is a particle containing a protein shell that contains a polypeptide encoded by a ringvirus ORF1 nucleic acid (e.g., the ORF1 nucleic acid of betatorquevirus, such as as described herein). In some embodiments, a protein shell surrounds the effector. In some embodiments, the effector is a therapeutic agent or a therapeutic effector, such as those described herein. In some cases, the effector is either an endogenous or exogenous effector, for example, with respect to wild-type ring virus or target cells. In some embodiments, the effector is exogenous with respect to wild-type ring virus or target cells. In some embodiments, a ring VLP can deliver the effector into a cell by contacting the cell and introducing the effector into the cell. In some cases, the effector is an endogenous effector (e.g., endogenous with respect to the target cell, but, for example, an endogenous effector provided by a ring VLP in a larger quantity). In other cases, the effector is an exogenous effector. In some cases, the effector can modulate cell function or modulate the activity or level of target molecules in the cell. For example, the effector can reduce the level of target proteins in the cell. In another instance, a ring VLP can deliver the effector, such as an exogenous protein, in vivo. For example, a ring VLP can be used to deliver the effector to target cells, tissues, or a subject; or to treat diseases and disorders, for example, by delivering an effector that can function as a therapeutic agent to the desired cells, tissues, or a subject. In some cases, ring-shaped VLPs are prepared via in vitro assembly. In vitro assembly of ring-shaped VLPs typically involves the formation of a protein coat associated with the effector (e.g., a protein coat surrounding the effector), which occurs outside the host cell (e.g., in a cell-free suspension, lysate, or supernatant). In some cases, in vitro assembly of ring-shaped VLPs may utilize components generated within the host cell, but typically does not require host cells for particle assembly.

[0009] The present invention also provides synthetic ring vectors and synthetic ring VLPs. The synthetic ring vectors or synthetic ring VLPs have at least one structural difference compared to wild-type viruses (e.g., wild-type ring viruses, as described herein), such as deletions, insertions, substitutions, or modifications (e.g., enzymatic modifications) relative to wild-type viruses. Generally, the synthetic ring vectors and synthetic ring VLPs comprise a protein shell that can be used to deliver effectors (e.g., exogenous or endogenous effectors) into eukaryotic (e.g., human) cells. In some embodiments, the ring vectors or ring VLPs do not induce detectable and / or unwanted immune or inflammatory responses, for example, they do not induce increases in one or more inflammatory molecular markers such as TNF-α, IL-6, IL-12, IFN exceeding 1%, 5%, 10%, or 15%, and do not induce B cell responses such as reactive or neutralizing antibodies; for example, the ring vectors or ring VLPs are substantially non-immunogenic to target cells, tissues, or subjects.

[0010] On one hand, the present invention is characterized by a ring carrier comprising: (i) a genetic element comprising a promoter element and a sequence encoding an effector (e.g., an endogenous or exogenous effector), and a protein-binding sequence (e.g., a capsid protein-binding sequence, such as a packaging signal); and (ii) a protein capsid; wherein the genetic element is enclosed within the protein capsid (e.g., a capsid); and wherein the ring carrier is capable of delivering the genetic element into eukaryotic (e.g., mammalian, such as human) cells. In some embodiments, the ring carrier comprises a surface portion (e.g., a surface portion having effector and / or targeting functions), for example, the surface portion being displayed on the capsid surface of the ring carrier (e.g., as described herein). In some embodiments, the surface portion comprises an effector.

[0011] In some embodiments, the genetic element is single-stranded and / or circular DNA. Alternatively or in combination, the genetic element has one, two, three, or all of the following characteristics: it is circular; it is single-stranded; it integrates into the cellular genome at a frequency less than about 0.0001%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the genetic element entering the cell; and / or it integrates into the genome of the target cell with fewer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 copies / genome. In some embodiments, the integration frequency is determined as described by Wang et al. (2004, GeneTherapy 11:711-721, incorporated herein by reference in its entirety). In some embodiments, the genetic element is enclosed within a protein shell. In some embodiments, the ring-shaped vector is capable of delivering the genetic element into eukaryotic cells. In some embodiments, the genetic element comprises a nucleic acid sequence (e.g., a nucleic acid sequence of 300-4000 nucleotides, such as 300-3500 nucleotides, 300-3000 nucleotides, 300-2500 nucleotides, 300-2000 nucleotides, 300-1500 nucleotides) that has at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with a wild-type ring virus sequence (e.g., a wild-type circovirus (TTV), small circovirus (TTMV), or TTMDV sequence, such as a wild-type ring virus sequence listed in any of Tables A1-A26 or N1-N26). In some embodiments, the genetic element comprises a nucleic acid sequence (e.g., at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity with a wild-type ring virus (e.g., a wild-type ring virus sequence as described herein, such as any one of Tables A1-A26 or N1-N26). In some embodiments, the nucleic acid sequence is codon-optimized, for example, for expression in mammalian (e.g., human) cells. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in the nucleic acid sequence are codon optimized, e.g., for expression in mammalian (e.g., human) cells.

[0012] In one aspect, the invention features a finger ring VLP comprising a protein shell (e.g., capsid) and an effector; wherein the finger ring VLP is capable of delivering the effector into a eukaryotic (e.g., mammalian, such as human) cell. In some embodiments, the effector is contained in a surface moiety, e.g., is expressed on the shell surface of a finger ring VLP (e.g., as described herein).

[0013] In one aspect, the invention features infectious (to human cells) particles comprising an anellovirus capsid (e.g., a capsid comprising an anellovirus ORF, such as an ORF1 polypeptide). In some embodiments, the infectious particle encapsulates a genetic element comprising a protein binding sequence that binds to the capsid and a heterologous (for the anellovirus) sequence encoding a therapeutic effector. In some embodiments, the particle is capable of delivering the genetic element into a cell of a mammal, such as a human. In some embodiments, the genetic element has less than about 6% (e.g., less than 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5% or less) identity to a wild-type anellovirus. In some embodiments, the genetic element has no more than 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5% or 6% identity to a wild-type anellovirus. In some embodiments, the genetic element has at least about 2% to at least about 5.5% (e.g., 2% to 5%, 3% to 5%, 4% to 5%) identity to a wild-type anellovirus. In some embodiments, the genetic element has a non-viral sequence (e.g., a non-anellovirus genomic sequence) of greater than about 2000, 3000, 4000, 4500, or 5000 nucleotides. In some embodiments, the genetic element has a non-viral sequence (e.g., a non-anellovirus genomic sequence) of greater than about 2000 to 5000, 2500 to 4500, 3000 to 4500, 2500 to 4500, 3500, or 4000, 4500 (e.g., about 3000 to 4500) nucleotides. In some embodiments, the genetic element is a single-stranded circular DNA. Alternatively or in combination, the genetic element has one, two or three of the following properties: is circular, is single-stranded, it integrates into the genome of the cell at a frequency of less than about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5% or 2% of the genetic elements that enter the cell, it integrates into the genome of the target cell at less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 copies per genome or integrates at a frequency of less than about 0.0001%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5% or 2% of the genetic elements that enter the cell. In some embodiments, integration frequency is determined as described in Wang et al. (2004, Gene Therapy 11:711-721, herein incorporated by reference in its entirety).

[0014] Also described herein are anellovirus-based viral vectors and viral particles that can be used to deliver agents (e.g., exogenous effectors or endogenous effectors, e.g., therapeutic effectors) to cells (e.g., cells in a subject to be treated). In some embodiments, anellovirus can be used as an effective delivery vehicle to introduce an agent (e.g., an effector described herein) into a target cell, e.g., a target cell in a subject to therapeutic or prophylactic treatment.

[0015] In one aspect, the invention features a polypeptide (e.g., a synthetic polypeptide, e.g., an ORF1 molecule) comprising (e.g., in tandem):

[0016] (i) a first region comprising a structural arginine-rich region, an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a structural arginine-rich region sequence described herein, or a sequence of at least about 40 amino acids containing at least 60%, 70%, or 80% basic residues (e.g., arginine, lysine, or a combination thereof),

[0017] (ii) a second region comprising a structural jelly-roll domain, such as an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a structural jelly-roll region sequence described herein, or a sequence comprising at least 6 beta strands,

[0018] (iii) a third region comprising an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a structural N22 domain sequence described herein,

[0019] (iv) a fourth region comprising an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an anellovirus ORF1 structural C-terminal domain (CTD) sequence described herein, and

[0020] (v) Optionally, the polypeptide has an amino acid sequence that is less than 100%, 99%, 98%, 95%, 90%, 85%, 80% identical to a wild-type anellovirus ORF1 protein described herein.

[0021] In some embodiments, the polypeptide has at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an Anellovirus ORF1 molecule as described herein (e.g., as listed in any one of Tables A1-A26). In some embodiments, the polypeptide comprises at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a subsequence of an Anellovirus ORF1 molecule as described herein (e.g., a structural arginine (Arg)-rich domain, a structural jelly coil domain, a hypervariable region (HVR), a structural N22 domain, or a structural C-terminal domain (CTD)). In one embodiment, the amino acid sequences of regions (i), (ii), (iii) and (iv) have at least 90% sequence identity to their respective references, and wherein the polypeptide has an amino acid sequence having less than 100%, 99%, 98%, 95%, 90%, 85%, 80% sequence identity to the wild-type anellovirus ORF1 protein described herein.

[0022] In one aspect, the invention features a complex comprising a polypeptide as described herein (e.g., an anellovirus ORF1 molecule as described herein) and a genetic element comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector) and a protein binding sequence.

[0023] The present disclosure also provides nucleic acid molecules (e.g., nucleic acid molecules comprising genetic elements as described herein, or nucleic acid molecules comprising sequences encoding protein coat proteins as described herein). The nucleic acid molecules of the present invention may comprise one or both of (a) genetic elements as described herein and (b) nucleic acid sequences encoding protein coat proteins as described herein.

[0024] In one aspect, the invention features an isolated nucleic acid molecule comprising a genetic element comprising a promoter element and an external protein binding sequence operably linked to a sequence encoding an effector, such as a payload. In some embodiments, the external protein binding sequence comprises a sequence that is at least 75% (at least 80%, 85%, 90%, 95%, 97%, 100%) identical to a 5'UTR sequence of an anellovirus as disclosed herein. In some embodiments, the genetic element is single-stranded DNA, is circular, integrates at a frequency of less than about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the genetic element that enters the cell, and / or integrates into the genome of the target cell at less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 copies per genome or integrates at a frequency of less than about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the genetic element that enters the cell. In some embodiments, the integration frequency is determined as described in Wang et al. (2004, Gene Therapy 11:711-721, incorporated herein by reference in its entirety). In embodiments, the effector is not derived from TTV and is not SV40-miR-S1. In embodiments, the nucleic acid molecule does not comprise a polynucleotide sequence of TTMV-LY2. In embodiments, the promoter element is capable of directing expression of the effector in a eukaryotic (eg, mammalian, eg, human) cell.

[0025] In some embodiments, the nucleic acid molecules are circular. In some embodiments, the nucleic acid molecules are linear. In some embodiments, the nucleic acid molecules described herein comprise one or more modified nucleotides (e.g., base modified, sugar modified, or backbone modified).

[0026] In some embodiments, the nucleic acid molecule comprises a sequence encoding an ORF1 molecule (e.g., an anellovirus ORF1 protein, e.g., as described herein). In some embodiments, the nucleic acid molecule comprises a sequence encoding an ORF2 molecule (e.g., an anellovirus ORF2 protein, e.g., as described herein). In some embodiments, the nucleic acid molecule comprises a sequence encoding an ORF3 molecule (e.g., an anellovirus ORF3 protein, e.g., as described herein). In one aspect, the invention features a genetic element comprising one, two, or three of: (i) a promoter element and a sequence encoding an effector (e.g., an exogenous or endogenous effector); (ii) at least 72 contiguous nucleotides (e.g., at least 72, 73, 74, 75, 76, 77, 78, 79, 80, 90, 100, or 150 nucleotides) having at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a wild-type anellovirus sequence; or (iii) at least 100 (e.g., at least 300, 500, 1000, 1500) contiguous nucleotides having at least 100% (e.g., at least 300, 500, 1000, 1500) sequence identity to a wild-type anellovirus sequence. at least 72% (e.g., at least 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity; and (iii) a protein binding sequence, such as a coat protein binding sequence, and wherein the nucleic acid construct is single-stranded DNA; and wherein the nucleic acid construct is circular, integrates at a frequency of less than about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the genetic elements entering the cell, and / or integrates into the genome of the target cell at less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 copies / genome. In some embodiments, the genetic element encoding an effector (e.g., an exogenous or endogenous effector, e.g., as described herein) is codon optimized. In some embodiments, the genetic element is circular. In some embodiments, the genetic element is linear. In some embodiments, the genetic element comprises an finger ring vector, e.g., as described herein. In some embodiments, the genetic element described herein comprises one or more modified nucleotides (e.g., base modifications, sugar modifications, or backbone modifications). In some embodiments, the genetic element comprises a sequence encoding an ORF1 molecule (e.g., an anellovirus ORF1 protein, e.g., as described herein). In some embodiments, the genetic element comprises a sequence encoding an ORF2 molecule (e.g., an anellovirus ORF2 protein, e.g., as described herein).In some embodiments, the genetic element comprises a sequence encoding an ORF3 molecule (eg, an anellovirus ORF3 protein, eg, as described herein).

[0027] In one aspect, the invention features a host cell or helper cell comprising: (a) a nucleic acid comprising a sequence encoding one or more of an ORF1 molecule, an ORF2 molecule, or an ORF3 molecule (e.g., a sequence encoding an anellovirus ORF1 polypeptide described herein), wherein the nucleic acid is a plasmid, a viral nucleic acid, or integrated into a helper cell chromosome; and (b) a genetic element, wherein the genetic element comprises (i) a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector) and (ii) a protein binding sequence that binds to the polypeptide of (a), wherein optionally, the genetic element does not encode an ORF1 polypeptide (e.g., an ORF1 protein). For example, the host cell or helper cell comprises (a) and (b) in cis (two parts of the same nucleic acid molecule) or in trans (each part of a different nucleic acid molecule). In embodiments, the genetic element of (b) is a circular single-stranded DNA. In some embodiments, the host cell is a production cell line. In some embodiments, the host cell or helper cell is adherent or suspended, or both. In some embodiments, the host cells or helper cells are grown in microcarriers. In some embodiments, the host cells or helper cells are compatible with cGMP production practices. In some embodiments, the host cells or helper cells are grown in a culture medium suitable for promoting cell growth. In certain embodiments, once the host cells or helper cells have grown sufficiently (e.g., to an appropriate cell density), the culture medium can be replaced with a culture medium suitable for the host cells or helper cells to produce the ring vector.

[0028] In one aspect, the invention features a pharmaceutical composition comprising a finger ring vector (e.g., a synthetic finger ring vector) as described herein. In embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. In embodiments, the pharmaceutical composition comprises a unit dose comprising about 10 5 -10 14 In some embodiments, the pharmaceutical composition comprises a genome equivalent of the ring vector per kilogram of target subject. In some embodiments, the pharmaceutical composition comprising the formulation is stable within an acceptable period and temperature range and / or is compatible with the desired route of administration and / or any device required for the route of administration (e.g., a needle or syringe). In some embodiments, the pharmaceutical composition is formulated for administration as a single dose or multiple doses. In some embodiments, the pharmaceutical composition is formulated at the site of administration, for example, by a healthcare professional. In some embodiments, the pharmaceutical composition comprises a desired concentration of ring vector genomes or genome equivalents (e.g., defined by the number of genomes per volume).

[0029] In one aspect, the invention features a method of treating a disease or disorder in a subject, the method comprising administering to the subject a finger ring vector, e.g., a synthetic finger ring vector, e.g., as described herein.

[0030] In one aspect, the invention features a method of delivering an effector or cargo (e.g., an endogenous or exogenous effector) to a cell, tissue, or subject, the method comprising administering to the subject a finger ring vector, e.g., a synthetic finger ring vector, e.g., as described herein, wherein the finger ring vector comprises a nucleic acid sequence encoding the effector. In embodiments, the cargo is a nucleic acid. In embodiments, the cargo is a polypeptide.

[0031] In one aspect, the invention features a method of delivering a finger ring vector to a cell, the method comprising contacting the finger ring vector (e.g., a synthetic finger ring vector, e.g., as described herein) with a cell (e.g., a eukaryotic cell, such as a mammalian cell), e.g., in vivo or ex vivo.

[0032] In one aspect, the invention features a method of treating a disease or disorder in a subject, the method comprising administering to the subject a finger ring VLP, e.g., a synthetic finger ring VLP, e.g., as described herein.

[0033] In one aspect, the invention features a method of delivering an effector or payload (e.g., an endogenous or exogenous effector) to a cell, tissue, or subject, the method comprising administering to the subject a finger ring VLP, e.g., a synthetic finger ring VLP, e.g., as described herein, wherein the finger ring VLP comprises an effector (e.g., wherein the protein shell of the finger ring VLP encapsulates the effector). In embodiments, the payload is a nucleic acid. In embodiments, the payload is a polypeptide (e.g., a protein).

[0034] In one aspect, the invention features a method of delivering a finger ring VLP to a cell, the method comprising contacting the finger ring VLP (e.g., a synthetic finger ring VLP, e.g., as described herein) with a cell (e.g., a eukaryotic cell, such as a mammalian cell), e.g., in vivo or ex vivo.

[0035] In one aspect, the invention features a method for preparing a finger ring vector, such as a synthetic finger ring vector. The method includes:

[0036] a) providing a host cell comprising:

[0037] (i) a first nucleic acid molecule comprising a nucleic acid sequence of a genetic element of a finger ring vector as described herein, e.g., a synthetic finger ring vector, and

[0038] (ii) the first nucleic acid or a second nucleic acid molecule encoding one or more of the following: an amino acid sequence selected from ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1 or ORF1 / 2 (e.g., as set forth in any one of Tables A1-A26), or an amino acid sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto; and

[0039] b) incubating the host cell under conditions suitable for preparing the finger ring vector.

[0040] In some embodiments, the method further comprises: before step (a), introducing the first nucleic acid molecule and / or the second nucleic acid molecule into the host cell. In some embodiments, the second nucleic acid molecule is introduced into the host cell before, simultaneously with, or after the first nucleic acid molecule. In other embodiments, the second nucleic acid molecule is integrated into the genome of the host cell. In some embodiments, the second nucleic acid molecule is an auxiliary (e.g., a helper plasmid, or the genome of a helper virus).

[0041] In another aspect, the invention features a method of producing a ring carrier composition, the method comprising:

[0042] a) providing a host cell comprising, for example, an expression ring vector, for example, a synthetic ring vector, for example, one or more components (e.g., all components) of a synthetic ring vector described herein. For example, the host cell comprises (a) a nucleic acid comprising a sequence encoding an anellovirus ORF1 polypeptide described herein, wherein the nucleic acid is a plasmid, a viral nucleic acid, or integrated into a helper cell chromosome; and (b) a genetic element, wherein the genetic element comprises (i) a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector) and (i) a protein binding sequence (e.g., a packaging sequence) that binds to the polypeptide of (a), wherein the host cell or helper cell comprises (a) and (b) in cis or in trans. In embodiments, the genetic element of (b) is a circular single-stranded DNA. In some embodiments, the host cell is a production cell line;

[0043] b) culturing the host cell under conditions suitable for producing a finger ring vector preparation from the host cell, wherein the finger ring vector of the preparation comprises a protein coat (e.g., comprising an ORF1 molecule) encapsulating the genetic element (e.g., as described herein), thereby preparing a finger ring vector preparation; and

[0044] Optionally, c) formulating the ring vector, for example, as a pharmaceutical composition suitable for administration to a subject.

[0045] In some embodiments, the components of the finger ring vector are introduced into the host cell at the time of production (e.g., by transient transfection). In some embodiments, the host cell stably expresses the components of the finger ring vector (e.g., wherein one or more nucleic acids encoding the finger ring vector components are introduced into the host cell or an ancestor thereof, e.g., by stable transfection).

[0046] In some embodiments, the method further comprises one or more purification steps (e.g., by sedimentation, chromatography, and / or ultrafiltration). In some embodiments, the purification step comprises removing one or more of serum, host cell DNA, host cell proteins, particles lacking genetic elements, and / or phenol red from the formulation. In some embodiments, the resulting formulation or pharmaceutical composition comprising the formulation is stable within an acceptable time period and temperature range and / or is compatible with the desired route of administration and / or any device desired for that route of administration, e.g., a needle or syringe.

[0047] In one aspect, the invention features a method of producing a finger ring vector composition, the method comprising: a) providing a plurality of finger ring vectors described herein, or a finger ring vector formulation described herein; and b) formulating the finger ring vector or its formulation, e.g., as a pharmaceutical composition suitable for administration to a subject.

[0048] In one aspect, the invention features a method of producing a Finger Ring VLP composition, the method comprising: a) providing a plurality of Finger Ring VLPs described herein, or a Finger Ring VLP formulation described herein; and b) formulating the Finger Ring VLP or its formulation, e.g., as a pharmaceutical composition suitable for administration to a subject.

[0049] In one aspect, the invention features a method of making a host cell, such as a first host cell or a producer cell (e.g., Figure 20 In some embodiments, the method further comprises introducing a helper, such as a helper virus, into the host cells. In some embodiments, the introduction comprises transfecting (e.g., chemically transfecting) or electroporating the host cells with the finger-ring vector.

[0050] In one aspect, the invention features a method of making a finger ring vector, the method comprising providing a host cell, e.g., a first host cell or a producer cell (e.g., Figure 20) and purifying the finger ring vector from the host cell. In some embodiments, the method further comprises, prior to the providing step, contacting the host cell with a finger ring vector, such as described herein, and incubating the host cell under conditions suitable for producing the finger ring vector. In some embodiments, the host cell is the first host cell or producer cell described in the above method for preparing a host cell. In some embodiments, purifying the finger ring vector from the host cell comprises lysing the host cell.

[0051] In some embodiments, the method further comprises a second step of combining the finger ring vector produced by the first host cell or producer cell with a second host cell, such as a permissive cell (e.g., Figure 20 In some embodiments, the method further comprises contacting the second host cell population with the finger ring vector, for example, a second host cell population. In some embodiments, the method further comprises incubating the second host cell under conditions suitable for producing the finger ring vector. In some embodiments, the method further comprises purifying the finger ring vector from the second host cell, for example, thereby producing a finger ring vector seed population. In some embodiments, the finger ring vector produced from the second host cell population is at least about 2-100 times more than the finger ring vector produced from the first host cell population. In some embodiments, purifying the finger ring vector from the second host cell comprises lysing the second host cell. In some embodiments, the method further comprises a second step of allowing the finger ring vector produced by the second host cell to interact with a third host cell, for example, a allowing cell (e.g., as Figure 20 In some embodiments, the method further comprises contacting the third host cell population with the third host cell population, for example, a third host cell population. In some embodiments, the method further comprises incubating the third host cell population under conditions suitable for producing the finger ring vector. In some embodiments, the method further comprises purifying the finger ring vector from the third host cell population, for example, thereby producing a stock population of finger ring vectors. In some embodiments, purifying the finger ring vector from the third host cell population comprises lysing the third host cell population. In some embodiments, the finger ring vector produced from the third host cell population is at least about 2-100 times more than the finger ring vector produced from the second host cell population.

[0052] In some embodiments, host cells are grown in a culture medium suitable for promoting cell growth. In certain embodiments, once the host cells have grown sufficiently (e.g., to an appropriate cell density), the culture medium can be replaced with a culture medium suitable for producing the finger ring vector from the host cells. In some embodiments, the finger ring vector produced by the host cell is separated from the host cell (e.g., by lysing the host cell) before contacting with the second host cell. In some embodiments, the finger ring vector produced by the host cell is contacted with the second host cell without an intervening purification step.

[0053] In one aspect, the invention features a method for preparing a pharmaceutical ring vector formulation. The method includes (a) preparing a ring vector formulation as described herein, (b) evaluating the formulation (e.g., a pharmaceutical ring vector formulation, a ring vector seed population, or a ring vector stock population) for one or more of the following pharmaceutical quality control parameters: identity, purity, titer, potency (e.g., in terms of genome equivalents per ring vector particle) and / or, for example, nucleic acid sequences of genetic elements contained in the ring vector, and (c) formulating the formulation for the evaluated pharmaceutical use to meet predetermined standards, e.g., meet pharmaceutical specifications. In some embodiments, the evaluation and identification includes evaluating (e.g., confirming) the sequence of the ring vector genetic element, e.g., the sequence encoding the effector. In some embodiments, evaluating purity includes evaluating the amount of impurities, such as mycoplasma, endotoxin, host cell nucleic acid (e.g., host cell DNA and / or host cell RNA), animal-derived process impurities (e.g., serum albumin or trypsin), replication-competent factors (RCA), such as replication-competent viruses or unnecessary ring vectors (e.g., ring vectors other than the desired ring vector, such as the synthetic ring vector described herein), free viral capsid proteins, exogenous factors, and aggregates. In some embodiments, evaluating potency includes evaluating the ratio of functional to non-functional (e.g., infectious to non-infectious) ring vectors in the formulation (e.g., evaluated by HPLC). In some embodiments, evaluating efficacy includes evaluating the level of detectable ring vector function (e.g., expression and / or function of the effector or genome equivalent encoded therein) in the formulation. In some embodiments, impurities include residual denaturants (e.g., urea) or cell substitutes (e.g., proteasomes or ferritin).

[0054] In some embodiments, the formulated formulation is substantially free of pathogens, host cell contaminants, or impurities; has a predetermined level of non-infectious particles or a predetermined ratio of particles:infectious units (e.g., <300:1, <200:1, <100:1, or <50:1). In some embodiments, multiple finger ring vectors can be produced in a single batch. In some embodiments, the levels of finger ring vectors produced in the batch can be evaluated (e.g., individually or collectively).

[0055] In one aspect, the invention features a method for preparing a pharmaceutical ring VLP formulation. The method comprises (a) preparing a ring VLP formulation as described herein, (b) evaluating the formulation (e.g., a pharmaceutical ring VLP formulation, a ring VLP seed population, or a ring VLP stock population) for one or more of the following pharmaceutical quality control parameters: identity, purity, titer, potency, and (c) formulating the formulation for the pharmaceutical use being evaluated to meet predetermined standards, e.g., meet pharmaceutical specifications. In some embodiments, evaluating purity includes evaluating the amount of impurities, such as mycoplasmas, endotoxins, host cell nucleic acids (e.g., host cell DNA and / or host cell RNA), animal-derived process impurities (e.g., serum albumin or trypsin), replication-competent factors (RCAs), such as replication-competent viruses or unwanted ring VLPs (e.g., ring VLPs other than the desired ring VLPs, e.g., synthetic ring VLPs described herein), free viral capsid proteins, exogenous factors, and aggregates. In some embodiments, evaluating potency comprises evaluating the ratio of functional to non-functional (e.g., infectious to non-infectious) ring VLPs in a formulation (e.g., evaluated by HPLC). In some embodiments, evaluating efficacy comprises evaluating the level of detectable ring VLP function (e.g., expression and / or function of an effector or genomic equivalent encoded therein) in a formulation. In some embodiments, impurities comprise residual denaturants (e.g., urea) or cell substitutes (e.g., proteasomes or ferritin).

[0056] In some embodiments, the formulated formulation is substantially free of pathogens, host cell contaminants, or impurities; has a predetermined level of non-infectious particles or a predetermined ratio of particles: infectious units (e.g., <300:1, <200:1, <100:1, or <50:1). In some embodiments, multiple finger ring VLPs can be produced in a single batch. In some embodiments, the levels of the finger ring VLPs produced in the batch can be evaluated (e.g., individually or collectively).

[0057] In one aspect, the invention features a host cell comprising:

[0058] (i) a first nucleic acid molecule comprising the nucleic acid sequence of a genetic element of a finger ring vector, such as a finger ring vector described herein; and

[0059] (ii) Optionally, a second nucleic acid molecule encoding one or more of the following: an amino acid sequence selected from ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1 or ORF1 / 2 (as listed in any one of Tables A1-A26), or an amino acid sequence having at least about 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto.

[0060] In one aspect, the invention features a reaction mixture comprising a finger ring vector described herein and a helper virus, wherein the helper virus comprises a polynucleotide, such as a polynucleotide encoding an external protein (e.g., an external protein capable of binding an external protein binding sequence and an optional lipid envelope), a polynucleotide encoding a replication protein (e.g., a polymerase), or any combination thereof.

[0061] In some embodiments, the finger ring vector (e.g., a synthetic finger ring vector) is isolated, for example, from a host cell and / or from other components of a solution (e.g., a supernatant). In some embodiments, the finger ring vector (e.g., a synthetic finger ring vector) is purified, for example, from a solution (e.g., a supernatant). In some embodiments, the finger ring vector is enriched in a solution relative to other components in the solution.

[0062] In some embodiments of any of the aforementioned finger ring vectors, compositions, or methods, providing the finger ring vector comprises isolating (e.g., harvesting) the finger ring vector from a composition comprising a finger ring vector-producing cell, e.g., as described herein. In other embodiments, providing the finger ring vector comprises obtaining the finger ring vector or a preparation thereof, e.g., from a third party.

[0063] In some embodiments of any of the foregoing finger-ring vectors, finger-ring vectors, compositions, or methods, the genetic element comprises a finger-ring vector genome, e.g., identified according to the methods described in Example 9. In embodiments, the finger-ring vector genome is a finger-ring vector genome capable of self-replication and / or self-amplification. In some embodiments, the finger-ring vector genome is not capable of self-replication and / or self-amplification. In some embodiments, the finger-ring vector genome is capable of replication and / or amplification in trans, e.g., in the presence of a helper (e.g., a helper virus).

[0064] Additional features of any of the foregoing finger-ring vectors, finger-ring VLPs, compositions, or methods include one or more of the embodiments listed below.

[0065] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.Such equivalent embodiments are intended to be encompassed by the examples listed below.

[0066] Examples

[0067] 1. A particle comprising:

[0068] a protein coat comprising about 40-80 (e.g., about 60), 100-140 (e.g., about 120), or 160-200 (e.g., about 180) copies of anellovirus ORF1 molecule,

[0069] The particles:

[0070] (i) does not comprise (e.g., does not surround) a polynucleotide (e.g., as determined using a nuclease protection assay as described herein),

[0071] (ii) does not comprise (e.g., does not encompass) a polynucleotide greater than 1000, 500, 200, or 100 nucleotides in length, or

[0072] (iii) comprises less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides.

[0073] 2. The particle of embodiment 1, wherein the anellovirus ORF1 molecule comprises an ORF1 domain and an exogenous surface portion.

[0074] 3. The particle of embodiment 1, wherein the anellovirus ORF1 molecule is bound to the exogenous surface moiety via non-covalent integration or a covalent bond other than a peptide bond.

[0075] 4. The particle of embodiment 1, wherein the anellovirus ORF1 molecule does not comprise a structural arginine-rich domain.

[0076] 5. The particle of embodiment 1, wherein the particle is a virus-like particle (VLP).

[0077] 6. A particle comprising:

[0078] (a) a protein coat comprising about 40-80 (e.g., about 60), 100 to 140 (e.g., about 120), or 160 to 200 (e.g., about 180) copies of an anellovirus ORF1 molecule and a foreign surface moiety, and

[0079] (b) A genetic element comprising a heterologous nucleic acid sequence encoding an exogenous effector.

[0080] 7. A particle comprising:

[0081] a protein coat comprising an anellovirus ORF1 molecule, wherein the ORF1 molecule comprises an ORF1 domain and a foreign surface portion;

[0082] One or more of the following:

[0083] a) the exogenous surface moiety is selected from a receptor, a ligand, an antibody molecule (e.g., scFv), an antigen (e.g., a viral antigen, a bacterial antigen, a fungal antigen, or a parasitic antigen), an adjuvant (e.g., a TLR agonist, e.g., bacterial flagellin);

[0084] b) wherein the ORF1 molecule comprises a hypervariable region (HVR);

[0085] c) wherein the particle comprises a genetic element encoding a peptide or polypeptide that enhances the immune response (e.g., an adjuvant, a TCR agonist (e.g., bacterial flagellin));

[0086] d) wherein the exogenous surface portion is 1-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-500, or 500-1000 amino acids in length;

[0087] e) wherein a polypeptide linker region is located between the exogenous surface moiety and the ORF1 molecule,

[0088] f) wherein the particle comprises 1-2, 2-5, 5-10, 10-20, 20-40, 40-60, 60-80, 80-100, 100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275, or 275-300 copies of the exogenous surface portion;

[0089] g) wherein the proteinaceous coat comprises: (i) a plurality of ORF1 molecules lacking the exogenous surface portion (e.g., wild-type ORF1 molecules), and (ii) a plurality of ORF1 molecules comprising the exogenous surface portion, wherein optionally the ratio of (i):(ii) is 10:1-5:1, 5:1-2:1, 2:1-1:2, 1:2-1:5 or 1:5-1:10; and / or

[0090] h) wherein the particle further comprises a second exogenous surface portion.

[0091] 8. The particle of embodiment 7, wherein the exogenous surface portion is located at the insertion point between the N-terminal portion of the ORF1 domain and the C-terminal portion of the ORF1 domain.

[0092] 9. The particle of embodiment 8, wherein the insertion point is located in the HVR.

[0093] 10. The particle of any one of embodiments 7-9, further comprising a genetic element comprising a heterologous nucleic acid sequence encoding an exogenous effector.

[0094] 11. The particle of any one of embodiments 7 to 10, wherein the particle does not comprise (e.g., does not encompass) a polynucleotide, or does not comprise (e.g., does not encompass) a polynucleotide that is greater than 1000, 500, 200, or 100 nucleotides in length, or comprises less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides.

[0095] 12. A particle comprising:

[0096] A protein coat comprising an anellovirus ORF1 molecule and a foreign surface moiety, wherein the foreign surface moiety is covalently bound to the ORF1 molecule using a bond other than a peptide bond.

[0097] 12a. The particle of embodiment 12, wherein the exogenous surface moiety is attached to an NHS moiety, and the exogenous surface moiety binds to the ORF1 molecule via the NHS moiety.

[0098] 13. The particle of embodiment 12, wherein a non-polypeptide linker is located between the exogenous surface moiety and the ORF1 molecule.

[0099] 14. The particle of embodiment 13, wherein the non-polypeptide linker comprises a click linkage.

[0100] 14a. The particle of embodiment 13, wherein the non-polypeptide linker is generated by a click reaction between a DBCO moiety and an azide moiety.

[0101] 14b. The particle of embodiment 14a, wherein the DBCO moiety is attached to the Anellovirus ORF1 molecule via an NHS moiety prior to the click reaction.

[0102] 14c. The particle of embodiment 14a or 14b, wherein the azide moiety is attached to the exogenous surface moiety via an NHS moiety prior to the click reaction.

[0103] 14c1. The particle of embodiment 14b or 14c, wherein the NHS portion of the DBCO moiety is attached to a lysine residue on the surface of the Anellovirus ORF1 molecule.

[0104] 14c2. The particle of any of embodiments 14b-14c1, wherein the NHS moiety of the azide moiety is attached to a lysine residue on the surface of the exogenous surface moiety.

[0105] 14d. The particle of embodiment 14a, wherein the DBCO moiety is attached to the exogenous surface moiety via an NHS moiety prior to the click reaction.

[0106] 14e. The particle of embodiment 14a or 14d, wherein the azide moiety is attached to the Anellovirus ORF1 molecule via an NHS moiety prior to the click reaction.

[0107] 14f. The particle of embodiment 14d or 14e, wherein the NHS portion of the DBCO moiety is attached to a lysine residue on the surface of the exogenous surface moiety.

[0108] 14g. The particle of any of embodiments 14d-14f, wherein the NHS portion of the azide moiety is attached to a lysine residue on the surface of the Anellovirus ORF1 molecule.

[0109] 15. A particle comprising:

[0110] A protein coat comprising an anellovirus ORF1 molecule and a foreign surface moiety, wherein the foreign surface moiety is non-covalently associated with the ORF1 molecule.

[0111] 16. The particle of embodiment 15, wherein the ORF1 molecule comprises an exogenous binding domain (e.g., MS2 coat protein or avidin), and the exogenous surface portion comprises a cognate binding moiety (e.g., MS2 hairpin or biotin) that binds to the exogenous binding domain.

[0112] 17. The particle of any of embodiments 12-16, wherein the exogenous surface portion comprises a polypeptide.

[0113] 18. The particle of any of embodiments 12-17, wherein the exogenous surface moiety comprises a small molecule or a nucleic acid molecule (eg, a polynucleotide).

[0114] 19. The particle of any preceding claim, wherein the ratio of the ORF1 molecules to the exogenous surface moieties is about 60:1-30:1, 30:1-20:1, 20:1-10:1 or 10:1-1:1.

[0115] 20. The particle of any preceding embodiment, wherein the antibody molecule is a bispecific antibody molecule.

[0116] 21. The particle of embodiment 20, wherein the bispecific antibody molecule comprises:

[0117] a first antigen binding domain that binds a first antigen on a first type of host cell, and

[0118] A second antigen binding domain that binds a second antigen on a second type of host cell.

[0119] 22. The particle of any preceding embodiment, which is capable of entering a target cell, for example by endocytosis.

[0120] 23. The particle of embodiment 22, wherein the exogenous surface moiety binds to a homologous moiety in the target cell.

[0121] 24. The particle of embodiment 22, wherein the particle comprises a genetic element encoding an exogenous effector to be delivered to the interior of the target cell.

[0122] 25. The particle of any preceding embodiment, wherein genetic elements are enclosed within the protein coat.

[0123] 26. The particle of any preceding embodiment, which comprises no polynucleotides, or no polynucleotides greater than 1000, 500, 200, or 100 nucleotides in length.

[0124] 27. The particle of any preceding embodiment, wherein the anellovirus ORF1 molecule comprises:

[0125] (b) a first region comprising an anellovirus ORF1 structural jellycoil region, such as an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an anellovirus ORF1 structural jellycoil region sequence described herein, or a sequence comprising at least 6 beta strands;

[0126] (c) a second region comprising an anellovirus ORF1 structural N22 domain, such as an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an anellovirus ORF1 structural N22 domain sequence described herein; and

[0127] (d) a third region comprising an anellovirus ORF1 structural C-terminal domain (CTD), such as an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an anellovirus ORF1 CTD sequence described herein; and

[0128] The anellovirus ORF1 molecule does not comprise the structural arginine-rich region of anellovirus ORF1, for example, a sequence of at least about 40 amino acids comprising at least 60%, 70% or 80% basic residues (eg, arginine, lysine or a combination thereof).

[0129] 28. A formulation comprising the particles of any preceding embodiment.

[0130] 29. The formulation of embodiment 28, wherein the formulation comprises less than 10 mg / kg of the subject to which the composition is to be administered. 10 -10 14 (For example, less than 10 10 -10 11 , 10 11 -10 12 , 10 12 -10 13 or 10 13 -10 14 ) viral genome equivalent of a nucleic acid molecule (e.g., a genetic element, e.g., a finger ring vector as described herein) (e.g., as determined by qPCR or by measuring optical density).

[0131] 30. A polypeptide, such as an anellovirus ORF1 molecule, comprising:

[0132] (b) a first region comprising an anellovirus ORF1 structural jellycoil region, such as an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an anellovirus ORF1 structural jellycoil region sequence described herein, or a sequence comprising at least 6 beta strands;

[0133] (c) a second region comprising an anellovirus ORF1 structural N22 domain, such as an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an anellovirus ORF1 structural N22 domain sequence described herein; and

[0134] (d) a third region comprising an anellovirus ORF1 structural C-terminal domain (CTD), such as an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an anellovirus ORF1 CTD sequence described herein; and

[0135] The finger polypeptide does not comprise the structural arginine-rich region of anellovirus ORF1, for example, a sequence of at least about 40 amino acids comprising at least 60%, 70% or 80% basic residues (eg, arginine, lysine or a combination thereof).

[0136] 31. A nucleic acid molecule encoding the polypeptide of embodiment 30.

[0137] 32. A particle comprising a protein coat comprising an anellovirus ORF1 molecule, wherein the ORF1 molecule comprises an ORF1 domain and an exogenous surface domain;

[0138] The particles are prepared by contacting a plurality of anellovirus ORF1 molecules in a cell-free solution under conditions suitable for forming a protein shell comprising the plurality of anellovirus ORF1 molecules.

[0139] 33. The particle of embodiment 32, wherein the particle does not comprise (e.g., does not surround) a polynucleotide, or does not comprise (e.g., does not surround) a polynucleotide that is greater than 1000, 500, 200, or 100 nucleotides in length.

[0140] 34. A method for preparing particles, the method comprising:

[0141] contacting the plurality of anellovirus ORF1 molecules in a cell-free solution under conditions suitable for formation of a protein coat comprising the plurality of anellovirus ORF1 molecules;

[0142] Thus, particles are prepared.

[0143] 35. A method for regulating the biological activity of a cell, the method comprising:

[0144] contacting the cell with a particle as described in any preceding embodiment;

[0145] wherein the cell comprises a moiety on its surface that binds to the exogenous surface moiety of the particle.

[0146] 36. A method of targeting particles to cells, the method comprising:

[0147] contacting the cell with a particle as described in any preceding embodiment;

[0148] wherein the cell comprises a moiety on its surface that binds to the exogenous surface moiety of the particle.

[0149] 37. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0150] (i) Structural jelly-coil region of anellovirus ORF1 molecule;

[0151] (ii) the structural N22 domain of the anellovirus ORF1 molecule; and

[0152] (iii) a portion of the structural C-terminal domain (CTD) of an anellovirus ORF1 molecule, the portion comprising a deletion of about 20-30, 30-40 (e.g., about 37), 40-50 (e.g., about 55), 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130 (e.g., about 129), 130-140 (e.g., about 131), 140-150 (e.g., about 148), or 150-160 (e.g., about 155) amino acids at the C-terminus of the structural CTD relative to the corresponding wild-type structural CTD of the anellovirus ORF1 molecule.

[0153] 38. A polypeptide comprising (e.g., in the N-terminal to C-terminal direction):

[0154] (i) optionally a first portion of the structural jelly-coil region of an Anatomic Ring Virus ORF1 molecule (e.g., beta strand BH comprising the structural jelly-coil region);

[0155] (ii) a first portion of the P1 domain of an anellovirus ORF1 molecule (e.g., a P1-1 domain as described herein);

[0156] (iii) the P2 domain of the anellovirus ORF1 molecule;

[0157] (iv) a second portion of the P1 domain of an anellovirus ORF1 molecule (e.g., a P1-2 domain as described herein);

[0158] (v) optionally a second portion of the structural jellycoil region of the Anellovirus ORF1 molecule (eg, beta strand 1 comprising the structural jellycoil region); and

[0159] (vi) a portion of the structural C-terminal domain (CTD) of an anellovirus ORF1 molecule, the portion comprising a deletion of about 20-30, 30-40 (e.g., about 37), 40-50 (e.g., about 55), 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130 (e.g., about 129), 130-140 (e.g., about 131), 140-150 (e.g., about 148), or 150-160 (e.g., about 155) amino acids at the C-terminus of the structural CTD relative to the corresponding wild-type structural CTD of the anellovirus ORF1 molecule.

[0160] 39. A polypeptide comprising (e.g., in the N-terminal to C-terminal direction):

[0161] (i) Structural jelly-coil region of anellovirus ORF1 molecule;

[0162] (ii) the structural N22 domain of the anellovirus ORF1 molecule; and

[0163] (iii) a portion of the structural CTD of an anellovirus ORF1 molecule, wherein the portion consists of the N-terminal-most 1-5, 5-10, (e.g., about 7), 10-20, 30-40, 40-50, 50-60 (e.g., about 52), 60-70 (e.g., about 69), 70-80, 80-90 (e.g., about 88), 90-100 (e.g., about 93) or 100-110 amino acids of the corresponding wild-type structural CTD of an anellovirus ORF1 molecule, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0164] 40. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0165] (i) optionally a first portion of the structural jelly-coil region of an Anatomic Ring Virus ORF1 molecule (e.g., beta strand BH comprising the structural jelly-coil region);

[0166] (ii) a first portion of the P1 domain of an anellovirus ORF1 molecule (e.g., a P1-1 domain as described herein);

[0167] (iii) the P2 domain of the anellovirus ORF1 molecule;

[0168] (iv) a second portion of the P1 domain of an anellovirus ORF1 molecule (e.g., a P1-2 domain as described herein);

[0169] (v) optionally a second portion of the structural jellycoil region of the Anellovirus ORF1 molecule (eg, beta strand 1 comprising the structural jellycoil region); and

[0170] (vi) a portion of the structural CTD of an anellovirus ORF1 molecule, wherein the portion consists of up to 1-5, 5-10, (e.g., about 7), 10-20, 30-40, 40-50, 50-60 (e.g., about 52), 60-70 (e.g., about 69), 70-80, 80-90 (e.g., about 88), 90-100 (e.g., about 93), or 100-110 amino acids from the N-terminus of the corresponding wild-type structural CTD of an anellovirus ORF1 molecule, or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0171] 41. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0172] (i) a structural jellycoil region of an anellovirus ORF1 molecule comprising the structural jellycoil sequence of a Loop 2, Loop 9, Loop 10, Loop 18, or Loop 19 anellovirus ORF1 protein (e.g., as described herein), or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and

[0173] (ii) a full-length structural N22 domain of an anellovirus ORF1 molecule comprising the structural N22 sequence of a Loop 2, Loop 9, Loop 10, Loop 18, or Loop 19 anellovirus ORF1 protein, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0174] The polypeptide does not comprise the amino acid sequence of the full-length structural C-terminal domain of loop 2, loop 9, loop 10, loop 18 or loop 19 anellovirus ORF1 protein.

[0175] 42. A polypeptide comprising (e.g., in the N-terminal to C-terminal direction):

[0176] (i) an amino acid sequence of beta chain BH of the structural jelly coil region of, optionally, Loop 2, Loop 9, Loop 10, Loop 18, or Loop 19 anellovirus ORF1 protein (e.g., as described herein), or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0177] (ii) the amino acid sequence of the full-length P1-1 domain of a Loop 2, Loop 9, Loop 10, Loop 18, or Loop 19 anellovirus ORF1 protein, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0178] (iii) the amino acid sequence of the full-length P2 domain of a Loop 2, Loop 9, Loop 10, Loop 18, or Loop 19 anellovirus ORF1 protein, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0179] (iv) the amino acid sequence of the full-length P1-2 domain of a Loop 2, Loop 9, Loop 10, Loop 18, or Loop 19 anellovirus ORF1 protein, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto; and

[0180] (v) the amino acid sequence of beta strand 1 of the structural jellycoil region of, optionally, Loop 2, Loop 9, Loop 10, Loop 18, or Loop 19 Anellovirus ORF1 protein (e.g., as described herein), or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0181] The polypeptide does not comprise the amino acid sequence of the full-length structural C-terminal domain of loop 2, loop 9, loop 10, loop 18 or loop 19 anellovirus ORF1 protein.

[0182] 43. The polypeptide of embodiment 41 or 42, comprising a fragment of the structural C-terminal domain.

[0183] 44. The polypeptide of embodiment 41 or 42, which does not comprise a fragment of the structural C-terminal domain.

[0184] 45. The polypeptide of any one of embodiments 37-44, further comprising (e.g., at the C-terminus of the C-terminal P1 subdomain sequence) a structural jelly coil I region of an Anellovirus ORF1 molecule comprising a structural jelly coil I sequence as listed in Table A2, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0185] 46. ​​The polypeptide of any one of embodiments 37-45, wherein the polypeptide does not comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the N-terminal-most amino acid residues of the structural jelly-coil BH region of the Anellovirus ORF1 molecule.

[0186] 47. The polypeptide of any preceding embodiment, further comprising a structural arginine-rich domain of an anellovirus ORF1 molecule, eg, wherein the structural arginine-rich domain is N-terminal to the structural jellycoil region.

[0187] 48. The polypeptide of any of embodiments 37-47, further comprising an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a structural arginine-rich region sequence described herein, or a sequence of at least about 40 amino acids comprising at least 60%, 70% or 80% basic residues.

[0188] 49. The polypeptide of any one of embodiments 37-48, wherein the polypeptide further comprises a deletion of the structural arginine-rich region of the Anellovirus ORF1 molecule.

[0189] 50. The polypeptide of any of embodiments 37-49, wherein the polypeptide comprises a deletion of 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, or 65-70 amino acids (e.g., 37, 40, 43, 44, 47, 49, 66, or 67 amino acids) relative to the corresponding wild-type arginine-rich region of the Anellovirus ORF1 molecule.

[0190] 51. The polypeptide of any one of embodiments 37-50, wherein the polypeptide does not comprise a deletion of the structural arginine-rich region of the Anellovirus ORF1 molecule.

[0191] 52. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise a sequence of at least 60 contiguous amino acids consisting of at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% (e.g., up to 80%) basic residues.

[0192] 53. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise a sequence of at least 60 consecutive amino acids consisting of at least 40%, 45%, 50%, 55%, 60%, 65% or 75% (e.g., up to 80%) arginine residues.

[0193] 54. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise a sequence of at least 60 contiguous amino acids consisting of at least 2%, 3%, 4%, 5%, 10% or 15% (e.g., up to 20%) lysine residues.

[0194] 55. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise a sequence of at least 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 consecutive amino acids consisting of at least 40%, 41%, 42%, 43%, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 consecutive amino acids.

[0195] 56. The polypeptide of any preceding embodiment, further comprising a structural hypervariable region (HVR) of an Anellovirus ORF1 molecule (eg, located between the structural jellycoil region and the structural N22 domain).

[0196] 57. A polypeptide comprising (e.g., in the N-terminal to C-terminal direction):

[0197] (i) the structural jelly-coil region of the anellovirus ORF1 molecule; and

[0198] (ii) the structural N22 domain of the anellovirus ORF1 molecule; and

[0199] The polypeptide lacks the structural C-terminal domain (CTD) of the anellovirus ORF1 molecule.

[0200] 58. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0201] (i) optionally a first portion of the structural jelly-coil region of an Anatomic Ring Virus ORF1 molecule (e.g., beta strand BH comprising the structural jelly-coil region);

[0202] (ii) a first portion of the P1 domain of an anellovirus ORF1 molecule (e.g., a P1-1 domain as described herein);

[0203] (iii) the P2 domain of the anellovirus ORF1 molecule;

[0204] (iv) a second portion of the P1 domain of an anellovirus ORF1 molecule (e.g., a P1-2 domain as described herein);

[0205] (v) optionally a second portion of the structural jellycoil region of the Anellovirus ORF1 molecule (eg, beta strand 1 comprising the structural jellycoil region); and

[0206] The polypeptide lacks the structural C-terminal domain (CTD) of the anellovirus ORF1 molecule.

[0207] 59. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0208] (i) the structural jelly-coil region of an Anellovirus ORF1 molecule, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto; and

[0209] (ii) the structural N22 domain of an anellovirus ORF1 molecule, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto; and

[0210] The polypeptide lacks the structural arginine-rich region of the anellovirus ORF1 molecule.

[0211] 60. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0212] (i) optionally a first portion of the structural jelly-coil region of an Anatomic Ring Virus ORF1 molecule (e.g., beta strand BH comprising the structural jelly-coil region);

[0213] (ii) a first portion of the P1 domain of an anellovirus ORF1 molecule (e.g., a P1-1 domain as described herein);

[0214] (iii) the P2 domain of the anellovirus ORF1 molecule;

[0215] (iv) a second portion of the P1 domain of an anellovirus ORF1 molecule (e.g., a P1-2 domain as described herein);

[0216] (v) optionally a second portion of the structural jellycoil region of the Anellovirus ORF1 molecule (eg, beta strand 1 comprising the structural jellycoil region); and

[0217] The polypeptide lacks the structural arginine-rich region of the anellovirus ORF1 molecule.

[0218] 61. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0219] (i) a portion of the full-length structural jelly coil region of an anellovirus ORF1 molecule, the portion not comprising the 1-10 (e.g., 7) most N-terminal amino acids of the full-length structural jelly coil region; and

[0220] (ii) the structural N22 domain of the anellovirus ORF1 molecule; and

[0221] The polypeptide lacks the structural arginine-rich region of the anellovirus ORF1 molecule.

[0222] 62. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0223] (i) a portion of a full-length β-chain BH of a structural jelly-coil region of an anellovirus ORF1 molecule, the portion not comprising the 1-10 (e.g., 7) most N-terminal amino acids of the full-length β-chain BH of the structural jelly-coil region;

[0224] (ii) a first portion of the P1 domain of an anellovirus ORF1 molecule (e.g., a P1-1 domain as described herein);

[0225] (iii) the P2 domain of the anellovirus ORF1 molecule;

[0226] (iv) a second portion of the P1 domain of an anellovirus ORF1 molecule (e.g., a P1-2 domain as described herein);

[0227] (v) optionally a second portion of the structural jellycoil region of the Anellovirus ORF1 molecule (eg, beta strand 1 comprising the structural jellycoil region); and

[0228] The polypeptide lacks the structural arginine-rich region of the anellovirus ORF1 molecule.

[0229] 63. The polypeptide of embodiment 61 or 62, wherein the Anellovirus ORF1 molecule is a loop 2 ORF1 molecule.

[0230] 64. The polypeptide of embodiment 61 or 62, wherein the Anellovirus ORF1 molecule is a loop 9 ORF1 molecule.

[0231] 65. The polypeptide of embodiment 61 or 62, wherein the Anellovirus ORF1 molecule is a loop 10 ORF1 molecule.

[0232] 66. The polypeptide of embodiment 61 or 62, wherein the Anellovirus ORF1 molecule is a loop 18 ORF1 molecule.

[0233] 67. The polypeptide of embodiment 61 or 62, wherein the Anellovirus ORF1 molecule is a Loop 19 ORF1 molecule.

[0234] 68. The polypeptide of any one of embodiments 59-67, wherein the polypeptide does not comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the N-terminal-most amino acid residues of the structural jelly-coil region of the Anellovirus ORF1 molecule.

[0235] 69. A polypeptide comprising:

[0236] (i) Structural jelly-coil region of anellovirus ORF1 molecule;

[0237] (ii) the structural N22 domain of the anellovirus ORF1 molecule; and

[0238] (iii) an amino acid sequence comprising substitutions of at least 50%, 60%, 70%, 80% or 90% of basic amino acids relative to the structural arginine-rich region of a wild-type anellovirus ORF1 molecule.

[0239] 70. A polypeptide comprising:

[0240] (i) optionally a first portion of the structural jelly-coil region of an Anatomic Ring Virus ORF1 molecule (e.g., beta strand BH comprising the structural jelly-coil region);

[0241] (ii) a first portion of the P1 domain of an anellovirus ORF1 molecule (e.g., a P1-1 domain as described herein);

[0242] (iii) the P2 domain of the anellovirus ORF1 molecule;

[0243] (iv) a second portion of the P1 domain of an anellovirus ORF1 molecule (e.g., a P1-2 domain as described herein);

[0244] (v) optionally a second portion of the structural jellycoil region of the Anellovirus ORF1 molecule (eg, beta strand 1 comprising the structural jellycoil region); and

[0245] (vi) an amino acid sequence comprising substitutions of at least 50%, 60%, 70%, 80% or 90% of basic amino acids relative to the structural arginine-rich region of a wild-type anellovirus ORF1 molecule.

[0246] 71. The polypeptide of embodiment 69 or 70, wherein (iii) comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) lysines mutated to histidines relative to the structural arginine-rich region of the wild-type anellovirus ORF1 molecule.

[0247] 72. The polypeptide of embodiment 69 or 71, comprising the amino acid sequence numbered 1721 in Table B4-2, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0248] 73. The polypeptide of embodiment 69, wherein the amino acid sequence of (iii) further comprises one or more substitutions at one or more acidic, hydrophobic, and / or non-polar amino acids in the structural arginine-rich region of the wild-type anellovirus ORF1 molecule.

[0249] 74. The polypeptide of any one of embodiments 59-73, further comprising a structural CTD, eg, a C-terminus of a structural N22 domain.

[0250] 75. The polypeptide of any of embodiments 59-74, further comprising a structural HVR, eg, located between the structural jelly-coil region and the structural N22 domain.

[0251] 76. The polypeptide of any one of embodiments 59-75, which does not substantially bind DNA.

[0252] 77. The polypeptide of any of embodiments 59-76, further comprising (e.g., at the N-terminus) an N-terminal portion of a capsid protein from a virus other than anellovirus, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto;

[0253] Optionally, the N-terminal region of the capsid protein from a virus other than anellovirus comprises at least the N-terminal 10, 20, 30, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55 or 60 amino acids of the capsid protein.

[0254] 78. The polypeptide of any of embodiments 59-77, further comprising (e.g., at the N-terminus) a structural arginine-rich motif from a capsid protein of a virus other than an Anellovirus, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0255] 79. The polypeptide of embodiment 78, wherein the virus other than anellovirus is beak and feather disease virus (BFDV).

[0256] 80. The polypeptide of any of embodiments 59-79, further comprising (e.g., at the N-terminus) the amino acid sequence MWGTSNCACAKFQIRRRYARPYRRRHIRRY RRRRRHFRRRRFTTNR, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0257] 81. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0258] (i) a structural arginine-rich region of a first anellovirus ORF1 molecule, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0259] (ii) a structural jelly-coil region of a second anellovirus ORF1 molecule, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto; and

[0260] (iii) the structural N22 domain of the second anellovirus ORF1 molecule, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0261] wherein the sequence of (i) comprises at least one amino acid sequence difference relative to the structural arginine-rich region of the second anellovirus ORF1 molecule.

[0262] 82. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0263] (i) a structural arginine-rich region of a first anellovirus ORF1 molecule, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0264] (ii) optionally a first portion of the structural jellycoil region of a second anellovirus ORF1 molecule (e.g., beta strand BH comprising the structural jellycoil region);

[0265] (iii) a first portion of the P1 domain of a second anellovirus ORF1 molecule (e.g., a P1-1 domain as described herein);

[0266] (iv) the P2 domain of the second anellovirus ORF1 molecule;

[0267] (v) a second portion of the P1 domain of a second anellovirus ORF1 molecule (e.g., a P1-2 domain as described herein);

[0268] (vi) optionally a second portion of the structural jellycoil region of a second anellovirus ORF1 molecule (eg, beta strand 1 comprising the structural jellycoil region); and

[0269] wherein the sequence of (i) comprises at least one amino acid sequence difference relative to the structural arginine-rich region of the second anellovirus ORF1 molecule.

[0270] 83. The polypeptide of embodiment 89 or 90, wherein (i) comprises the structural arginine-rich region of the first anellovirus ORF1 molecule, or an amino acid sequence at least 90% identical thereto.

[0271] 84. The polypeptide of embodiment 89 or 90, wherein (i) comprises the structural arginine-rich region of the first anellovirus ORF1 molecule, or an amino acid sequence at least 95% identical thereto.

[0272] 85. The polypeptide of embodiment 89 or 90, wherein (i) comprises the structural arginine-rich region of the first anellovirus ORF1 molecule, or an amino acid sequence at least 97% identical thereto.

[0273] 86. The polypeptide of embodiment 89 or 90, wherein (i) has 100% sequence identity to the structural arginine-rich region of the first anellovirus ORF1 molecule.

[0274] 87. The polypeptide of any one of embodiments 89-94, wherein the first Anellovirus ORF1 molecule is loop 9 and the second Anellovirus ORF1 molecule is loop 2.

[0275] 88. The polypeptide of any one of embodiments 89-95, wherein the first Anellovirus ORF1 molecule is loop 2 and the second Anellovirus ORF1 molecule is loop 9.

[0276] 89. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0277] (i) the N-terminal portion of a first anellovirus ORF1 molecule, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, wherein the N-terminal portion of the first anellovirus ORF1 molecule has a length of 30-40, 40-50, 50-60, 60-70, 70-80, or 80-90 amino acids; and

[0278] (ii) the C-terminal portion of a second anellovirus ORF1 molecule, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, wherein the C-terminal portion of the second anellovirus ORF1 molecule has a length of 590-600, 600-610, 610-620, 620-630, 630-640, 640-650, 650-660, 660-670, 670-680, 680-690, 690-700, 700-710, 710-720 or 720-730 amino acids; and

[0279] wherein the sequence of (i) comprises at least one amino acid sequence difference relative to the structural arginine-rich region of the second anellovirus ORF1 molecule.

[0280] 90. The polypeptide of embodiment 97, wherein the N-terminal portion of the first anellovirus ORF1 molecule comprises a structural arginine-rich domain.

[0281] 91. The polypeptide of embodiment 97 or 98, wherein the N-terminal portion of the first Anellovirus ORF1 molecule further comprises an N-terminal portion of a structural jellycoil domain (e.g., having a length of between 1-10, 10-20, 20-30, 30-40, or 40-50 amino acids).

[0282] 92. The polypeptide of any one of embodiments 97-99, wherein the N-terminal portion of the first anellovirus ORF1 molecule further comprises one or more beta strands of a structural jellycoil domain (e.g., beta strands 1 and / or 2 of a structural jellycoil domain).

[0283] 93. The polypeptide of any of embodiments 97-100, wherein the C-terminal portion of the second Anellovirus ORF1 molecule comprises the C-terminal portion of a structural jellycoil domain (e.g., having a length of between 100-110, 110-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, or 190-200 amino acids).

[0284] 94. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0285] (i) a first portion of the structural jellycoil region of a first anellovirus ORF1 molecule, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto; and

[0286] (ii) a second portion of the structural jellycoil region of a second anellovirus ORF1 molecule, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto; and

[0287] wherein the sequences of (i) and (ii) together comprise at least one amino acid sequence difference relative to each of the sequences of the structural jellycoil regions of the first and second anellovirus ORF1 molecules.

[0288] 95. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0289] (i) the structural jelly-coil region of the anellovirus ORF1 molecule; and

[0290] (ii) the structural N22 domain of the anellovirus ORF1 molecule; and

[0291] wherein the polypeptide lacks the structural arginine-rich region of the anellovirus ORF1 molecule; and

[0292] The polypeptide lacks the structural C-terminal domain of the anellovirus ORF1 molecule.

[0293] 96. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0294] (i) optionally a first portion of the structural jelly-coil region of an Anatomic Ring Virus ORF1 molecule (e.g., beta strand BH comprising the structural jelly-coil region);

[0295] (ii) a first portion of the P1 domain of an anellovirus ORF1 molecule (e.g., a P1-1 domain as described herein);

[0296] (iii) the P2 domain of the anellovirus ORF1 molecule;

[0297] (iv) a second portion of the P1 domain of an anellovirus ORF1 molecule (e.g., a P1-2 domain as described herein);

[0298] (v) optionally a second portion of the structural jellycoil region of the Anellovirus ORF1 molecule (eg, beta strand 1 comprising the structural jellycoil region); and

[0299] wherein the polypeptide lacks the structural arginine-rich region of the anellovirus ORF1 molecule; and

[0300] The polypeptide lacks the structural C-terminal domain of the anellovirus ORF1 molecule.

[0301] 97. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 2-45 of the amino acid sequence of the loop 2ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0302] 98. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 518-666 of the amino acid sequence of the loop 2ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0303] 99. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 538-666 of the amino acid sequence of the loop 2ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0304] 100. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 545-666 of the amino acid sequence of the RING2ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0305] 101. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 594-666 of the amino acid sequence of the loop 2ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0306] 102. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 611-666 of the amino acid sequence of the loop 2ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0307] 103. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 630-666 of the amino acid sequence of the loop 2ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0308] 104. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 2-45 of the amino acid sequence of the loop 10ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0309] 105. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 2-51 of the amino acid sequence of the loop 10ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0310] 106. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 542-672 of the amino acid sequence of the ring 10ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0311] 107. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 552-672 of the amino acid sequence of the loop 10ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0312] 108. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 557-672 of the amino acid sequence of the ring 10ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0313] 109. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 560-672 of the amino acid sequence of the loop 10ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0314] 110. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 566-672 of the amino acid sequence of the loop 10ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0315] 111. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 569-672 of the amino acid sequence of the ring 10ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0316] 112. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 610-672 of the amino acid sequence of the ring 10ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0317] 113. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 2-69 of the amino acid sequence of the loop 18ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0318] 114. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 582-736 of the amino acid sequence of loop 18ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0319] 115. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 651-736 of the amino acid sequence of loop 18ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0320] 116. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 2-49 of the amino acid sequence of loop 19ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0321] 117. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 527-655 of the amino acid sequence of loop 19ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0322] 118. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 537-655 of the amino acid sequence of loop 19ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0323] 119. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 583-655 of the amino acid sequence of the loop 19ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0324] 120. The polypeptide of any preceding embodiment, wherein the polypeptide does not comprise amino acids 600-655 of the amino acid sequence of loop 19ORF1 protein as described herein, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0325] 121. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0326] (i) a mutant ORF1 structural jelly-coil region; and

[0327] (ii) the structural N22 domain of the anellovirus ORF1 molecule;

[0328] The mutant ORF1 structural jelly coil region comprises one or more mutations (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations) in the beta chain relative to the amino acid sequence of the wild-type anellovirus ORF1 structural jelly coil region.

[0329] 122. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0330] (i) a mutant ORF1 structural jelly-coil region; and

[0331] (ii) a first portion of the P1 domain of an anellovirus ORF1 molecule (e.g., a P1-1 domain as described herein);

[0332] (iii) the P2 domain of the anellovirus ORF1 molecule;

[0333] (iv) a second portion of the P1 domain of an anellovirus ORF1 molecule (e.g., a P1-2 domain as described herein);

[0334] (v) optionally a second portion of the structural jellycoil region of the Anellovirus ORF1 molecule (eg, beta strand 1 comprising the structural jellycoil region); and

[0335] The mutant ORF1 structural jelly coil region comprises one or more mutations (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations) in the beta chain relative to the amino acid sequence of the wild-type anellovirus ORF1 structural jelly coil region.

[0336] 123. The polypeptide of embodiment 121 or 122, wherein the one or more mutations in the beta chain comprise one or more mutations (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations) of a basic residue in the amino acid sequence of the wild-type anellovirus ORF1 structural jelly coil region, such as a mutation to a residue other than a basic residue (e.g., a non-polar residue or an acidic residue).

[0337] 124. The polypeptide of any one of embodiments 121-123, wherein the beta chain is selected from beta chain B, D, G, or I of the structural jelly coil region of wild-type anellovirus ORF1.

[0338] 125. The polypeptide of any of embodiments 121-124, wherein the one or more mutations reduce binding of the polypeptide to a nucleic acid molecule relative to an otherwise similar polypeptide comprising a wild-type anellovirus ORF1 structural jellycoil region.

[0339] 126. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0340] (i) optionally a structural arginine-rich region of an Anellovirus ORF1 (e.g., the full-length arginine-rich region or a portion of the structural arginine-rich region),

[0341] (ii) the structural jelly-coil region of the anellovirus ORF1 molecule,

[0342] (iii) the P1 domain of the anellovirus ORF1 molecule,

[0343] (iv) the P2 domain of anellovirus ORF1 molecule, and

[0344] (v) optionally a structural C-terminal domain (CTD) of an Anellovirus ORF1 molecule (e.g., a full-length structural CTD or a portion of a structural CTD);

[0345] in:

[0346] (a) the P1 domain is from an anellovirus that is different from one or more (e.g., 1, 2, 3, or 4) of the structural arginine-rich region, the structural jellycoil region, the P2 domain, and / or the structural CTD;

[0347] (b) the P2 domain is from an anellovirus that is different from one or more (e.g., 1, 2, 3, or 4) of the structural arginine-rich region, the structural jelly-coil region, the Pl domain, and / or the structural CTD; or

[0348] (c) the Pl domain and the P2 domain are from an anellovirus that differs from one or more (e.g., 1, 2, or 3) of the structural arginine-rich region, the structural jellycoil region, and / or the structural CTD, optionally wherein the Pl domain and the P2 domain are from the same anellovirus.

[0349] 127. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0350] (i) optionally a structural arginine-rich region of an Orovirus ORF1 molecule (e.g., the full-length arginine-rich region or a portion of the structural arginine-rich region),

[0351] (ii) the structural jelly-coil region of the anellovirus ORF1 molecule,

[0352] (iii) the P1 domain of the anellovirus ORF1 molecule,

[0353] (iv) the P2 domain of a viral capsid protein, wherein the viral capsid protein is not an anatomic virus ORF1 molecule, and

[0354] (v) optionally the structural C-terminal domain (CTD) of an Anellovirus ORF1 molecule (eg, a full-length structural CTD or a portion of a structural CTD).

[0355] 128. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0356] (i) optionally a structural arginine-rich region of an Anellovirus ORF1 (e.g., the full-length arginine-rich region or a portion of the structural arginine-rich region),

[0357] (ii) the structural jelly-coil region of the anellovirus ORF1 molecule,

[0358] (iii) the P1 domain of a viral capsid protein, wherein the viral capsid protein is not an Alphavirus ORF1 molecule,

[0359] (iv) the P2 domain of anellovirus ORF1 molecule, and

[0360] (v) optionally the structural C-terminal domain (CTD) of an Anellovirus ORF1 molecule (eg, a full-length structural CTD or a portion of a structural CTD).

[0361] 129. The polypeptide of embodiment 127 or 128, wherein the viral capsid protein is a hepatitis virus capsid protein (e.g., a hepatitis E virus capsid protein).

[0362] 131. A polypeptide comprising (e.g., in an N-terminal to C-terminal direction):

[0363] (i) a portion (e.g., an N-terminal portion) of a viral capsid protein, wherein the viral capsid protein is not from an anellovirus; and

[0364] (ii) the P2 domain of an anellovirus ORF1 molecule (e.g., a wild-type anellovirus ORF1 protein), or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0365] 132. The polypeptide of embodiment 131, wherein the viral capsid protein is a hepatitis virus capsid protein (e.g., hepatitis E virus (HEV) capsid protein), or a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0366] 133. The polypeptide of embodiment 131 or 132, wherein the portion of the viral capsid protein comprises the amino acid sequence MAVAPAPDTAPVPDVDSRGAILRRQYNLSTSPLTSTIATGTNLVLYAAPLSSLLPLQDGTNTHIMATEASNYAQYRVVRATIRYRPLVPSAVGGYAISISFWPQTTTTPTSVDMNSITSTDVRILVQPGIASELVIPSERLHYRNQGWRSVETSGVAEEEATSGLVMLCIHGSPVNSYTNTPYT GALGLLDFALELEFRNLTPGNTNTRVSRYSSSARHKLRRGPDGTAELTTTAATRFMKDLHFTGTNDVGEVGRGIALTLFNLADTLLGGLPTELISSAGGQLFYSRPVVSANGEPTVKLYTSVENAQQDKGIAIPHDIDLGESRVVIQDYDNQHEQDRPTPSPAPSRP, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0367] 134. The polypeptide of any of embodiments 131-133, wherein the P2 domain of the amino acid sequence comprises the amino acid sequence LNTTYIQNRDWGDRNKTYYCQTLGTQRYFLYGTHSTAQNINDIKLQELIPLTNTQDYVQGFDWTEKDKHNITTYKEFLTKGAGNPFHAEWITAQNPVIHTANSPTQIEQIYTASTTTFQNKKLTDLPTPGYIFITPTV, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0368] 137. The polypeptide of embodiment 135 or 136, wherein the P2 domain of the amino acid sequence comprises the amino acid sequence LNTTYIQNRDWGDRNKTYYCQTLGTQRYFLYGTHST AQNINDIKLQELIPLTNTQDYVQGFDWTEKDKHNITTYKEFLTKGAGNPF HAEWITAQNPVIHTANSPTQIEQIYTASTTTFQNKKLTDLPTPGYIFITPTV, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto.

[0369] 138. The polypeptide of any one of embodiments 131-137, further comprising one or more additional portions of the viral capsid protein (e.g., at the C-terminus of the polypeptide).

[0370] 139. The polypeptide of embodiment 138, wherein the additional portion of the viral capsid protein comprises the amino acid sequence HHHHH (e.g., at the C-terminus of the polypeptide).

[0371] 140. The polypeptide of any one of embodiments 126-139, wherein the amino acid sequence of the P1 domain is discontinuous (e.g., wherein the amino acid sequence of the P2 domain is located between a first portion and a second portion of the amino acid sequence of the P1 domain).

[0372] 141. A polypeptide comprising (e.g., in the N-terminal to C-terminal direction) one or more:

[0373] (i) Structural arginine-rich region of anellovirus ORF1 molecule,

[0374] (ii) the structural jelly-coil region of the anellovirus ORF1 molecule,

[0375] (iii) the structural N22 domain of anellovirus ORF1 molecule, and / or

[0376] (iv) the structural C-terminal domain of the anellovirus ORF1 molecule;

[0377] wherein the polypeptide further comprises (e.g., between the structural jelly coil region and the structural N22 domain, e.g., in a structural hypervariable region (HVR) as described herein) the amino acid sequence EQI, e.g., the amino acid sequence SPTQIEQIYT; and

[0378] wherein the anellovirus ORF1 molecule of (i)-(iv) comprises at least one difference relative to the loop 10 ORF1 protein.

[0379] 142. A polypeptide comprising (e.g., in the N-terminal to C-terminal direction) one or more:

[0380] (i) Structural arginine-rich region of anellovirus ORF1 molecule,

[0381] (ii) the jelly-coil BH chain subdomain of the anellovirus ORF1 molecule,

[0382] (iii) the P1-1 subdomain of the anellovirus ORF1 molecule,

[0383] (iv) the P1-2 subdomain of the anellovirus ORF1 molecule,

[0384] (v) the jelly coil I strand subdomain of anellovirus ORF1 molecule, and / or

[0385] (vi) the structural C-terminal domain of the anellovirus ORF1 molecule;

[0386] wherein the polypeptide further comprises (e.g., between the P1-1 subdomain and the P1-2 subdomain) the amino acid sequence EQI, e.g., the amino acid sequence SPTQIEQIYT (e.g., in a P2 domain as described herein); and

[0387] wherein the anellovirus ORF1 molecule of (i)-(vi) comprises at least one difference relative to the loop 10 ORF1 protein.

[0388] 143. A polypeptide comprising a structural hypervariable region (HVR) having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the structural HVR sequence of a loop 2ORF1 protein;

[0389] The polypeptide comprises one or more (eg, 1, 2, or all 3) amino acid substitutions relative to the sequence of the loop 2ORF1 protein, the substitutions being selected from K357E, N358Q, and E359I.

[0390] 144. A polypeptide comprising a P2 domain having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the P2 domain sequence of a RING2ORF1 protein;

[0391] The polypeptide comprises one or more (eg, 1, 2, or all 3) amino acid substitutions relative to the sequence of the loop 2ORF1 protein, the substitutions being selected from K357E, N358Q, and E359I.

[0392] 145. The polypeptide of embodiment 143 or 144, wherein the polypeptide comprises all three of the substitutions selected from K357E, N358Q, and E359I.

[0393] 146. A method of enriching the polypeptide of any one of embodiments X1-X3b, the method comprising:

[0394] (a) providing the polypeptide (e.g., wherein the polypeptide is contained in a finger-ring VLP or particle);

[0395] (b) contacting the polypeptide with a binding moiety (e.g., an antibody molecule) that binds an epitope comprising the amino acid sequence EQI, and

[0396] (c) enriching material bound by the binding moiety relative to material not bound by the binding moiety, thereby enriching the polypeptide.

[0397] 147. A finger-ring carrier, comprising:

[0398] (i) a proteinaceous coat comprising the polypeptide of any preceding embodiment; and

[0399] (ii) a genetic element surrounded by the protein coat, wherein the genetic element comprises a sequence encoding an exogenous effector.

[0400] 148. A finger-ring carrier, comprising:

[0401] (i) a protein coat comprising a plurality of anellovirus ORF1 molecules, wherein the anellovirus ORF1 molecules lack part or all of the structural C-terminal domain (CTD); and

[0402] (ii) a genetic element surrounded by the protein coat, wherein the genetic element comprises a sequence encoding an exogenous effector.

[0403] 149. A finger-ring carrier, comprising:

[0404] (i) a protein coat comprising a plurality of anellovirus ORF1 molecules, wherein the anellovirus ORF1 molecules lack part or all of a structural arginine-rich region; and

[0405] (ii) a genetic element surrounded by the protein coat, wherein the genetic element comprises a sequence encoding an exogenous effector.

[0406] 150. A composition comprising a plurality of the finger-ring carriers of any one of embodiments S1-X.

[0407] 151. A particle comprising:

[0408] A protein coat comprising multiple anellovirus ORF1 molecules, wherein the anellovirus ORF1 molecules lack part or all of a structural C-terminal domain (CTD);

[0409] The particles:

[0410] (i) does not comprise (e.g., does not surround) a polynucleotide (e.g., as determined using a nuclease protection assay as described herein),

[0411] (ii) does not comprise (e.g., does not encompass) a polynucleotide greater than 1000, 500, 200, or 100 nucleotides in length, or

[0412] (iii) comprises less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides.

[0413] 152. A particle comprising:

[0414] A protein shell comprising a plurality of anellovirus ORF1 molecules, wherein the anellovirus ORF1 molecules lack some or all of the structural arginine-rich region;

[0415] The particles:

[0416] (i) does not comprise (e.g., does not surround) a polynucleotide (e.g., as determined using a nuclease protection assay as described herein),

[0417] (ii) does not comprise (e.g., does not encompass) a polynucleotide greater than 1000, 500, 200, or 100 nucleotides in length, or

[0418] (iii) comprises less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides.

[0419] 153. A particle comprising:

[0420] A protein coat comprising a plurality of anellovirus ORF1 molecules, wherein the anellovirus ORF1 molecules comprise the polypeptide of any one of the preceding embodiments;

[0421] The particles:

[0422] (i) does not comprise (e.g., does not surround) a polynucleotide (e.g., as determined using a nuclease protection assay as described herein),

[0423] (ii) does not comprise (e.g., does not encompass) a polynucleotide greater than 1000, 500, 200, or 100 nucleotides in length, or

[0424] (iii) comprises less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides.

[0425] 154. The particle of any of embodiments 151-153, wherein the protein coat comprises about 40-80 (e.g., about 60), 100-140 (e.g., about 120), or 160-200 (e.g., about 180) copies of an Anellovirus ORF1 molecule.

[0426] 155. The particle of any of embodiments 151-154, wherein the particle further comprises an exogenous effector or a nucleic acid sequence encoding an exogenous effector.

[0427] 156. A composition comprising a plurality of particles as described in any of embodiments 151-155.

[0428] 157. A nucleic acid molecule encoding the polypeptide of any preceding embodiment.

[0429] 158. A method of delivering an exogenous effector to a cell, the method comprising contacting the cell with the finger ring vector or particle of any preceding embodiment, or with a particle (e.g., a finger ring vector) comprising the polypeptide of any preceding embodiment, thereby delivering the exogenous effector to the cell.

[0430] 159. A method of delivering an exogenous effector to a subject, the method comprising administering to the subject the ring vector or particle of any one of the preceding embodiments, or a particle (e.g., a ring vector) comprising the polypeptide of any one of the preceding embodiments, thereby delivering the exogenous effector to the subject.

[0431] 160. A method for treating or preventing a disease or disorder in a subject, the method comprising administering to a subject in need thereof the ring vector or particle of any one of the preceding embodiments, or a particle (e.g., a ring vector) comprising the polypeptide of any one of the preceding embodiments, thereby treating or preventing the disease or disorder in the subject.

[0432] 161. A method for preparing a ring carrier, the method comprising:

[0433] (a) providing a host cell comprising:

[0434] (i) a plurality of anellovirus ORF1 molecules, wherein the anellovirus ORF1 molecules comprise the polypeptide of any one of the preceding embodiments, and

[0435] (ii) a genetic element, wherein the genetic element comprises a sequence encoding an exogenous effector; and

[0436] (b) maintaining the host cell under conditions that allow production of a protein coat comprising the plurality of anellovirus ORF1 molecules, wherein the protein coat surrounds the genetic element,

[0437] One or more of the finger ring vectors are thereby produced from the host cell.

[0438] 162. A method for preparing a ring carrier, the method comprising:

[0439] (a) providing a host cell comprising:

[0440] (i) a plurality of anellovirus ORF1 molecules, wherein the anellovirus ORF1 molecules lack part or all of the structural C-terminal domain (CTD), and

[0441] (ii) a genetic element, wherein the genetic element comprises a sequence encoding an exogenous effector; and

[0442] (b) maintaining the host cell under conditions that allow production of a protein coat comprising the plurality of anellovirus ORF1 molecules, wherein the protein coat surrounds the genetic element;

[0443] One or more of the finger ring vectors are thereby produced from the host cell.

[0444] 163. A method for preparing a ring carrier, the method comprising:

[0445] (a) providing a host cell comprising:

[0446] (i) a plurality of anellovirus ORF1 molecules, wherein the anellovirus ORF1 molecules lack part or all of the structural arginine-rich region, and

[0447] (ii) a genetic element, wherein the genetic element comprises a sequence encoding an exogenous effector; and

[0448] (b) maintaining the host cell under conditions that allow production of a protein coat comprising the plurality of anellovirus ORF1 molecules, wherein the protein coat surrounds the genetic element;

[0449] One or more of the finger ring vectors are thereby produced from the host cell.

[0450] 164. The method of any one of embodiments 161-163, wherein a host cell comprising the plurality of anellovirus ORF1 molecules is provided, the method comprising maintaining the host cell under conditions permissive for expression of the anellovirus ORF1 molecules.

[0451] 165. A method for preparing a ring carrier, the method comprising:

[0452] (a) providing a mixture comprising a plurality of anellovirus ORF1 molecules, wherein the anellovirus ORF1 molecules comprise a polypeptide as described in any of the preceding embodiments; optionally subjecting the mixture to denaturing conditions (e.g., providing a denaturing agent as part of the mixture or contacting the mixture with a denaturing agent), wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the plurality of ORF1 molecules are not contained in particles comprising about 40-80 (e.g., about 60) copies of an ORF1 molecule;

[0453] (b) subjecting the mixture to non-denaturing conditions (e.g., reducing the concentration of the denaturant to a level) suitable for in vitro assembly of the anellovirus ORF1 molecule (e.g., by dialysis); and

[0454] (c) incubating the anellovirus ORF1 molecule with a plurality of genetic elements under conditions suitable for assembling the anellovirus ORF1 molecule into one or more finger ring vectors each encompassing one or more of the genetic elements.

[0455] 166. A method for preparing a ring carrier, the method comprising:

[0456] (a) providing a mixture comprising a plurality of anellovirus ORF1 molecules, wherein the anellovirus ORF1 molecules lack part or all of a structural C-terminal domain (CTD); optionally subjecting the mixture to denaturing conditions (e.g., providing a denaturing agent as part of the mixture or contacting the mixture with a denaturing agent), wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the plurality of ORF1 molecules are not contained in particles comprising about 40-80 (e.g., about 60) copies of the ORF1 molecule;

[0457] (b) subjecting the mixture to non-denaturing conditions (e.g., reducing the concentration of the denaturant to a level) suitable for in vitro assembly of the anellovirus ORF1 molecule (e.g., by dialysis); and

[0458] (c) incubating the anellovirus ORF1 molecule with a plurality of genetic elements under conditions suitable for assembling the anellovirus ORF1 molecule into one or more finger ring vectors each encompassing one or more of the genetic elements.

[0459] 167. A method for preparing a ring carrier, the method comprising:

[0460] (a) providing a mixture comprising a plurality of anellovirus ORF1 molecules, wherein the anellovirus ORF1 molecules lack some or all of a structural arginine-rich region; optionally subjecting the mixture to denaturing conditions (e.g., providing a denaturing agent as part of the mixture or contacting the mixture with a denaturing agent), wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the plurality of ORF1 molecules are not contained in particles comprising about 40-80 (e.g., about 60) copies of the ORF1 molecule;

[0461] (b) subjecting the mixture to non-denaturing conditions (e.g., reducing the concentration of the denaturant to a level) suitable for in vitro assembly of the anellovirus ORF1 molecule (e.g., by dialysis); and

[0462] (c) incubating the anellovirus ORF1 molecule with a plurality of genetic elements under conditions suitable for assembling the anellovirus ORF1 molecule into one or more finger ring vectors each encompassing one or more of the genetic elements.

[0463] 168. A method for preparing a ring VLP, the method comprising:

[0464] (a) providing a mixture comprising a plurality of anellovirus ORF1 molecules, wherein the anellovirus ORF1 molecules comprise a polypeptide as described in any of the preceding embodiments, and wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the plurality of ORF1 molecules are not contained in particles comprising about 40-80 (e.g., about 60) copies of the ORF1 molecule;

[0465] (b) subjecting the mixture to conditions suitable for in vitro assembly of the anellovirus ORF1 molecule; and

[0466] (c) incubating the anellovirus ORF1 molecule with multiple effectors (eg, exogenous effectors) under conditions suitable for the anellovirus ORF1 molecule to assemble into one or more anellovirus VLPs that each enclose one or more of the effectors.

[0467] 169. A method for preparing a ring VLP, the method comprising:

[0468] (a) providing a mixture comprising a plurality of anellovirus ORF1 molecules, wherein the anellovirus ORF1 molecules lack part or all of a structural C-terminal domain (CTD), and wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the plurality of ORF1 molecules are not contained in particles comprising about 40-80 (e.g., about 60) copies of the ORF1 molecule;

[0469] (b) subjecting the mixture to conditions suitable for in vitro assembly of the anellovirus ORF1 molecule; and

[0470] (c) incubating the anellovirus ORF1 molecule with multiple effectors (eg, exogenous effectors) under conditions suitable for the anellovirus ORF1 molecule to assemble into one or more anellovirus VLPs that each enclose one or more of the effectors.

[0471] 170. A method for preparing a ring VLP, the method comprising:

[0472] (a) providing a mixture comprising a plurality of anellovirus ORF1 molecules, wherein the anellovirus ORF1 molecules lack some or all of a structural arginine-rich region, and wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the plurality of ORF1 molecules are not contained in particles comprising about 40-80 (e.g., about 60) copies of the ORF1 molecule;

[0473] (b) subjecting the mixture to conditions suitable for in vitro assembly of the anellovirus ORF1 molecule; and

[0474] (c) incubating the anellovirus ORF1 molecule with multiple effectors (eg, exogenous effectors) under conditions suitable for the anellovirus ORF1 molecule to assemble into one or more anellovirus VLPs that each enclose one or more of the effectors.

[0475] 171. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or Anellovirus ORF1 molecule comprises an exogenous surface moiety.

[0476] 172. The polypeptide, particle, nucleic acid molecule, or method of embodiment 171, wherein the exogenous surface moiety is fused to the N-terminus of the Anellovirus ORF1 molecule.

[0477] 173. The polypeptide, particle, nucleic acid molecule, or method of embodiment 171, wherein the exogenous surface moiety is fused to the C-terminus of the Anellovirus ORF1 molecule.

[0478] 174. The polypeptide, particle, nucleic acid molecule, or method of embodiment 171, wherein the exogenous surface moiety is inserted within the amino acid sequence of the Anellovirus ORF1 molecule.

[0479] 175. The polypeptide, particle, nucleic acid molecule or method of any of embodiments 172-174, wherein the anellovirus ORF1 molecule comprises a portion of the structural C-terminal domain (CTD) of an anellovirus ORF1 molecule comprising a deletion of about 20-30, 30-40 (e.g., about 37), 40-50 (e.g., about 55), 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130 (e.g., about 129), 130-140 (e.g., about 131), 140-150 (e.g., about 148) or 150-160 (e.g., about 155) amino acids at the C-terminus of the structural CTD relative to the corresponding wild-type structural CTD of the anellovirus ORF1 molecule.

[0480] 176. The polypeptide, particle, nucleic acid molecule or method of any of embodiments 172-174, wherein the anellovirus ORF1 molecule comprises a portion of the structural CTD of an anellovirus ORF1 molecule, wherein the portion consists of up to 1-5, 5-10, (e.g., about 7), 10-20, 30-40, 40-50, 50-60 (e.g., about 52), 60-70 (e.g., about 69), 70-80, 80-90 (e.g., about 88), 90-100 (e.g., about 93) or 100-110 amino acids from the N-terminus of the corresponding wild-type structural CTD of an anellovirus ORF1 molecule, or a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0481] 177. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or Anellovirus ORF1 molecule comprises a detectable marker or effector (eg, an exogenous effector).

[0482] 178. The polypeptide, particle, nucleic acid molecule, or method of embodiment 177, wherein the detectable marker or effector is fused to the N-terminus of the Anellovirus ORF1 molecule.

[0483] 179. The polypeptide, particle, nucleic acid molecule, or method of embodiment 177, wherein the detectable marker or effector is fused to the C-terminus of the Anellovirus ORF1 molecule.

[0484] 180. The polypeptide, particle, nucleic acid molecule or method of embodiment 177, wherein the detectable marker or effector is inserted within the amino acid sequence of the Anellovirus ORF1 molecule.

[0485] 181. The polypeptide, particle, nucleic acid molecule or method of any one of embodiments 177-180, wherein the detectable marker comprises an epitope tag, e.g., a His tag or a FLAG tag.

[0486] 182. The polypeptide, particle, nucleic acid molecule or method of any of embodiments 177-181, wherein the detectable marker comprises a fluorescent protein (e.g., GFP).

[0487] 183. The polypeptide, particle, nucleic acid molecule or method of any of embodiments 177-182, wherein the polypeptide or Anellovirus ORF1 molecule comprises a protease recognition sequence (e.g., a 3C protease recognition sequence) between the detectable marker or effector and the remainder of the polypeptide or Anellovirus ORF1 molecule.

[0488] 184. The polypeptide, particle, nucleic acid molecule or method of any of embodiments 177-183, wherein the effector is a therapeutic effector (e.g., a therapeutic polypeptide or therapeutic nucleic acid molecule).

[0489] 185. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule comprises a methionine residue at its N-terminus that corresponds to the N-terminal methionine residue of the anellovirus structural arginine-rich region.

[0490] 186. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the anellovirus ORF1 molecule is a loop 2 ORF1 molecule (e.g., as described herein), or an ORF1 molecule having at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0491] 187. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the anellovirus ORF1 molecule is the ORF1 molecule of SEQ ID NO:58, or an ORF1 molecule having at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0492] 188. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the anellovirus ORF1 molecule is a loop9 ORF1 molecule (e.g., as described herein), or an ORF1 molecule having at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0493] 189. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the anellovirus ORF1 molecule is the ORF1 molecule of SEQ ID NO: 1005, or an ORF1 molecule having at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0494] 190. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the anellovirus ORF1 molecule is a loop 10 ORF1 molecule (e.g., as described herein), or an ORF1 molecule having at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0495] 191. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the anellovirus ORF1 molecule is the ORF1 molecule of SEQ ID NO: 1012, or an ORF1 molecule having at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0496] 192. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the anellovirus ORF1 molecule is a loop 18 ORF1 molecule (e.g., as described herein), or an ORF1 molecule having at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0497] 193. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the anellovirus ORF1 molecule is the ORF1 molecule of SEQ ID NO: 1100, or an ORF1 molecule having at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0498] 194. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the anellovirus ORF1 molecule is a loop 19 ORF1 molecule (e.g., as described herein), or an ORF1 molecule having at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0499] 195. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the anellovirus ORF1 molecule is an ORF1 molecule of Table Bl-lla, or an ORF1 molecule having at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0500] 196. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or Anellovirus ORF1 molecule comprises an amino acid sequence as shown in any one of Tables Bl-1 to B4-5, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0501] 197. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or Anellovirus ORF1 molecule comprises an amino acid sequence as shown in any one of Tables Bl-1 to B4-5, or an amino acid sequence having at least 90% sequence identity thereto.

[0502] 198. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or Anellovirus ORF1 molecule comprises an amino acid sequence as shown in any one of Tables Bl-1 to B4-5, or an amino acid sequence having at least 95% sequence identity thereto.

[0503] 199. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or Anellovirus ORF1 molecule comprises an amino acid sequence as shown in any one of Tables Bl-1 to B4-5, or an amino acid sequence having at least 99% sequence identity thereto.

[0504] 200. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule comprises a structural arginine-rich region sequence of Loop 2, Loop 9, Loop 10, Loop 18 or Loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0505] 201. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule comprises a structural jellycoil domain sequence of Loop 2, Loop 9, Loop 10, Loop 18 or Loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0506] 202. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule comprises a loop 2, loop 9, loop 10, loop 18 or loop 19 structural hypervariable domain sequence, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0507] 203. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule comprises a structural N22 domain sequence of Loop 2, Loop 9, Loop 10, Loop 18 or Loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0508] 204. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule comprises a structural C-terminal domain sequence of Loop 2, Loop 9, Loop 10, Loop 18 or Loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0509] 205. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule does not comprise the structural arginine-rich region sequence of Loop 2, Loop 9, Loop 10, Loop 18 or Loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0510] 206. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule does not comprise the structural jellycoil domain sequence of Loop 2, Loop 9, Loop 10, Loop 18 or Loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0511] 207. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule does not comprise a loop 2, loop 9, loop 10, loop 18 or loop 19 structural hypervariable domain sequence, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0512] 208. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule does not comprise a structural N22 domain sequence of Loop 2, Loop 9, Loop 10, Loop 18 or Loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0513] 209. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule does not comprise a Loop 2, Loop 9, Loop 10, Loop 18 or Loop 19 structural C-terminal domain sequence, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0514] 210. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule comprises a structural arginine-rich region sequence of Loop 2, Loop 9, Loop 10, Loop 18 or Loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0515] 211. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule comprises a beta chain BH of a structural jelly-coil domain sequence of loop 2, loop 9, loop 10, loop 18 or loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0516] 212. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule comprises a P1-1 domain sequence of Loop 2, Loop 9, Loop 10, Loop 18 or Loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0517] 213. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule comprises a P2 domain sequence of Loop 2, Loop 9, Loop 10, Loop 18 or Loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0518] 214. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule comprises a P1-2 domain sequence of Loop 2, Loop 9, Loop 10, Loop 18 or Loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0519] 215. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule comprises beta strand I of the structural jellycoil domain sequence of loop 2, loop 9, loop 10, loop 18 or loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0520] 216. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule comprises a structural C-terminal domain sequence of Loop 2, Loop 9, Loop 10, Loop 18 or Loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0521] 217. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule does not comprise the structural arginine-rich region sequence of Loop 2, Loop 9, Loop 10, Loop 18 or Loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0522] 218. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule does not comprise the beta chain BH of the structural jellycoil domain sequence of loop 2, loop 9, loop 10, loop 18 or loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0523] 219. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule does not comprise a P1-1 domain sequence of Loop 2, Loop 9, Loop 10, Loop 18 or Loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0524] 220. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule does not comprise a P2 domain sequence of Loop 2, Loop 9, Loop 10, Loop 18 or Loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0525] 221. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule does not comprise a P1-2 domain sequence of Loop 2, Loop 9, Loop 10, Loop 18 or Loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0526] 222. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule does not comprise beta strand I of the structural jellycoil domain sequence of loop 2, loop 9, loop 10, loop 18 or loop 19, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0527] 223. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule does not comprise a Loop 2, Loop 9, Loop 10, Loop 18 or Loop 19 structural C-terminal domain sequence, or an amino acid sequence having at least 75%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0528] 224. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or anellovirus ORF1 molecule comprises one or more (e.g., 1, 2, or all 3) amino acid substitutions relative to the sequence of the loop 2 ORF1 protein selected from the group consisting of K357E, N358Q, and E359I.

[0529] 225. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or Anellovirus ORF1 molecule (eg, within a structural HVR domain as described herein) comprises the amino acid sequence SPTQIEQIYT.

[0530] 226. The polypeptide, particle, nucleic acid molecule or method of any preceding embodiment, wherein the polypeptide or Anellovirus ORF1 molecule (eg, in a P2 domain as described herein) comprises the amino acid sequence SPTQIEQIYT.

[0531] 227. A finger-ring carrier, comprising:

[0532] (i) a protein coat comprising: an Anellovirus ORF1 protein listed in Table A26, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and

[0533] (ii) a genetic element surrounded by the protein coat, wherein the genetic element comprises a promoter element operably linked to a nucleic acid sequence (eg, a DNA sequence) encoding an exogenous effector.

[0534] 228. A finger-ring carrier, comprising:

[0535] (i) a protein coat comprising: an Anellovirus ORF1 protein listed in Table A26, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and

[0536] (ii) a genetic element surrounded by the proteinaceous coat, wherein the genetic element comprises a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector);

[0537] wherein the protein coat and / or the genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type anellovirus ORF1 protein and / or a wild-type anellovirus genome (e.g., as described herein), respectively, such as an insertion, substitution, chemical or enzymatic modification, and / or deletion, such as a deletion of a domain (e.g., one or more of a structural arginine-rich region, a structural jelly-coil domain, a structural HVR, a structural N22, a structural CTD, a P1 domain, or a P2 domain, e.g., as described herein) or a genomic region (e.g., one or more of a TATA box, a capping site, a transcription start site, a 5'UTR, an open reading frame (ORF), a poly(A) signal, or a GC-rich region, e.g., as described herein).

[0538] 229. A finger-ring carrier, comprising:

[0539] (i) a protein coat comprising a polypeptide encoded by an Anellovirus ORF1 nucleic acid sequence listed in Table N24, or a polypeptide encoded by a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the Anellovirus ORF1 nucleic acid sequence, and

[0540] (ii) a genetic element surrounded by the protein coat, wherein the genetic element comprises a promoter element operably linked to a nucleic acid sequence (eg, a DNA sequence) encoding an exogenous effector.

[0541] 230. A finger-ring carrier, comprising:

[0542] (i) a protein coat comprising a polypeptide encoded by an Anellovirus ORF1 nucleic acid sequence listed in Table N24, or a polypeptide encoded by a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the Anellovirus ORF1 nucleic acid sequence, and

[0543] (ii) a genetic element surrounded by the proteinaceous coat, wherein the genetic element comprises a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector);

[0544] wherein the protein coat and / or the genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type anellovirus ORF1 protein and / or a wild-type anellovirus genome (e.g., as described herein), respectively, such as an insertion, substitution, chemical or enzymatic modification, and / or deletion, such as a deletion of a domain (e.g., one or more of a structural arginine-rich region, a structural jelly-coil domain, a structural HVR, a structural N22, a structural CTD, a P1 domain, or a P2 domain, e.g., as described herein) or a genomic region (e.g., one or more of a TATA box, a capping site, a transcription start site, a 5'UTR, an open reading frame (ORF), a poly(A) signal, or a GC-rich region, e.g., as described herein).

[0545] 231. A finger-ring carrier, comprising:

[0546] (i) a protein coat (e.g., a protein coat comprising, for example, an Anellovirus ORF1 molecule as described herein, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto), and

[0547] (ii) a genetic element surrounded by the protein coat, wherein the genetic element comprises: (a) a 5'UTR conserved domain as listed in Table N24, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto and (b) a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an exogenous effector.

[0548] 232. A finger-ring carrier, comprising:

[0549] (i) a protein coat (e.g., a protein coat comprising, for example, an Anellovirus ORF1 molecule as described herein, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto), and

[0550] (ii) a genetic element surrounded by the protein coat, wherein the genetic element comprises: (a) a 5'UTR conserved domain as listed in Table N24, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto and (b) a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector);

[0551] wherein the protein coat and / or the genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type anellovirus ORF1 protein and / or a wild-type anellovirus genome (e.g., as described herein), respectively, such as an insertion, substitution, chemical or enzymatic modification, and / or deletion, such as a deletion of a domain (e.g., one or more of a structural arginine-rich region, a structural jelly-coil domain, a structural HVR, a structural N22, a structural CTD, a P1 domain, or a P2 domain, e.g., as described herein) or a genomic region (e.g., one or more of a TATA box, a capping site, a transcription start site, a 5'UTR, an open reading frame (ORF), a poly(A) signal, or a GC-rich region, e.g., as described herein).

[0552] 233. A finger-ring carrier, comprising:

[0553] (i) a protein coat (e.g., a protein coat comprising, for example, an Anellovirus ORF1 molecule as described herein, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto), and

[0554] (ii) a genetic element surrounded by the protein coat, wherein the genetic element comprises a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an exogenous effector, and wherein the genetic element has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an Anellovirus genomic sequence listed in Table N24.

[0555] 234. A finger-ring carrier, comprising:

[0556] (i) a protein coat (e.g., a protein coat comprising, for example, an Anellovirus ORF1 molecule as described herein, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto), and

[0557] (ii) a genetic element surrounded by the proteinaceous coat, wherein the genetic element comprises a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector), and wherein the genetic element has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an Anellovirus genomic sequence listed in Table N24;

[0558] wherein the protein coat and / or the genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type anellovirus ORF1 protein and / or a wild-type anellovirus genome (e.g., as described herein), such as an insertion, substitution, chemical or enzymatic modification, and / or deletion, such as a deletion of a structural domain (e.g., one or more of a structural arginine-rich region, a structural jelly-coil domain, a structural HVR, a structural N22, or a structural CTD, such as described herein) or a genomic region (e.g., one or more of a TATA box, a capping site, a transcription start site, a 5'UTR, an open reading frame (ORF), a poly(A) signal, or a GC-rich region, such as described herein).

[0559] 235. An isolated ORF1 molecule comprising an amino acid sequence of ORF1 listed in Table A26, or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto;

[0560] wherein the ORF1 molecule comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type ORF1 protein (e.g., as described herein), such as an insertion, substitution, chemical or enzymatic modification, and / or deletion, such as a deletion of a domain (e.g., one or more of a structural arginine-rich region, a structural jelly-coil domain, a structural HVR, a structural N22, a structural CTD, a P1 domain, or a P2 domain, e.g., as described herein).

[0561] 236. An isolated ORF1 molecule comprising the amino acid sequence of the structural jellycoil domain of ORF1 listed in Table A26, or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto;

[0562] wherein the ORF1 molecule comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type ORF1 protein (e.g., as described herein), such as an insertion, substitution, chemical or enzymatic modification, and / or deletion, such as a deletion of a domain (e.g., one or more of a structural arginine-rich region, a jelly-coil domain, a structural HVR, a structural N22, a structural CTD, a P1 domain, or a P2 domain, e.g., as described herein).

[0563] 237. An isolated ORF2 molecule comprising the amino acid sequence of ORF2 listed in Table A26, or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto;

[0564] wherein the ORF2 molecule comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type ORF2 protein (e.g., as described herein), such as an insertion, substitution, chemical or enzymatic modification, and / or deletion, such as a deletion of a domain.

[0565] 238. An isolated nucleic acid molecule (e.g., a genetic element construct or genetic element) comprising a nucleic acid sequence of a 5'UTR conserved domain listed in Table N24, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0566] 239. An isolated nucleic acid molecule (e.g., a genetic element construct or construct for providing, in trans, an ORF1 molecule, e.g., as described herein) comprising a nucleic acid sequence of an ORF1 gene listed in Table N24, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0567] 240. An isolated nucleic acid molecule (e.g., a genetic element construct or construct for providing an ORF2 molecule, e.g., as described herein, in trans) comprising a nucleic acid sequence of an ORF2 gene listed in Table N24, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0568] 241. An isolated nucleic acid molecule (e.g., a genetic element construct, genetic element, or construct for providing, in trans, an ORF1 or ORF2 molecule, e.g., as described herein) comprising an Anellovirus genomic sequence listed in Table N24, or a nucleic acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0569] 242. A genetic element comprising:

[0570] (a) a 5'UTR conserved domain as listed in Table N24, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, and

[0571] (b) A promoter element operably linked to a nucleic acid sequence (eg, a DNA sequence) encoding an exogenous effector.

[0572] 243. A method for preparing a ring carrier composition, the method comprising:

[0573] (a) providing a cell, such as a host cell as described herein;

[0574] (b) introducing into the cell a nucleic acid molecule encoding an ORF1 polypeptide as listed in Table A26 (or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereof);

[0575] (c) introducing a genetic element construct into the cell (e.g., before, after, or simultaneously with (b)),

[0576] (d) incubating the cell under conditions that allow the cell to produce the ring vector; and

[0577] (e) formulating the ring carrier into a pharmaceutical composition suitable for administration to a subject,

[0578] Thus, the finger ring carrier composition was prepared.

[0579] 244. A method for preparing a ring carrier composition, the method comprising:

[0580] (a) providing a cell, such as a host cell as described herein;

[0581] (b) introducing a nucleic acid molecule encoding an ORF1 polypeptide into the cell;

[0582] (c) introducing into the cell (e.g., before, after, or simultaneously with (b)) a genetic element construct as listed in Table N24 (or a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto),

[0583] (d) incubating the cell under conditions that allow the cell to produce the ring vector; and

[0584] (e) formulating the ring carrier into a pharmaceutical composition suitable for administration to a subject,

[0585] Thus, the finger ring carrier composition was prepared.

[0586] 245. A method for preparing a finger ring vector, such as a synthetic finger ring vector, comprising:

[0587] (a) providing a host cell comprising:

[0588] (i) a nucleic acid molecule, e.g., a first nucleic acid molecule, comprising a nucleic acid sequence of an Anellovirus genome listed in Table N24 (or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto), and

[0589] (ii) a nucleic acid molecule, e.g., a second nucleic acid molecule, encoding one or more of the following: an amino acid sequence selected from, e.g., ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, or ORF1 / 2 as listed in Table A1, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and

[0590] (b) culturing the host cell under conditions suitable for producing the finger ring vector.

[0591] 246. A method of delivering an effector to an eye of a subject, the method comprising administering to the eye of the subject the finger ring vector of any one of embodiments 227-234.

[0592] 247. A method of modulating a biological function in an eye of a subject, the method comprising administering to the subject the finger ring vector of any one of embodiments 227-234.

[0593] 248. A method of treating an eye disease or disorder in a subject in need thereof, the method comprising administering to the subject the finger ring vector of any one of embodiments 227-234.

[0594] 249. An ORF1 molecule comprising an exogenous surface moiety, wherein the exogenous surface moiety is fused to, replaces, and / or is located within an insertion point of an ORF1 domain (eg, within an HVR or P2).

[0595] 250. An ORF1 molecule comprising an exogenous surface moiety, wherein the exogenous surface moiety is fused to, replaces, and / or is located at an insertion point between residues corresponding to positions 284 to 285 of loop 10 ORF1, e.g., in an ORF1 domain (e.g., within an HVR or P2 domain).

[0596] 251. An ORF1 molecule comprising an exogenous surface moiety, wherein the exogenous surface moiety is fused to, replaces, and / or is located at an insertion point between residues corresponding to positions 328 to 329 in a loop 10 ORF1, e.g., an ORF1 domain (e.g., within an HVR or P2 domain).

[0597] 252. An ORF1 molecule comprising an exogenous surface moiety, wherein the exogenous surface moiety is fused to, replaces, and / or is located at an insertion point between residues corresponding to positions 256 to 383 of loop 10 ORF1, e.g., in an ORF1 domain (e.g., within an HVR or P2 domain).

[0598] 253. An ORF1 molecule comprising an exogenous surface moiety, wherein the exogenous surface moiety is fused to, replaces, and / or is located at an insertion point between residues corresponding to positions 251 to 383 of loop 10 ORF1, e.g., in an ORF1 domain (e.g., within an HVR or P2 domain).

[0599] 254. An ORF1 molecule comprising an exogenous surface moiety, wherein the exogenous surface moiety is fused to, replaces, and / or is located at an insertion point between residues corresponding to positions 251 to 384 of loop 10 ORF1, e.g., in an ORF1 domain (e.g., within an HVR or P2 domain).

[0600] 255. An ORF1 molecule comprising an exogenous surface moiety, wherein the exogenous surface moiety is attached to (e.g., conjugated to) positions 254, 263, 264, 265, 272, 273, 274, 276, 283, 284, 285, 287, 288, 290, 291, 292, 293, 294, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332 374, 376, 378, 380, or 381 corresponding to an amino acid residue (e.g., a cysteine ​​residue).

[0601] 256. The ORF1 molecule of any one of embodiments 249 to 255, wherein the exogenous surface portion forms a pentamer when the ORF1 molecule is complexed with four other ORF1 molecules (eg, four other copies of the ORF1 molecule).

[0602] 257. The ORF1 molecule of any one of embodiments 249 to 256, wherein the exogenous surface portion forms a trimer when the ORF1 molecule is complexed with four other ORF1 molecules (eg, four other copies of the ORF1 molecule).

[0603] 258. The ORF1 molecule of any one of embodiments 249 to 257, wherein the exogenous surface moiety forms a dimer when the ORF1 molecule is complexed with four other ORF1 molecules (eg, four other copies of the ORF1 molecule).

[0604] 259. A protein complex comprising five ORF1 molecules, wherein each of the ORF1 molecules comprises:

[0605] (i) ORF1 domain, and

[0606] (ii) exogenous surface portion;

[0607] The exogenous surface portions of the five ORF1 molecules form a pentamer.

[0608] 260. The protein complex of embodiment 259, wherein each of the exogenous surface moieties is fused to, replaces, and / or is located within an insertion point of the ORF1 domain (e.g., within an HVR or P2) of the corresponding ORF1 molecule.

[0609] 261. The protein complex of embodiment 259, wherein each of the exogenous surface moieties is fused to, replaces, and / or is located within an insertion point between residues corresponding to positions 284 to 285 in the ORF1 domain of loop 10ORF1 (e.g., within an HVR or P2 domain).

[0610] 262. The protein complex of embodiment 259, wherein each of the exogenous surface moieties is fused to, replaces, and / or is located within an insertion point between residues corresponding to positions 328 to 329 in the ORF1 domain of loop 10ORF1 (e.g., within an HVR or P2 domain).

[0611] 263. The protein complex of embodiment 259, wherein each of the exogenous surface moieties is fused to, replaces, and / or is located within an insertion point between residues corresponding to positions 256 to 383 in the ORF1 domain of loop 10ORF1 (e.g., within an HVR or P2 domain).

[0612] 264. The protein complex of embodiment 259, wherein each of the exogenous surface moieties is fused to, replaces, and / or is located within an insertion point between residues corresponding to positions 251 to 383 in the ORF1 domain of loop 10ORF1 (e.g., within an HVR or P2 domain).

[0613] 265. The protein complex of embodiment 259, wherein each of the exogenous surface moieties is fused to, replaces, and / or is located within an insertion point between residues corresponding to positions 251 to 384 in the ORF1 domain of loop 10ORF1 (e.g., within an HVR or P2 domain).

[0614] 266. The protein complex of embodiment 259, wherein each of the exogenous surface moieties is attached to (e.g., conjugated to) positions 254, 263, 264, 265, 272, 273, 274, 276, 283, 284, 285, 287, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332 1, 308, 311, 312, 313, 314, 316, 317, 318, 319, 321, 324, 328, 329, 341, 343, 354, 358, 361, 362, 363, 364, 365, 368, 369, 371, 374, 376, 378, 380 or 381 corresponding amino acid residues (e.g., cysteine ​​residues).

[0615] 267. The protein complex of any one of embodiments 259-266, wherein the exogenous surface portions of the ORF1 molecules have the same amino acid sequence.

[0616] 268. The protein complex of any one of embodiments 259 to 267, wherein at least two (eg, at least 2, 3, 4, or 5) of the exogenous surface portions of the ORF1 molecule have different amino acid sequences.

[0617] 269. A protein complex comprising three ORF1 molecules, wherein each of the ORF1 molecules comprises:

[0618] (i) ORF1 domain, and

[0619] (ii) exogenous surface portion;

[0620] The exogenous surface portions of the three ORF1 molecules form a trimer.

[0621] 270. The protein complex of embodiment 269, wherein each of the exogenous surface moieties is fused to, replaces, and / or is located within an insertion point of the ORF1 domain (e.g., within an HVR or P2) of the corresponding ORF1 molecule.

[0622] 271. The protein complex of embodiment 269, wherein each of the exogenous surface moieties is fused to, replaces, and / or is located within an insertion point between positions 284 and 285 in the ORF1 domain (e.g., within an HVR or P2 domain) of the corresponding ORF1 molecule.

[0623] 272. The protein complex of embodiment 269, wherein each of the exogenous surface moieties is fused to, replaces, and / or is located within an insertion point between positions 328 and 329 in the ORF1 domain (e.g., within an HVR or P2 domain) of the corresponding ORF1 molecule.

[0624] 273. The protein complex of embodiment 269, wherein each of the exogenous surface moieties is fused to, replaces, and / or is located within an insertion point between positions 256 to 383 in the ORF1 domain (e.g., within an HVR or P2 domain) of the corresponding ORF1 molecule.

[0625] 274. The protein complex of embodiment 269, wherein each of the exogenous surface moieties is fused to, replaces, and / or is located within an insertion point between positions 251 to 383 in the ORF1 domain (e.g., within an HVR or P2 domain) of the corresponding ORF1 molecule.

[0626] 275. The protein complex of embodiment 269, wherein each of the exogenous surface moieties is fused to, replaces, and / or is located within an insertion point between positions 251 to 384 in the ORF1 domain (e.g., within an HVR or P2 domain) of the corresponding ORF1 molecule.

[0627] 276. The protein complex of embodiment 269, wherein each of the exogenous surface moieties is attached to (e.g., conjugated to) a corresponding ORF1 molecule, e.g., at position 254, 263, 264, 265, 272, 273, 274, 276, 283, 284, 285, 287, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331 374, 376, 378, 380, or 381.

[0628] 277. The protein complex of any one of embodiments 269-276, wherein the exogenous surface portions of the ORF1 molecules have the same amino acid sequence.

[0629] 278. The protein complex of any one of embodiments 269 to 277, wherein at least two (eg, at least 2 or 3) of the exogenous surface portions of the ORF1 molecule have different amino acid sequences.

[0630] 279. A protein complex comprising two ORF1 molecules, wherein each of the ORF1 molecules comprises:

[0631] (i) ORF1 domain, and

[0632] (ii) exogenous surface portion;

[0633] The exogenous surface portions of the two ORF1 molecules form a dimer.

[0634] 280. The protein complex of embodiment 279, wherein each of the exogenous surface moieties is fused to, replaces, and / or is located within an insertion point of the ORF1 domain (e.g., within an HVR or P2) of the corresponding ORF1 molecule.

[0635] 281. The protein complex of embodiment 279, wherein each of the exogenous surface moieties is fused to, replaces, and / or is located within an insertion point between residues corresponding to positions 284 to 285 in the ORF1 domain of loop 10ORF1 (e.g., within an HVR or P2 domain).

[0636] 282. The protein complex of embodiment 279, wherein each of the exogenous surface moieties is fused to, replaces, and / or is located within an insertion point between residues corresponding to positions 328 to 329 in the ORF1 domain of loop 10ORF1 (e.g., within an HVR or P2 domain).

[0637] 283. The protein complex of embodiment 279, wherein each of the exogenous surface moieties is fused to, replaces, and / or is located within an insertion point between residues corresponding to positions 256 to 383 in the ORF1 domain of loop 10ORF1 (e.g., within an HVR or P2 domain).

[0638] 284. The protein complex of embodiment 279, wherein each of the exogenous surface moieties is fused to, replaces, and / or is located within an insertion point between residues corresponding to positions 251 to 383 in the ORF1 domain of loop 10ORF1 (e.g., within an HVR or P2 domain).

[0639] 285. The protein complex of embodiment 279, wherein each of the exogenous surface moieties is fused to, replaces, and / or is located within an insertion point between residues corresponding to positions 251 to 384 in the ORF1 domain of loop 10ORF1 (e.g., within an HVR or P2 domain).

[0640] 286. The protein complex of embodiment 279, wherein each of the exogenous surface moieties is attached to (e.g., conjugated to) positions 254, 263, 264, 265, 272, 273, 274, 276, 283, 284, 285, 287, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331 1, 308, 311, 312, 313, 314, 316, 317, 318, 319, 321, 324, 328, 329, 341, 343, 354, 358, 361, 362, 363, 364, 365, 368, 369, 371, 374, 376, 378, 380 or 381 corresponding amino acid residues (e.g., cysteine ​​residues).

[0641] 287. The protein complex of any one of embodiments 279-286, wherein the exogenous surface portions of the two ORF1 molecules have the same amino acid sequence.

[0642] 288. The protein complex of any one of embodiments 279-287, wherein the exogenous surface portions of the two ORF1 molecules have different amino acid sequences.

[0643] 289. The polypeptide, particle, nucleic acid molecule, method or protein complex of any preceding embodiment, wherein the polypeptide or ORF1 molecule comprises one or more substitutions of a cysteine ​​residue (e.g., one or more cysteine ​​to alanine substitutions, or one or more cysteine ​​to serine substitutions).

[0644] 290. The polypeptide, particle, nucleic acid molecule, method or protein complex of any preceding embodiment, wherein the polypeptide or ORF1 molecule comprises a cysteine ​​to serine mutation at one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) residues corresponding to positions 63, 70, 137, 269, 403, 460, 503 and / or 515 of loop 10 ORF1 protein (e.g., as described herein).

[0645] 291. The polypeptide, particle, nucleic acid molecule, method or protein complex of any preceding embodiment, wherein the polypeptide or ORF1 molecule comprises a cysteine ​​to alanine mutation at one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) residues corresponding to positions 63, 137, 269, 403, 460, 503 and / or 515 of loop 10 ORF1 protein (e.g., as described herein).

[0646] 292. The polypeptide, particle, nucleic acid molecule, method or protein complex of any preceding embodiment, wherein the polypeptide or ORF1 molecule comprises a cysteine ​​to serine mutation at the residue corresponding to position 70 of loop 10 ORF1 protein (eg, as described herein).

[0647] 293. The polypeptide, particle, nucleic acid molecule, method or protein complex of any preceding embodiment, wherein the polypeptide or ORF1 molecule comprises a substitution of an amino acid residue (e.g., a threonine, serine, asparagine, alanine, glutamine or lysine residue) to cysteine.

[0648] 294. The polypeptide, particle, nucleic acid molecule, method, or protein complex of embodiment 293, wherein the polypeptide or ORF1 molecule comprises a threonine to cysteine ​​substitution (e.g., at a position corresponding to position 365 of a ring 10 ORF1 protein (e.g., as described herein)).

[0649] 295. The polypeptide, particle, nucleic acid molecule, method or protein complex of embodiment 293, wherein the polypeptide or ORF1 molecule comprises a serine to cysteine ​​substitution (e.g., at a position corresponding to position 284 of a ring 10 ORF1 protein (e.g., as described herein)).

[0650] 296. The polypeptide, particle, nucleic acid molecule, method or protein complex of embodiment 293, wherein the polypeptide or ORF1 molecule comprises an asparagine to cysteine ​​substitution (e.g., at a position corresponding to position 290 of a loop 10 ORF1 protein (e.g., as described herein)).

[0651] 297. The polypeptide, particle, nucleic acid molecule, method or protein complex of embodiment 293, wherein the polypeptide or ORF1 molecule comprises a lysine to cysteine ​​substitution (e.g., at a position corresponding to position 317 of a ring 10 ORF1 protein (e.g., as described herein)).

[0652] 298. The polypeptide, particle, nucleic acid molecule, method or protein complex of embodiment 293, wherein the polypeptide or ORF1 molecule comprises a lysine to cysteine ​​substitution (e.g., at a position corresponding to position 324 of a ring 10 ORF1 protein (e.g., as described herein)).

[0653] 299. The polypeptide, particle, nucleic acid molecule, method or protein complex of embodiment 293, wherein the polypeptide or ORF1 molecule comprises an alanine to cysteine ​​substitution (e.g., at a position corresponding to position 362 of a ring 10 ORF1 protein (e.g., as described herein)).

[0654] 300. The polypeptide, particle, nucleic acid molecule, method, or protein complex of embodiment 293, wherein the polypeptide or ORF1 molecule comprises a serine to cysteine ​​substitution (e.g., at a position corresponding to position 363 of a ring 10 ORF1 protein (e.g., as described herein)).

[0655] 301. The polypeptide, particle, nucleic acid molecule, method, or protein complex of embodiment 293, wherein the polypeptide or ORF1 molecule comprises an asparagine to cysteine ​​substitution (e.g., at a position corresponding to position 369 of a ring 10 ORF1 protein (e.g., as described herein)).

[0656] 302. The polypeptide, particle, nucleic acid molecule, method, or protein complex of embodiment 293, wherein the polypeptide or ORF1 molecule comprises a lysine to cysteine ​​substitution (e.g., at a position corresponding to position 371 of a ring 10 ORF1 protein (e.g., as described herein).

[0657] 303. The polypeptide, particle, nucleic acid molecule, method, or protein complex of embodiment 293, wherein the polypeptide or ORF1 molecule comprises a glutamine to cysteine ​​substitution (e.g., at a position corresponding to position 287 of a ring 10 ORF1 protein (e.g., as described herein)).

[0658] 304. The polypeptide, particle, nucleic acid molecule, method or protein complex of embodiment 293, wherein the polypeptide or ORF1 molecule (e.g., as described herein) comprises one or more substitutions to cysteine ​​at one or more positions corresponding to Y254, R263, N264, K265, L272, G273, T274, R276, H283, T285, N288, D291, Q308, D311, W312, T313, E314, D316, H318, N319, T321, T328, K329, T341, Q343, T354, Q358, T361, T364, Q368, D374, P376, P378, Y380 and / or 1381 of RING10 ORF1 protein.

[0659] 305. A particle comprising:

[0660] (a) a protein coat comprising an ORF1 molecule; and

[0661] (b) a genetic element comprising a heterologous nucleic acid sequence encoding an exogenous effector;

[0662] wherein the genetic element is enclosed within the protein coat; and

[0663] The particles have one or more of the following characteristics:

[0664] (i) the genetic element (e.g., a DNA genetic element) does not comprise anellovirus 5'UTR or an origin of replication;

[0665] (ii) the sequence encoding the exogenous effector occupies at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the genetic element (e.g., DNA genetic element);

[0666] (iii) the heterologous nucleic acid sequence occupies at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the genetic element (e.g., DNA genetic element);

[0667] (iv) the particle does not comprise a detectable amount of (e.g., any) a polypeptide from the host cell, or comprises fewer than 5, 10, 15, 20, 25, 30, 40, or 50 copies of a polypeptide from the host cell;

[0668] (v) the particle contains no detectable amount (e.g., any) of a nucleic acid molecule from the host cell, or contains fewer than 2, 3, 4, or 5 copies of a nucleic acid molecule from the host cell;

[0669] (vi) the particle comprises a denaturant at a concentration of less than about 0.01 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, 1.1 M, 1.2 M, 1.3 M, 1.5 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, or 2 M;

[0670] (vii) does not substantially replicate when introduced into a cell (e.g., a human cell); and / or

[0671] (viii) It has a symmetrical shape.

[0672] 306. The particle of embodiment 305, wherein the heterologous nucleic acid sequence is about 60-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1500, or 1500-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides in length.

[0673] 307. A population of particles as described in embodiment 305, wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the particles in the population comprise at least 50, 55 or 60 copies of the Anellovirus ORF1 molecule.

[0674] 308. A population of particles as described in embodiment 305, wherein at least 90% of the particles in the population have a diameter of at least 30, 31, 32, 33, 34, or 35 nm.

[0675] 309. A population of particles as described in embodiment 305, wherein at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the particles have a symmetrical morphology.

[0676] 310. A population of particles as described in embodiment 305, wherein the population does not contain a detectable amount of a polypeptide from a host cell, or contains fewer than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, or 50 copies of a polypeptide from a host cell per particle.

[0677] 311. A population of particles as described in embodiment 305, wherein the population does not contain a detectable amount of a nucleic acid molecule from a host cell, or contains fewer than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, or 50 copies of a nucleic acid molecule from a host cell per particle.

[0678] 312. A population of particles as described in embodiment 305, wherein the population comprises less than 10 ng of nucleic acid.

[0679] 313. A population of particles as described in embodiment 305, wherein the population does not contain a detectable amount of a nucleic acid molecule from a host cell, or contains fewer than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, or 50 copies per particle of a nucleic acid molecule from a host cell that is 200 bp or less in length.

[0680] 314. A particle comprising:

[0681] a protein coat comprising about 40-80 (e.g., about 60) copies of ORF1 molecules; and

[0682] The particles:

[0683] (i) does not contain (e.g., does not surround) a polynucleotide,

[0684] (ii) does not contain (e.g., does not surround) a detectable level of a polynucleotide,

[0685] (iii) does not comprise (e.g., does not encompass) a polynucleotide greater than 1000, 500, 200, or 100 nucleotides in length,

[0686] (iv) does not comprise (e.g., does not encompass) a polynucleotide comprising any contiguous nucleic acid sequence of at least 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides in length that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a contiguous sequence in a wild-type anellovirus genome (e.g., as described herein), and / or

[0687] (v) does not contain a polynucleotide containing an anellovirus 5'UTR or origin of replication.

[0688] 315. The particle of embodiment 314, further comprising an exogenous effector.

[0689] 316. The particle of embodiment 315, wherein the exogenous effector is enclosed within the protein shell.

[0690] 317. The particle of embodiment 314 or 315, wherein the exogenous effector is a polypeptide.

[0691] 318. The particle of any of embodiments 314-317, wherein the exogenous effector is a small molecule.

[0692] 319. A composition comprising a plurality of particles comprising a protein shell comprising about 40-80 (e.g., about 60) copies of an ORF1 molecule;

[0693] wherein at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% of the particles do not contain (e.g., do not surround):

[0694] (i) a polynucleotide,

[0695] (ii) a nucleic acid molecule greater than 1000, 500, 200 or 100 nucleotides in length,

[0696] (iii) a plurality of polynucleotides,

[0697] (iv) circular nucleic acid molecules,

[0698] (v) single-stranded nucleic acid molecules, and / or

[0699] (vi) a genetic element (e.g., a genetic element of a ring vector), e.g., as described herein; or

[0700] wherein the composition comprises less than 10 10 -10 14 (For example, less than 10 10 -10 11 , 10 11 -10 12 , 10 12 -10 13 or 10 13 -10 14 ) viral genome equivalents of nucleic acid molecules (e.g., genetic elements, e.g., genetic elements of a ring vector as described herein) per kilogram of the subject to be administered the composition (e.g., as determined by qPCR or by measuring optical density).

[0701] 320. The composition of embodiment 319, further comprising a denaturant (e.g., urea), e.g., at a concentration of less than about 0.01 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, 1.1 M, 1.2 M, 1.3 M, 1.5 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, or 2 M; a proteasome; or ferritin.

[0702] 321. The composition of embodiment 319 or 320, wherein the composition comprises 0.01-100 mg of the particles (e.g., 0.01-1, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 mg of the particles).

[0703] 322. A method of decomposing particles, the method comprising:

[0704] (a) providing a mixture comprising particles and a denaturant, wherein the particles comprise:

[0705] (i) a protein coat comprising multiple anellovirus ORF1 molecules, and

[0706] (ii) a nucleic acid molecule (e.g., a nucleic acid endogenous to the host cell or a nucleic acid exogenous to the host cell, such as an anellovirus genome); and

[0707] (b) incubating the mixture under conditions suitable for:

[0708] The protein coat breaks down, and

[0709] The nucleic acid molecule dissociates from the protein coat.

[0710] 323. The method of embodiment 322, wherein the anellovirus ORF1 molecule is produced in mammalian cells.

[0711] 324. The method of embodiment 322 or 323, wherein conditions suitable for disassembly of the protein coat comprise one or more of: a predetermined conductivity, a detergent (e.g., SDS (e.g., 0.1% SDS), Tween, or Triton), a chaotropic agent (e.g., urea), a high salt solution (e.g., a solution comprising NaCl, e.g., at a concentration of at least about 1 M, e.g., at least about 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 2 M, 3 M, 4 M, or 5 M), or conditions involving a predetermined temperature.

[0712] 325. The method of any of embodiments 322-324, wherein the mixture comprises a population of the particles.

[0713] 326. The method of any one of embodiments 322-325, wherein the incubation of (b) results in at least 50%, 60%, ... 95%, or 100% of the particle population being broken down.

[0714] 327. The method of any one of embodiments 322-326, further comprising step (c) removing (partially or completely) the nucleic acid molecule from the mixture, for example by washing.

[0715] 328. The method of any one of embodiments 322-327, wherein the host cell is a human cell.

[0716] 329. A method for preparing a ring carrier, the method comprising:

[0717] (a) providing a mixture comprising a plurality of anellovirus ORF1 molecules, wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the plurality of ORF1 molecules are not contained in particles comprising about 40-80 (e.g., about 60) copies of the ORF1 molecule;

[0718] (b) subjecting the mixture to conditions suitable for in vitro assembly of the anellovirus ORF1 molecule; and

[0719] (c) incubating the anellovirus ORF1 molecule with a plurality of genetic elements under conditions suitable for assembling the anellovirus ORF1 molecule into one or more finger ring vectors each encompassing one or more of the genetic elements.

[0720] 330. The method of embodiment 329, wherein the mixture provided in (a) is under denaturing conditions, e.g., wherein the mixture comprises a denaturant at a level sufficient to disaggregate a complex (e.g., a protein coat) comprising at least about 20, 30, 40, 50, or 60 copies, or 20-30, 30-40, 40-50, or 50-60 copies of the Anellovirus ORF1 molecule.

[0721] 331. The method of embodiment 329 or 330, wherein the conditions suitable for in vitro assembly comprise reducing the concentration of the denaturing agent or removing the mixture from the denaturing conditions.

[0722] 332. A method for preparing a ring carrier, the method comprising:

[0723] (a) providing a mixture comprising a plurality of anellovirus ORF1 molecules, and subjecting the mixture to denaturing conditions (e.g., providing a denaturing agent as part of the mixture, e.g., contacting the mixture with a denaturing agent), wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the plurality of ORF1 molecules are not contained in particles comprising about 40-80 (e.g., about 60) copies of the ORF1 molecule;

[0724] (b) subjecting the mixture to non-denaturing conditions (e.g., reducing the concentration of the denaturant to a level) suitable for in vitro assembly of the anellovirus ORF1 molecule (e.g., by dialysis); and

[0725] (c) incubating the anellovirus ORF1 molecule with a plurality of genetic elements under conditions suitable for assembling the anellovirus ORF1 molecule into one or more finger ring vectors each encompassing one or more of the genetic elements.

[0726] 333. The method of embodiment 332, wherein (b) and (c) are performed simultaneously.

[0727] 334. The method of embodiment 332, wherein (b) is performed before (c).

[0728] 335. The method of any of embodiments 332-334, wherein the genetic elements are introduced into the mixture comprising the Anellovirus ORF1 molecules before, simultaneously with, or after (b).

[0729] 336. The method of any one of embodiments 332 to 335, wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the plurality of ORF1 molecules in the mixture of (a) are contained in capsomers (e.g., decamers or particles of 25-40 nm in diameter, e.g., 25-30, 30-32, 32-35, or 35-40 nm, or about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nm in diameter).

[0730] 337. The method of any one of embodiments 332 to 3336, wherein the ratio of ORF1 molecules contained in capsomeres (e.g., decamers) in the mixture of (a) compared to ORF1 molecules contained in particles in the mixture of (a) is at least 2:1, 3:1, 4:1, 5:1, 10:1, 50:1, 100:1, 500:1, 1000:1, 5000:1, or 10,000:1.

[0731] 338. The method of any one of embodiments 332 to 3337, wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the plurality of ORF1 molecules are contained within a ring carrier (e.g., a 60-mer or particle having a diameter of at least 30, 31, 32, 33, 34, or 35 nm) following the incubation of (c).

[0732] 339. The method of any one of embodiments 332-338, wherein the genetic element encodes an exogenous effector.

[0733] 340. The method of any one of embodiments 332-339, wherein the genetic element is an oligonucleotide.

[0734] 341. The method of any one of embodiments 332-340, wherein the genetic element does not encode a polypeptide or a functional nucleic acid.

[0735] 342. The method of any one of embodiments 332-341, wherein the concentration of the denaturant after step (b) is no more than about 0.01 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, 1.1 M, 1.2 M, 1.3 M, 1.5 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, or 2 M.

[0736] 343. The method of any of embodiments 332-342, wherein after incubation (c), at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the particles in the mixture contain at least 50, 55 or 60 copies of the Anellovirus ORF1 molecule.

[0737] 344. The method of any one of embodiments 332-343, wherein after incubating (c), at least 90% of the particles in the mixture have a diameter of at least 30, 31, 32, 33, 34, or 35 nm.

[0738] 345. The method of any one of embodiments 332-344, wherein after incubating (c), at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the particles in the mixture have a symmetrical morphology.

[0739] 346. The method of any one of embodiments 332-41, wherein the denaturant is selected from a chaotropic agent (e.g., urea), heat (e.g., a temperature greater than about 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C), or pH (e.g., an acidic pH or a basic pH).

[0740] 347. A method for preparing a ring VLP, the method comprising:

[0741] (a) providing a mixture comprising a plurality of anellovirus ORF1 molecules, wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the plurality of ORF1 molecules are not contained in particles comprising about 40-80 (e.g., about 60) copies of the ORF1 molecule;

[0742] (b) subjecting the mixture to conditions suitable for in vitro assembly of the anellovirus ORF1 molecule; and

[0743] (c) incubating the anellovirus ORF1 molecule with multiple effectors (eg, exogenous effectors) under conditions suitable for the anellovirus ORF1 molecule to assemble into one or more anellovirus VLPs that each enclose one or more of the effectors.

[0744] 348. The method of embodiment 347, wherein the mixture provided in (a) is under denaturing conditions, e.g., wherein the mixture comprises a denaturant at a level sufficient to disaggregate a complex (e.g., a protein coat) comprising at least about 20, 30, 40, 50, or 60 copies, or 20-30, 30-40, 40-50, or 50-60 copies of the Anellovirus ORF1 molecule.

[0745] 349. The method of embodiment 347 or 348, wherein the conditions suitable for in vitro assembly comprise reducing the concentration of the denaturing agent or removing the mixture from the denaturing conditions.

[0746] 350. A method for preparing a ring VLP, the method comprising:

[0747] (a) providing a mixture comprising a plurality of anellovirus ORF1 molecules and a denaturing agent, wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the plurality of ORF1 molecules are not contained in particles comprising about 40-80 (e.g., about 60) copies of the ORF1 molecule;

[0748] (b) reducing the concentration of the denaturing agent to a level suitable for the in vitro assembly of the anellovirus ORF1 molecules; and

[0749] (c) incubating the anellovirus ORF1 molecule with multiple effectors (eg, exogenous effectors) under conditions suitable for the in vitro assembly of the anellovirus ORF1 molecule into one or more anellovirus VLPs each enclosing one or more of the effectors.

[0750] 351. The method of embodiment 350, wherein the effectors are introduced into the mixture comprising the Anellovirus ORF1 molecules before, simultaneously with, or after (b).

[0751] 352. The method of embodiment 350 or 351, wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the plurality of ORF1 molecules in the mixture of (a) are contained in capsomeres (e.g., decamers or particles having a diameter of at most 25-40 nm, e.g., 25-30, 30-32, 32-35, or 35-40 nm, or about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nm).

[0752] 353. The method of any one of embodiments 350 to 352, wherein the ratio of ORF1 molecules contained in capsomeres (e.g., decamers) in the mixture of (a) compared to ORF1 molecules contained in particles in the mixture of (a) is at least 2:1, 3:1, 4:1, 5:1, 10:1, 50:1, 100:1, 500:1, 1000:1, 5000:1, or 10,000:1.

[0753] 354. The method of any one of embodiments 350 to 353, wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the plurality of ORF1 molecules are contained within ring VLPs (e.g., 60-mers or particles having a diameter of at least 30, 31, 32, 33, 34 or 35 nm) following the incubation of (c).

[0754] 355. The method of any one of embodiments 350-354, wherein the finger-ring VLP has one or more of the following features:

[0755] (i) does not contain (e.g., does not surround) a polynucleotide,

[0756] (ii) does not contain (e.g., does not surround) a detectable level of a polynucleotide,

[0757] (iii) does not comprise (e.g., does not encompass) a polynucleotide greater than 1000, 500, 200, or 100 nucleotides in length,

[0758] (iv) does not comprise (e.g., does not encompass) a polynucleotide comprising any contiguous nucleic acid sequence of at least 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides in length that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a contiguous sequence in a wild-type anellovirus genome (e.g., as described herein), and / or

[0759] (v) does not contain a polynucleotide containing an anellovirus 5'UTR or an origin of replication.

[0760] 356. The method of any one of embodiments 350-355, wherein the concentration of the denaturant after step (b) is no more than about 0.01 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, 1.1 M, 1.2 M, 1.3 M, 1.5 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, or 2 M.

[0761] 357. The method of any of embodiments 350-356, wherein after incubation (c), at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the particles in the mixture contain at least 50, 55 or 60 copies of the Anellovirus ORF1 molecule.

[0762] 358. The method of any of embodiments 350-357, wherein after incubating (c), at least 90% of the particles in the mixture have a diameter of at least 30, 31, 32, 33, 34, or 35 nm.

[0763] 359. The method of any one of embodiments 350-358, wherein after incubating (c), at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the particles in the mixture have a symmetrical morphology.

[0764] 360. The method of any one of embodiments 350-359, wherein the denaturant is selected from a chaotropic agent (e.g., urea), heat (e.g., a temperature greater than about 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C), or pH (e.g., an acidic pH or a basic pH).

[0765] 361. A method for preparing a ring VLP, the method comprising:

[0766] (a) providing a mixture comprising particles and a denaturant, wherein the particles comprise:

[0767] (i) a protein coat comprising multiple anellovirus ORF1 molecules, and

[0768] (ii) a nucleic acid molecule (e.g., a host cell nucleic acid molecule); and

[0769] (b) incubating the mixture under conditions suitable for:

[0770] The protein coat breaks down, and

[0771] The nucleic acid molecule dissociates from the protein coat;

[0772] (c) providing a mixture comprising a plurality of anellovirus ORF1 molecules and a denaturing agent, wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the plurality of ORF1 molecules are not contained in particles comprising about 40-80 (e.g., about 60) copies of the ORF1 molecule;

[0773] (d) reducing the concentration of the denaturing agent to a level suitable for the in vitro assembly of the anellovirus ORF1 molecules; and

[0774] (e) incubating the anellovirus ORF1 molecules with multiple effectors (eg, exogenous effectors) under conditions suitable for assembling the anellovirus ORF1 molecules into one or more anellovirus VLPs, each anellovirus VLP enclosing one or more effectors.

[0775] 362. A polypeptide, such as an ORF1 molecule, comprising one or more of:

[0776] (a) a first region comprising an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an arginine-rich region sequence described herein, such as MPYYYRRRRYNYRRPRWYGRGWIRRPFRRRFRRKRRVR (SEQ ID NO:216) or MAWGWWKRRRRWWFRKRWTRGRLRRRWPRSARRRPRRRRVRRRWRRGRRKTRTYRRRRRFRRRGRK (SEQ ID NO:186), or as listed in any one of Tables A1-A26, or a sequence of at least about 40 amino acids containing at least 60%, 70%, or 80% basic residues (e.g., arginine, lysine, or a combination thereof),

[0777] (b) a second region comprising a jelly coil sequence as described herein (e.g., PTYTTIPLKQWQPPYKRTCYIKGQDCLIYYSNLRLGMNSTMYEKSIVPVHWPGGGSFSVSMLTLDALYDIHKLCRNWWTSTNQDLPLVRYKGCKITFYQSTFTDYIVRIHTELPANSNKLTYPNTHPLMMMMSKYKHIIPSRQTRRKKKPYTKIFVKPPPQFENKWYFATDLYKIPLLQIHCTACNLQNPFVKPDKLSNNVTLWSLNT (SEQ ID NO: 217), or as listed in any one of Tables A1-A26), or an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence of at least 6 (e.g., at least 6, 7, 8, 9, 10, 11, or 12) beta strands;

[0778] (c) a third region comprising an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an N22 domain sequence described herein, e.g., TMALTPFNEPIFTQIQYNPDRDTGEDTQLYLLSNATGTGWDPPGIPELILEGFPLWLIYWGFADFQKNLKKVTNIDTNYMLVAKTKFTQKPGTFYLVILNDTFVEGNSPYEKQPLPEDNIKWYPQVQYQLEAQNKLLQTGPFTPNIQGQLSDNISMFYKFYFK (SEQ ID NO: 219), or as listed in any one of Tables A1-A26);

[0779] (d) a fourth region comprising an amino acid sequence having at least 30% (e.g., at least about 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an anellovirus ORF1 C-terminal domain (CTD) sequence described herein (e.g., WGGSPPKAINVENPAHQIQYPIPRNEHETTSLQSPGEAPES ILYSFDYRHGNYTTTALSRISQDWALKDTVSKITEPDRQQLLKQALECLQI SEETQEKKEKEVQQLISNLRQQQQLYRERIISLLKDQ (SEQ ID NO: 220), or as listed in any one of Tables A1-A26);

[0780] wherein the ORF1 molecule comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type ORF1 protein (e.g., as described herein), such as an insertion, substitution, chemical or enzymatic modification, and / or deletion, such as a deletion of a domain (e.g., one or more of an arginine-rich region, a jelly-coil domain, an HVR, N22, or a CTD, such as described herein).

[0781] 363. The polypeptide of embodiment 362, wherein the amino acid sequences of regions (a), (b), (c), and (d) have at least 90% sequence identity to their respective references.

[0782] 364. The polypeptide of embodiment 362, wherein the polypeptide comprises:

[0783] (i) the first region and the second region;

[0784] (ii) the first region and the third region;

[0785] (iii) the first region and the fourth region;

[0786] (iv) the second region and the third region;

[0787] (v) the second region and the fourth region;

[0788] (vi) the third region and the fourth region;

[0789] (vii) the first region, the second region, and the third region;

[0790] (viii) the first region, the second region, and the fourth region;

[0791] (ix) the first region, the third region, and the fourth region; or

[0792] (x) the second region, the third region, and the fourth region.

[0793] 365. The polypeptide of any one of embodiments 362-364, wherein:

[0794] The first region comprises an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an arginine-rich region sequence as listed in any one of Tables A1-A26;

[0795] the second region comprises an amino acid sequence having at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a jelly coil region sequence as listed in any one of Tables A1-A26;

[0796] The third region comprises an amino acid sequence that has at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an N22 domain sequence as listed in any one of Tables A1-A26; and / or

[0797] The fourth region comprises an amino acid sequence that has at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a CTD sequence as listed in any one of Tables A1-A26.

[0798] 366. The polypeptide of embodiment 365, wherein the amino acid sequences of the first, second, third, and fourth regions have at least 90% sequence identity to their respective references.

[0799] 367. The polypeptide of any one of the preceding embodiments, wherein the polypeptide comprises the first region, the second region, the third region, and the fourth region in order from N-terminus to C-terminus.

[0800] 368. The polypeptide of any preceding embodiment, wherein the at least one difference comprises at least one difference in the first region relative to the arginine-rich region of wild-type ORF1 protein.

[0801] 369. The polypeptide of any preceding embodiment, wherein the first region comprises an arginine-rich region of an ORF1 protein from an anellovirus other than a wild-type anellovirus having greatest sequence identity to the polypeptide or the portion thereof excluding the first region.

[0802] 370. The polypeptide of any preceding embodiment, wherein the first region comprises an amino acid sequence that has at least 70% sequence identity to an arginine-rich region from an Anellovirus other than the wild-type Anellovirus with which the polypeptide has greatest sequence identity.

[0803] 371. The polypeptide of any preceding embodiment, wherein the second region comprises a jellycoil region of an ORF1 protein from an anellovirus other than a wild-type anellovirus having greatest sequence identity to the polypeptide or portion thereof excluding the second region.

[0804] 372. The polypeptide of any preceding embodiment, wherein the second region comprises an amino acid sequence that has at least 70% sequence identity to a jellycoil region from an anellovirus other than the wild-type anellovirus with which the polypeptide has greatest sequence identity.

[0805] 373. The polypeptide of any preceding embodiment, wherein the third region comprises the N22 domain of the ORF1 protein from an anellovirus other than a wild-type anellovirus having greatest sequence identity to the polypeptide or the portion thereof excluding the third region.

[0806] 374. The polypeptide of any preceding embodiment, wherein the third region comprises an amino acid sequence that has at least 70% sequence identity to the N22 region from an Anellovirus other than the wild-type Anellovirus with which the polypeptide has greatest sequence identity.

[0807] 375. The polypeptide of any preceding embodiment, wherein the fourth region comprises a CTD domain of an ORF1 protein from an anellovirus other than a wild-type anellovirus having maximum sequence identity to the polypeptide or the portion thereof excluding the fourth region.

[0808] 376. The polypeptide of any preceding embodiment, wherein the fourth region comprises an amino acid sequence that has at least 70% sequence identity to a CTD region from an Anellovirus other than the wild-type Anellovirus with which the polypeptide has greatest sequence identity.

[0809] 377. The polypeptide of any of embodiments 362-376, wherein the HVR sequence is located between the second region and the third region.

[0810] 378. The polypeptide of embodiment 377, wherein the HVR sequence comprises an amino acid sequence that has at least 70% (e.g., at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to an HVR from an Anellovirus other than the wild-type Anellovirus that has maximum sequence identity to the ORF1 protein.

[0811] 379. The polypeptide of any of embodiments 377 or 378, wherein the HVR sequence is heterologous to one or more of the first, second, third, and / or fourth regions.

[0812] 380. The polypeptide of any of embodiments 377-379, wherein the HVR sequence comprises an HVR from an ORF1 protein of an anellovirus other than a wild-type anellovirus having maximum sequence identity to the polypeptide or the portion thereof excluding the HVR sequence.

[0813] 381. The polypeptide of any of embodiments 377-380, wherein the HVR sequence comprises an amino acid sequence that has at least about 70% sequence identity to an HVR from an Anellovirus other than the wild-type Anellovirus with which the polypeptide shares greatest sequence identity.

[0814] 382. The ring vector of any preceding embodiment, wherein the protein shell comprises the polypeptide of any one of embodiments 362-381.

[0815] 383. The particle of any preceding embodiment, wherein the protein shell comprises the polypeptide of any one of embodiments 362-381.

[0816] 384. The finger-ring VLP of any preceding embodiment, wherein the protein shell comprises the polypeptide of any one of embodiments 362-381.

[0817] 385. A method for preparing two or more different anellovirus ORF molecules, the method comprising:

[0818] (i) providing an insect cell comprising a nucleic acid construct encoding two or more different anellovirus ORF molecules (e.g., two or more of ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, and / or ORF1 / 2 molecules);

[0819] (ii) incubating the insect cells under conditions suitable for expressing the two or more different anellovirus ORF molecules.

[0820] 386. The method of embodiment 385, wherein the nucleic acid construct comprises sequences encoding all of the ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1 and / or ORF1 / 2 molecules.

[0821] 387. The method of embodiment 385, further comprising incubating the insect cell under conditions suitable for secretion of the anellovirus ORF molecule.

[0822] 388. The method of embodiment 385, further comprising isolating the anellovirus ORF molecule from the insect cell.

[0823] 389. The method of embodiment 388, wherein the isolating step comprises lysing the insect cells.

[0824] 390. The method of any one of embodiments 385-389, wherein the Anellovirus ORF comprises an Anellovirus ORF1 molecule.

[0825] 391. A method for preparing anellovirus ORF1 molecule, the method comprising:

[0826] (i) providing an insect cell comprising a nucleic acid construct encoding an anellovirus ORF1 molecule, wherein:

[0827] (a) the molecular weight of the anellovirus ORF1 molecule is at least 101 kDa,

[0828] (b) the anellovirus ORF1 molecule is a full-length anellovirus ORF1 protein,

[0829] (c) a plurality of the anellovirus ORF1 molecules surround the anellovirus genetic element when the anellovirus genetic element is present,

[0830] (d) the anellovirus ORF1 molecule is not a TTV ORF1 protein,

[0831] (e) the anellovirus ORF1 molecule is a beta-type parvovirus or a gamma-type parvovirus ORF1 molecule; or

[0832] (f) the anellovirus ORF1 molecule comprises an anellovirus ORF1 arginine-rich region and an anellovirus C-terminal domain;

[0833] (ii) incubating the insect cells under conditions suitable for expressing the anellovirus ORF1 molecule.

[0834] 392. The method of embodiment 391, further comprising incubating the insect cell under conditions suitable for secretion of the anellovirus ORF1 molecule.

[0835] 393. The method of embodiment 391, further comprising isolating the anellovirus ORF1 molecule from the insect cell.

[0836] 394. The method of embodiment 393, wherein the isolating step comprises lysing the insect cells.

[0837] 395. The method of any preceding embodiment, wherein the incubating step produces an amount of the Anellovirus ORF1 molecule detectable by Western blot, eg, as described herein.

[0838] 396. A method for preparing anellovirus ORF molecules (e.g., ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, and / or ORF1 / 2 molecules), the method comprising:

[0839] (i) providing an insect cell (e.g., Sf9 cell) comprising a nucleic acid construct encoding the anellovirus ORF molecule;

[0840] (ii) incubating the insect cell under conditions suitable for expressing a plurality of the anellovirus ORF molecules; and

[0841] (iii) optionally isolating, purifying and / or enriching a plurality of anellovirus ORF molecules from the insect cell, or other components or constituents of the insect cell;

[0842] Thus, the anellovirus ORF molecule is prepared.

[0843] 397. The method of embodiment 396, wherein the anellovirus ORF molecule is fused to a marker (e.g., a His tag), e.g., at its N-terminus or at its C-terminus (e.g., as described in Table E1 and / or Example 9).

[0844] 398. The method of embodiment 396 or 397, wherein the insect cell further comprises a nucleic acid construct encoding one or more additional anellovirus ORF molecules (e.g., one or more of ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, and / or ORF1 / 2 molecules), and wherein the method further comprises:

[0845] incubating the insect cell under conditions suitable for expression of a plurality of the one or more additional anellovirus ORF molecules, e.g., before, simultaneously with, or after step (ii); and

[0846] The one or more additional anellovirus ORF molecules are optionally isolated, purified and / or enriched from the insect cell or other components or constituents of the insect cell, eg, before, simultaneously with or after step (iii).

[0847] 399. The method of embodiment 398, wherein the nucleic acid construct encoding the one or more additional Anellovirus ORF molecules is the same as the nucleic acid construct of (i).

[0848] 400. The method of embodiment 399, wherein the nucleic acid construct of (i) comprises sequences encoding 2, 3, 4, 5, or all 6 of the Anellovirus ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, and / or ORF1 / 2 molecules.

[0849] 401. The method of embodiment 399, wherein the nucleic acid construct of (i) encodes anellovirus ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, and ORF1 / 2 molecules.

[0850] 402. The method of embodiment 399, wherein the nucleic acid construct of (i) comprises the entire open reading frame region of an Anellovirus genome.

[0851] 403. The method of embodiment 398, wherein the nucleic acid construct encoding the one or more additional Anellovirus ORF molecules is different from the nucleic acid construct of (i).

[0852] 404. The method of any one of embodiments 398-403, wherein the anellovirus ORF molecules are from the same anellovirus genome.

[0853] 405. The method of any one of embodiments 398-403, wherein the anellovirus ORF molecules are from multiple anellovirus genomes (eg, wherein the ORF1 molecule is from one anellovirus genome and the ORF2 molecule is from a different anellovirus genome).

[0854] 406. The method of any one of embodiments 398-405, wherein one or more of the anellovirus ORF molecules are from an Alpha-type parvovirus (e.g., as listed in Table E2).

[0855] 407. The method of any one of embodiments 398-406, wherein one or more of the anellovirus ORF molecules are from a Betavirus (e.g., as listed in Table E2).

[0856] 408. The method of any one of embodiments 398-407, wherein one or more of the anellovirus ORF molecules are from a Gamma-type leucovirus (e.g., as listed in Table E2).

[0857] 409. The method of any one of embodiments 398-408, wherein the one or more nucleic acid constructs each comprise a promoter (e.g., a promoter that controls expression of one or more of the anellovirus ORF molecules, e.g., a baculovirus polyhedron promoter).

[0858] 410. The method of any one of embodiments 398-409, further comprising incubating the insect cell under conditions suitable for secretion of the anellovirus ORF molecules.

[0859] 411. The method of any one of embodiments 398-410, wherein the isolating step comprises lysing the insect cells.

[0860] 412. The method of any one of embodiments 398-411, wherein the incubating step produces an amount of the Anellovirus ORF molecule (eg, ORF1 molecule) detectable by Western blot, eg, as described herein.

[0861] 413. The method of any one of embodiments 398-412, wherein the incubating step produces at least 1, 2, 3, 4, 5, or 6 mg of the anellovirus ORF1 molecule per 1 L of cell culture (eg, Sf9 culture).

[0862] 414. The method of any preceding embodiment, wherein the anellovirus ORF molecules are isolated, enriched or purified by isopycnic centrifugation.

[0863] 415. The method of any preceding embodiment, wherein the anellovirus ORF molecule is an anellovirus ORF1 molecule, and wherein the method further comprises:

[0864] The isolated, purified, or enriched anellovirus ORF1 molecule is contacted in vitro with a genetic element under conditions suitable to enclose the genetic element within a protein coat comprising the anellovirus ORF1 molecule, eg, as described herein.

[0865] Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0866] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods and examples are illustrative only and not intended to be limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0867] The following detailed description of embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For purposes of illustrating the invention, the accompanying drawings show embodiments, which are now described by way of example. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0868] Figure 1 The series of figures shows the generation of exemplary malarial peptide conjugation constructs comprising the C-terminal region of the CS protein.

[0869] Figure 2 The series of figures shows the structures of exemplary malarial peptide conjugated constructs on the surface of the ring vector capsid.

[0870] Figure 3 The figure shows that after denaturation, in vitro circularized DNA separates into positive-sense and negative-sense circularized single-stranded DNA (ssDNA).

[0871] Figure 4A and 4B A series of figures show that anellovirus ORF1 molecules were successfully expressed in cells. (A) Anellovirus strains expressing ORF1 including loops 2, 3, 4, 5, 6, 9, and 10 (e.g., as described herein) were detected from Sf9 cells. (B) Virus-like particles (VLPs) were observed by electron microscopy after production of anellovirus ORF1 proteins of loops 2 and 10, as well as chicken anemia virus (CAV) VP1.

[0872] Figures 5A-5C A series of figures shows the successful expression and purification of loop2ORF1 and ORF2 proteins from Sf9 cells. (A) Exemplary workflow for purification of ORF1 and ORF2 proteins from Sf9 cells. (B) Western blot using an anti-ORF1 antibody showing detection of loop2ORF1 protein produced in Sf9 cells, as described. (C) Electron micrograph showing the formation of virus-like particles (VLPs) from loop2ORF1 protein produced in Sf9 cells, as described.

[0873] Figures 6A-6C A series of figures showing the disassembly of loop 2 VLPs using urea as a chaotropic denaturant. (A) Loop 2 ORF1 protein was produced and purified from Sf9 cells as described, and VLPs were examined by electron microscopy. (B) After treatment with urea, VLPs were no longer observed, and the solution contained small ORF1 capsomers. (C) Figure 6B Magnified view of the boxed area shown in , showing details of the capsomeres.

[0874] Figures 7A-7BA series of electron micrographs showing exemplary symmetric (A) and asymmetric (B) particle morphologies.

[0875] Figure 8 Depicted is the expression of loop2ORF1 with a C-terminal His tag in insect cells.

[0876] Figure 9 Depicted is the expression of loop 1 ORF1 and ORF1 / 1 with a C-terminal His tag in insect cells.

[0877] Figure 10 Depicted is the expression of loop2ORF1 with an N-terminal His tag in insect cells with or without the PreScission cleavage sequence.

[0878] Figure 11 Depicted is the expression of loop 1 ORFs 1 / 1, 1 / 2, 2, 2 / 2, and 2 / 3 as C-terminally tagged recombinant proteins in insect cells.

[0879] Figure 12 Depicts the expression of individual loop 2 ORFs in insect cells. Two exposures of the same blot are shown in the middle and right panels. The left panel shows the structure of the loop 2 constructs tested as indicated.

[0880] Figure 13 Depicted are baculovirus-mediated co-expression of loop 2 ORF1 + "full-length ORF," ORF1 + ORF2, ORF1 + ORF2 / 2, and ORF1 + ORF2 / 3 in insect cells.

[0881] Figure 14 Depicted is the simultaneous co-expression of multiple RING 2 proteins in insect cells using baculovirus.

[0882] Figure 15 Depicted is the expression of ORFs from anellovirus genome delivered into insect cells by baculovirus and by transfection.

[0883] Figure 16 It is shown that loop 1 ORF2 is expressed independently of the polyhedron promoter (arrow marked pH) in Sf9 cells.

[0884] Figure 17 Depicts co-delivery of loop2ORF1-His and loop2 genomic DNA into Sf9 cells, followed by incubation and fractionation on a CsCl linear density gradient. The top panel shows an anti-His tag Western blot of the fractions, along with qPCR analysis of each fraction. The bottom panel shows transmission electron microscopy images of two individual fractions and a pool of fractions, as indicated by the boxes on the Western blots. The inset in the middle panel is a magnified view showing proteasome-like structures.

[0885] Figure 18 Depicted is the characterization of Sf9 isopycnic fractions by immunogold electron microscopy.

[0886] Figure 19 Expression of ORF1 from additional anellovirus strains is depicted.

[0887] Figure 20 The schematic diagram shows an exemplary workflow for generating a finger ring vector (eg, a replication-competent or replication-deficient finger ring vector as described herein).

[0888] Figure 21 The figure shows the primer specificity for primer sets designed to quantify TTV and TTMV genome equivalents. Quantitative PCR based on SYBR Green chemistry shows a distinct peak for each amplification product using TTMV or TTV specific primer sets (as indicated) on plasmids encoding the respective genomes.

[0889] Figure 22 The figure shows an exemplary amplification plot for linear amplification of TTMV (target 1) or TTV (target 2) using 7 genome equivalent concentrations log10. Genome equivalents were quantified with high PCR efficiency and linearity using seven 10-fold dilutions (R 2 TTMV: 0.996; R 2 TTV: 0.997).

[0890] Figure 23A and 23B A series of figures show that tandem anellovirus plasmids can increase the production of anelloviruses or anellovirus vectors. (A) Plasmid map of an exemplary tandem anellovirus plasmid. (B) Transfection of HEK293T cells with tandem anellovirus plasmids produces four times the number of viral genomes compared to plasmids carrying a single copy.

[0891] Figure 23C Gel electrophoresis images show circularization of TTMV-LY2 plasmids pVL46-063 and pVL46-240.

[0892] Figure 23D The chromatograms show the copy numbers of linear and circular TTMV-LY2 constructs as determined by size exclusion chromatography (SEC).

[0893] Figure 24 Schematic diagram showing the domains of the anellovirus ORF1 molecule and the hypervariable regions that will be replaced by structural hypervariable domains from different anelloviruses.

[0894] Figure 25Schematic diagram showing the domains of ORF1 and the hypervariable region to be replaced by a protein or peptide of interest (POI) from a non-anellovirus source.

[0895] Figure 26 A series of figures illustrate the design of exemplary anellovirus vector genetic elements based on the anellovirus genome. The protein coding region is deleted from the anellovirus genome (left), leaving the anellovirus non-coding region (NCR), including the viral promoter, 5'UTR conserved domain (5CD) and GC-rich region. The load DNA is inserted into the non-coding region of the protein-coding locus (right). The resulting finger ring vector contains the load DNA (including open reading frames, genes, non-coding RNA, etc.) and the necessary anellovirus cis-replication and packaging elements, but lacks the necessary protein elements for replication and packaging.

[0896] Figure 27 is a graph showing an alignment of 36 nucleotide GC-rich regions from nine anellovirus genome sequences (SEQ ID NOs 818-827, respectively, in order of appearance) and a consensus sequence based thereon.

[0897] Figure 28 A series of figures shows the structures of ORF1 from anellovirus strains LY2 and CBD203. Putative domains are labeled: arginine-rich region (arg-rich), core region, which contains the structural jelly-coil domain, hypervariable region (HVR), structural N22 region, and structural C-terminal domain (CTD), as indicated.

[0898] Figure 29 is a graph showing the ability of an in vitro circularized (IVC) TTV-tth8 genome (IVC TTV-tth8) to produce TTV-tth8 genome copies at the expected density in HEK293T cells compared to TTV-tth8 genome in a plasmid.

[0899] Figure 30 The series of graphs shows the ability of an in vitro circularized (IVC) LY2 genome (WT LY2 IVC) and a wild-type LY2 genome in a plasmid (WT LY2 plasmid) to produce LY2 genome copies at the expected density in Jurkat cells.

[0900] Figure 31ASchematic diagram of full-length loop 10ORF1 (also referred to herein as Ly1) labeled and colored by domain. The structural arginine-rich motif (ARM) is shown in purple, the structural jelly roll (JR) domain is shown in red, the spike P1 domain is shown in blue, the spike P2 domain is shown in green, and the C-terminal domain is shown in cyan. The residue numbering of each domain and the beginning of the structural C-terminal domain (also referred to as the C-terminus) is as shown above.

[0901] Figure 31B is the sequence of the full-length loop 10ORF1, and its color is Figure 31A The residues are numbered as above. Residues included in the loop 10delARM construct are in bold, including the labeled initial residue K46. Dashed lines above the sequence indicate residues not observed in the density. Secondary structural elements are shown as above, with β-strands as arrows and α-helices as zigzag lines. The JR β-strand is conventionally labeled BI, while other secondary structures are numbered according to their domains. The three peptides used to generate the polyclonal antibodies are underlined.

[0902] Figure 31C Show expression (expression) after and purification and storage (purification) after the Western blot analysis of ring 10delARM.On the left side of these gels, be marked with molecular weight marker, and the arrow indication ring 10delARM on the right side is before proteolysis (ring 10delARM) and the band of (ring 10delARM fragment) after proteolysis.The polyclonal antibody for detecting Western blot is as follows, and is painted by the peptide for generating them.

[0903] Figure 31D is a superposition of the electron density of the ring 10delARM VLP and the 3D reconstruction of the 60-mer VLP molecular structure, as shown Figure 31A Medium coloring. The spike P1 and P2 domains are labeled.

[0904] Figure 31E Depicted is an ORF1 protomer, shown in its electron density, where the domains are shown in Figure 2. Figure 31A Marking and coloring.

[0905] Figure 31F The electron density of the ring 10delARM VLP is depicted, colored by its local resolution. The bar graph (left) represents the resolution (in angstroms) by color. The orientation of the particle (right) is shown in Figure 31D shown.

[0906] Figure 32A Sixty loop 10 structural jelly-coil (JR) domains are depicted, with one domain uniquely colored red. Sixty loop 10 structural jelly-coil (JR) domains form the core of the anellovirus particle.

[0907] Figure 32B The two JR domains are depicted, shown in red, and the observed C-terminal domain backbone is colored in cyan. The JR domains are arbitrarily labeled JR 1 and JR 2 , For clarity, the first (K48) and last (V562) observed residues of each protomer are labeled with corresponding numbers.

[0908] Figure 32C A single JR domain is depicted, oriented to show the β-sheets within the particle core. Side chains of basic residues at positions contacting the viral genome are shown and labeled.

[0909] Figure 32D The structural arginine-rich region, JR, and structural C-terminal domain of loop 10 are depicted, aligned with corresponding ORF1 sequences from different anellovirus genera (indicated in brackets). Figures 31A-31D Basic residues of loop 10 that may contact the viral genome are indicated by asterisks.

[0910] Figure 33A The structure of an anellovirus particle is depicted as a surface rendering. The particle is shown in gray with five spikes forming a crown structure, numbered and colored for clarity, as shown in Figure 2. Figures 31A-31D The spike domain extends from the core along a 5-fold axis.

[0911] Figure 33B The exterior of the crown structure is depicted, shown from the side. The five spike domains are colored as Figure 33A The hydrophobic and hydrophilic conserved residues are colored in light blue and magenta, respectively.

[0912] Figure 33C Depicts the Figure 33B The same spike domain, rotated to visualize residues within the crown structure.

[0913] Figure 33D Depicted is the spike domain of loop 10 (colored as Figures 31A-31D Magenta and black asterisks indicate surface exposed residues in the P1 and P2 domains, respectively. Below the alignment are >30% shared residues, or or γ indicates that the residue is >70% hydrophobic or >60% hydrophilic, respectively.

[0914] Figure 34A Schematic representation of full-length ring 10 (top) with negative stain electron microscopy (bottom) showing highly heterogeneous particles. Scale bar = 100 nm.

[0915] Figure 34B Schematic representation of loop 10delARM (arginine-rich motif; top) showing structurally homologous virus-like particles (VLPs) as seen by negative stain electron microscopy (bottom). Scale bar = 100 nm.

[0916] Figure 34C Schematic representation of loop 10delARMdelCTD (top), where further truncation of this structural C-terminal domain (Δ552-672) retains structured VLPs as shown by negatively stained electron microscopy (bottom). Scale bar = 100 nm.

[0917] Figure 35 Figure 1. Data processing of the Ring 10delARM cryo-electron microscopy (cryo-EM) reconstruction. In brief, crYOLO selected 58,391 particles from 11,083 micrographs. Several rounds of 2D classification yielded 11,185 particles. After the initial model reconstruction in Relion de novo, Relion 3D refinement was performed to obtain orientation parameters. All particles with parameters were input into the 3D classification. The class with the most abundant particle population was generated. Resolution.

[0918] Figure 36A Representative negative staining micrographs of ring 10delARM. Micrographs were taken by NanoImaging Service.

[0919] Figure 36B Representative cryo-EM micrographs of ring 10delARM. Micrographs were taken by NanoImaging Service.

[0920] Figure 37A and 37B Circular dichroism (CD) results of the TTMV-loop 10 C-terminal peptide (CSEEEEESNLFERLLRQRTKQLQLKRRIIQTLKDLQKLE) are depicted. Figure 37A is a table showing the average values ​​of secondary structure scores estimated by different CDPro packages. α-Helices dominate the secondary structure assignments of CD spectra. Figure 37B Shown are the experimental spectra of this C-terminal peptide (shown in red) overlaid with calculated and averaged reference set spectra (shown in blue) from three different packages: SELCON3, CDSSTR, and CONTINLL.

[0921] Figure 38 depicts a sequence alignment of 15 known anelloviruses within different genera shown in parentheses. Conserved amino acids are shown in the first blue row below the sequence. If conservation is greater than 30%, the top blue row below the sequence alignment indicates a homologous sequence. The bottom blue row shows the hydrophobic amino acids ( Similarity within 70%) or 60% positively charged amino acids (γ, similarity within 60%). Alignments were performed using Clustal Omega as implemented by Geneious.

[0922] Figures 39A-39D Loop 10 or α-sheet predicted for the spike of JA20 and MN779270.1 are depicted. Figure 39A A spherical representation of the ring 10 spike (same as Figure 36C) is depicted. Figure 39B and 39C Figure 2. Representation of the spike protein of JA20 and MN779270.1, respectively, using the predicted α-fold. The P1 and P2 domains are shown in blue and green, respectively. Conserved hydrophobic and basic residues are shown in light blue and magenta, respectively. Figure 39D Shown is the sequence alignment between loop 10, JA20 and MN779210.1.

[0923] Figure 40 is a series of graphs showing that a polyclonal antibody (i.e., AB3725) that recognizes the surface-exposed Loop 10 HVR helix consisting of amino acid residues 352-361 of Loop 10 ORF1 specifically binds to Loop 10 finger-loop VLPs, but polyclonal antibodies raised against HVRs of different strains (as shown in strains 1 and 2) do not bind to Loop 10 ORF1.

[0924] Figures 41A-41B AB3725 was shown to recognize a loop 2 ORF1 mutant in which the point mutations K357E, N358Q, and E359I from loop 10 ORF1 were introduced.

[0925] Figures 42A-42B is a series of images showing Coomassie staining of loop 2 virus-like particles (VLPs) ( Figure 42A ) and Western blot ( Figure 42B ).

[0926] Figure 43 is an electron micrograph showing loop 2 VLPs obtained after Capto400 purification.

[0927] Figures 44A-44B A series of figures show Coomassie staining of ring 19 VLPs ( Figure 44A ) and Western blot ( Figure 44B ).

[0928] Figure 45is an electron micrograph showing loop 19 VLPs obtained after Capto400 purification.

[0929] Figure 46 The figure shows an exemplary workflow for conjugating NHS ester moieties to surface lysines of finger ring VLPs using click chemistry.

[0930] Figures 47A-47B A series of figures show Coomassie staining of loop 2 VLPs conjugated with NHS ester 647 ( Figure 47A ) and Western blot ( Figure 47B ).

[0931] Figures 48A-48B Figure 2 shows the expression of ring 2 finger VLPs conjugated to NHS ester biotin (using streptavidin CV800 antibody ( Figure 48A ) or ring 19HVR3 primary antibody and goat anti-rabbit secondary antibody ( Figure 48B A series of images of Western blots of ) marked).

[0932] Figure 49 The figure shows an exemplary two-step method for conjugating surface effector moieties to the surface of finger-ring VLPs.

[0933] Figure 50 Figures show Coomassie staining, Western blot, and UV labeling of the SARS-CoV-2 receptor binding domain (RBD) attached to DBCO, CalFluor 488, azide, or Alexa488, as indicated.

[0934] Figure 51 The figure shows Coomassie staining and Western blot of pRTx-2652 (loop 2 ORF1 variant) generated using SE-FPLC.

[0935] Figure 52 is a graph showing Coomassie staining and Western blot showing conjugation of an RBD attached to an azide moiety and a pRTx-2652 ORF1 polypeptide attached to DBCO to generate RBD-pRTx-2652 conjugate finger ring VLPs.

[0936] Figure 53A It is a 60-mer icosahedral structure of ring 10delARM produced by Sf9 cells.

[0937] Figure 53B The 60-mer icosahedral structure of the loop 10-ORF1 delC term helix produced by Expi293 cells.

[0938] Figure 53Cis a superposition of the ORF1 protomers derived from the loop10delARM and loop10-ORF1delCterm helices. DETAILED DESCRIPTION

[0939] definition

[0940] The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims.Unless otherwise indicated, the terms set out below should generally be understood in their common sense.

[0941] When the term "comprising" is used in this description and the claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered a preferred embodiment of the term "comprising..." If a group is defined below as comprising at least a certain number of embodiments, this should be understood as preferably also disclosing a group consisting only of these embodiments.

[0942] When referring to a singular noun where an indefinite or definite article is used, for example "a", "an" or "the", this includes a plural of that noun unless something else is specifically stated.

[0943] The phrase "a compound, composition, product, etc. for use in therapy, regulation, etc." is to be understood as meaning a compound, composition, product, etc. that is suitable per se for the specified purpose of therapy, regulation, etc. The phrase "a compound, composition, product, etc. for use in therapy, regulation, etc." additionally discloses, as an example, that such a compound, composition, product, etc. is for use in therapy, regulation, etc.

[0944] The words "compounds, compositions, products, etc. for use in...", "use of compounds, compositions, products, etc. in the production of a medicament, pharmaceutical composition, veterinary composition, diagnostic composition, etc. for..." or "compounds, compositions, products, etc. for use as medicaments..." indicate that these compounds, compositions, products, etc. are to be used in a method of treatment that can be practiced on the human or animal body. They are to be considered as equivalent disclosures of the examples and claims relating to methods of treatment, etc. If an example or claim thus refers to "a compound for use in treating a human or animal suspected of having a disease", this is also to be considered as disclosing "the use of a compound in the manufacture of a medicament for treating a human or animal suspected of having a disease" or "a method of treatment by administering a compound to a human or animal suspected of having a disease". The words "compounds, compositions, products, etc. for treatment, regulation, etc." are to be understood as referring to compounds, compositions, products, etc. that are themselves suitable for the specified purpose of treatment, regulation, etc.

[0945] If examples of terms, values, quantities, etc. are provided in parentheses below, this should be understood to mean that the examples mentioned in the parentheses may constitute embodiments. For example, if it is stated that "In embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an Anellovirus ORF1 encoding nucleotide sequence of Table 1 (e.g., nucleotides 571-2613 of the nucleic acid sequence of Table 1)," some embodiments relate to nucleic acid molecules comprising a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to nucleotides 571-2613 of the nucleic acid sequence of Table 1.

[0946] As used herein, the term "ring vector" refers to a vector comprising genetic elements, such as episomes, such as circular DNA, enclosed in a protein coat. As used herein, "synthetic ring vector" generally refers to a non-naturally occurring ring vector, for example, one having a sequence different from that of a wild-type virus (e.g., a wild-type ring virus as described herein). In some embodiments, the protein coat comprises an ORF1 molecule (e.g., an anellovirus ORF1 protein), for example, as described herein. In some embodiments, the protein coat comprises a plurality of ORF1 molecules (e.g., an anellovirus ORF1 protein), for example, at least about 40, 45, 50, 55, 60, 65, or 70 ORF1 molecules. In some embodiments, a synthetic ring vector is engineered or recombinant, for example, comprising genetic elements comprising differences or modifications relative to a wild-type viral genome (e.g., a wild-type ring virus genome as described herein). In some embodiments, being enclosed within a protein shell encompasses 100% protein shell coverage, as well as coverage less than 100%, such as 95%, 90%, 85%, 80%, 70%, 60%, 50% or less. For example, the protein shell may contain gaps or interruptions (e.g., which render the protein shell permeable to water, ions, peptides or small molecules), as long as the genetic element is retained within the protein shell, e.g., prior to entry into the host cell. In some embodiments, the finger ring vector is purified, e.g., separated from its original source and / or is substantially free (>50%, >60%, >70%, >80%, >90%) of other components.

[0947] In some embodiments, the finger ring vector can comprise a nucleic acid vector comprising sufficient nucleic acid sequence (the nucleic acid sequence is derived from or is highly similar (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical) to an anellovirus genomic sequence or a contiguous portion thereof to allow packaging into a protein coat (e.g., capsid)) and further comprising a heterologous sequence. In some embodiments, the finger ring vector is a viral vector or naked nucleic acid. In some embodiments, the anellovirus vector comprises at least about 50, 60, 70, 71, 72, 73, 74, 75, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, or 3500 contiguous nucleotides of a native anellovirus sequence, or a sequence highly similar thereto (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical). In some embodiments, the anellovirus vector further comprises one or more of anellovirus ORF1, ORF2, or ORF3. In some embodiments, the heterologous sequence comprises a multiple cloning site, comprises a heterologous promoter, comprises a coding region for a therapeutic protein, or encodes a therapeutic nucleic acid. In some embodiments, the capsid is a wild-type anellovirus capsid. In embodiments, the anellovirus vector comprises genetic elements as described herein, e.g., comprising a genetic element comprising a promoter, a sequence encoding a therapeutic effector, and a capsid binding sequence.

[0948] As used herein, the term "ring VLP" refers to a vehicle (e.g., virus-like particle) comprising a protein shell and an effector (e.g., an exogenous effector). In some cases, the ring VLP does not contain a large amount of nucleic acid. In some embodiments, the protein shell comprises an ORF1 molecule (e.g., an anellovirus ORF1 protein), for example, as described herein. In some embodiments, the protein shell comprises a plurality of ORF1 molecules (e.g., an anellovirus ORF1 protein), for example, at least about 40, 45, 50, 55, 60, 65, or 70 ORF1 molecules. In some embodiments, the effector is enclosed in the protein shell. In some embodiments, the effector is located on the surface of the protein shell (e.g., contained in a surface portion as described herein). In some embodiments, the Anellovirus VLP does not comprise a polynucleotide greater than 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 nucleotides in length. In some embodiments, the Anellovirus VLP does not comprise a polynucleotide comprising an Anellovirus 5'UTR or an Anellovirus origin of replication. In some embodiments, the Anellovirus VLP does not comprise a polynucleotide comprising any contiguous nucleic acid sequence of at least 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides in length that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a contiguous sequence in a wild-type Anellovirus genome (e.g., as described herein).

[0949] As used herein, the term "antibody molecule" refers to a protein comprising at least one immunoglobulin variable domain sequence, such as an immunoglobulin chain or its fragment. The term "antibody molecule" encompasses full-length antibodies and antibody fragments (e.g., scFv). In certain embodiments, the antibody molecule is a multispecific antibody molecule, for example, the antibody molecule comprises multiple immunoglobulin variable domain sequences, wherein the multiple first immunoglobulin variable domain sequence has binding specificity to the first epi-position, and the multiple second immunoglobulin variable domain sequence has binding specificity to the second epi-position. In an embodiment, the multispecific antibody molecule is a bispecific antibody molecule. The feature of a bispecific antibody molecule is generally that the first immunoglobulin variable domain sequence has binding specificity to the first epi-position and the second immunoglobulin variable domain sequence has binding specificity to the second epi-position.

[0950] As used herein, the term "deletion" with respect to an amino acid sequence or a nucleic acid sequence refers to a portion of a sequence that is absent relative to a reference sequence. In some embodiments, deletions are actively removed from the sequence (e.g., by cutting and / or by an enzyme). In some embodiments, a sequence is generated de novo without the deletion (e.g., a de novo synthesized nucleic acid molecule without the deletion sequence, a nucleic acid molecule generated using a template sequence in which the deletion sequence has been removed, or a polypeptide translated from a nucleic acid sequence that does not encode the deletion sequence).

[0951] As used herein, the term "disassembly" with respect to a particle (such as a virus-like particle (VLP)) or a protein coat refers to the dissociation of one or more components of the particle (e.g., a capsid protein, e.g., an ORF1 molecule as described herein) from the rest of the particle. In some cases, disassembly of a particle (e.g., a VLP) comprises separating enough ORF1 molecules from one another so that they no longer form a protein coat. In some cases, ORF1 molecules are separated from one another via disassembly of particle-forming capsomers (e.g., decamer capsomers), e.g., as described herein. In some embodiments, disassembly reduces the particle to individual monomers. In some embodiments, after disassembly, multimers, e.g., decamers, monomers, and / or pentamers, remain. In some cases, disassembly comprises denaturation of a protein complex of the particle (e.g., breaking non-covalent bonds between ORF1 molecules of the protein coat). In some cases, disassembly is driven by a denaturant as described herein.

[0952] As used herein, the term "in vitro assembly" with respect to a ring vector or ring VLP refers to the formation of a protein shell comprising ORF1 molecules, wherein the formation does not occur inside a cell (e.g., occurs in a cell-free system such as a cell-free suspension, lysate, or supernatant). In some cases, the in vitro assembly of the ring vector comprises enclosing a genetic element (e.g., as described herein) within a protein shell outside the cell. In some cases, the in vitro assembly of the ring VLP comprises the association of an effector (e.g., an exogenous effector, e.g., as described herein) with the protein shell outside the cell (e.g., enclosed within the protein shell). In some cases, the in vitro assembly of the protein shell can occur under conditions (e.g., non-denaturing conditions) suitable for the multimerization of multiple ORF1 molecules (e.g., to form a multimer of more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ORF1 molecules). In some cases, the in vitro assembly results in the formation of a protein coat comprising at least about 20, 30, 40, 50, or 60 ORF1 molecules, or about 20 to 30, 30 to 40, 40 to 50, 50 to 60, or 60 to 70 ORF1 molecules. In some cases, the protein coat is formed from ORF1 molecules produced in cells and subsequently purified therefrom. In some cases, the in vitro assembly occurs in a solution that does not contain cells or their components. In other cases, the in vitro assembly occurs in a solution that contains cell debris (e.g., from lysed cells). In some cases, the in vitro assembly occurs in a solution that is substantially free of cellular nucleic acid molecules (e.g., genomic DNA, mitochondrial DNA, mRNA, and / or non-coding RNA from cells). As used herein, a nucleic acid that "encodes..." refers to a nucleic acid sequence that encodes an amino acid sequence or a functional polynucleotide (e.g., a non-coding RNA, such as an siRNA or miRNA).

[0953] As used herein, "exogenous" agents (e.g., effectors, nucleic acids (e.g., RNA), genes, loads, proteins) refer to agents that are not contained or encoded by a corresponding wild-type virus, e.g., an anellovirus as described herein. In some embodiments, the exogenous agent is not naturally occurring, e.g., a protein or nucleic acid with a sequence that is altered (e.g., by insertion, deletion, or substitution) relative to a naturally occurring protein or nucleic acid. In some embodiments, the exogenous agent is not naturally present in the host cell. In some embodiments, the exogenous agent is naturally present in the host cell, but is exogenous to the virus. In some embodiments, the exogenous agent is naturally present in the host cell, but is not present at a desired level or at a desired time.

[0954] As used herein, a "heterologous" agent or element (e.g., an effector, a nucleic acid sequence, an amino acid sequence) with respect to another agent or element (e.g., an effector, a nucleic acid sequence, an amino acid sequence) refers to an agent or element that does not naturally occur together, for example, in a wild-type virus such as an anellovirus. In some embodiments, a heterologous nucleic acid sequence can be present in the same nucleic acid as a naturally occurring nucleic acid sequence (e.g., a sequence naturally occurring in an anellovirus). In some embodiments, a heterologous agent or element is exogenous relative to the anellovirus on which the other (e.g., remaining) elements of the anellovirus vector are based.

[0955] As used herein, the term "genetic element" refers to a nucleic acid sequence typically within a ring vector. It should be understood that the genetic element can be produced as naked DNA and optionally further assembled into a protein coat. It should also be understood that a ring vector can insert its genetic element into a cell, with the result that the genetic element is present in the cell, without the protein coat necessarily entering the cell.

[0956] As used herein, the term "ORF1 molecule" refers to a polypeptide having the activity and / or structural characteristics of an anellovirus ORF1 protein (e.g., an anellovirus ORF1 protein as described herein, e.g., as listed in any one of Tables A1-A26), or a functional fragment thereof. In some cases, the ORF1 molecule can comprise one or more (e.g., 1, 2, 3, or 4) of the following: a first region comprising at least 60% basic residues (e.g., at least 60% arginine residues), a second region comprising at least about six beta strands (e.g., at least 4, 5, 6, 7, 8, 9, 10, 11, or 12 beta strands), a third region comprising the structure or activity of an anellovirus structural N22 domain (e.g., as described herein, e.g., from an anellovirus ORF1 protein as described herein), and / or a fourth region comprising the structure or activity of an anellovirus structural C-terminal domain (CTD) (e.g., as described herein, e.g., from an anellovirus ORF1 protein as described herein). In some cases, the ORF1 molecule comprises the first, second, third and fourth regions in order from the N-terminus to the C-terminus. In some cases, the ring vector comprises an ORF1 molecule comprising the first, second, third and fourth regions in order from the N-terminus to the C-terminus. In some cases, the ORF1 molecule may comprise a polypeptide encoded by an anellovirus ORF1 nucleic acid (e.g., as listed in any one of Tables N1-N26). In some cases, the ORF1 molecule may further comprise a heterologous sequence, such as a hypervariable region (HVR), such as an HVR from an anellovirus ORF1 protein, such as, for example, as described herein. As used herein, an "anellovirus ORF1 protein" refers to an ORF1 protein encoded by an anellovirus genome (e.g., a wild-type anellovirus genome, such as, for example, as described herein), such as an ORF1 protein having an amino acid sequence as listed in any one of Tables A1-A26, or an ORF1 protein encoded by an ORF1 gene listed in any one of Tables N1-N26.

[0957] As used herein, the term "ORF1 domain" with respect to an ORF1 molecule refers to a portion of the ORF1 molecule that has the structure or function of an anellovirus ORF1 protein. The ORF1 domain is typically capable of forming a multimer with other copies of the ORF1 domain (e.g., in other ORF1 molecules) or with other ORF1 molecules, for example, to form a protein coat (e.g., a ring vector or ring VLP as described herein). In some cases, the ORF1 molecule may comprise one or more additional domains other than the ORF1 domain (e.g., a domain comprising or attached to a surface effector, e.g., as described herein). In some cases, the amino acid sequence of the ORF1 domain comprises an insertion (e.g., between the N-terminus and the C-terminus of the ORF1 domain) (e.g., an insertion encoding a surface portion or a domain capable of binding to a surface portion). In some cases, the insertion does not substantially destroy the structure and / or function of the ORF1 domain, for example, such that the ORF1 domain is still capable of forming a multimer with other ORF1 domains or ORF1 molecules. The location within the ORF1 domain sequence where insertion is performed is referred to herein as the "insertion point". Inserts can be inserted into the ORF1 domain by any genetic or polypeptide engineering method known in the art. In some embodiments, the ORF1 molecule consists of the ORF1 domain. In other embodiments, the ORF1 molecule comprises the ORF1 domain and a heterologous domain (e.g., a surface moiety as described herein). In some embodiments, the ORF1 domain is connected to the surface moiety via a polypeptide linker region.

[0958] As used herein, the term "ORF2 molecule" refers to a polypeptide having the activity and / or structural characteristics of an anellovirus ORF2 protein (e.g., an anellovirus ORF2 protein as described herein, e.g., as listed in any one of Tables A1-A26), or a functional fragment thereof. As used herein, an "anellovirus ORF2 protein" refers to an ORF2 protein encoded by an anellovirus genome (e.g., a wild-type anellovirus genome, e.g., as described herein), e.g., an ORF2 protein having an amino acid sequence as listed in any one of Tables A1-A26, or encoded by an ORF2 gene listed in any one of Tables N1-N26.

[0959] As used herein, the term "particle" refers to a vehicle having a diameter of less than 100 nm (e.g., about 20-25, 25-30, 30-35, or 35-40 nm) and comprising a protein shell. In some cases, the particle comprises a plurality of ORF1 molecules. The protein shell of the particle typically forms an enclosing shell that can limit or prevent certain molecules from moving between the inside and outside of the protein shell. In some embodiments, gaps or discontinuities (e.g., such that the protein shell is permeable to water, ions, peptides, or small molecules) may be present in the protein shell. In certain embodiments, these gaps or discontinuities have a sufficiently small size (e.g., diameter) such that the protein shell limits or prevents one or more large macromolecules (e.g., peptides, polypeptides, polynucleotides, lipids, or polysaccharides) from passing through the protein shell.

[0960] As used herein, the term "proteinaceous coat" refers to a coat component that is predominantly (eg, >50%, >60%, >70%, >80%, >90%) protein.

[0961] As used herein, the term "regulatory nucleic acid" refers to a nucleic acid sequence that modifies the expression of a DNA sequence encoding an expression product, such as a transcribed and / or translated nucleic acid sequence. In embodiments, the expression product comprises RNA or protein.

[0962] As used herein, the term "regulatory sequence" refers to a nucleic acid sequence that modifies the transcription of a target gene product. In some embodiments, the regulatory sequence is a promoter or enhancer.

[0963] As used herein, the term "replication protein" refers to a protein, such as a viral protein, used during infection, viral genome replication / expression, viral protein synthesis, and / or assembly of viral components.

[0964] When observed by electron microscopy, the ring vector or ring VLP particle typically adopts one of two conformations: a symmetrical morphology (e.g., Figure 7A ) and asymmetric or less symmetric morphologies (e.g., Figure 7B ). Thus, as used herein with respect to the morphology of a ring vector or ring VLP particle, the term "symmetrical morphology" refers to a particle having a shape that is primarily symmetrical. In some cases, a particle having a symmetrical morphology may be approximately circular. In some cases, a particle having a symmetrical morphology may not be perfectly circular or spherical (e.g., may be oval). In some cases, a particle having a symmetrical morphology may include one or more deviations from a circular or spherical shape (e.g., one or more protrusions or indentations from its surface).

[0965] As used herein, the term "structural arginine-rich region" refers to a domain of an anellovirus ORF1 molecule having a structural arginine-rich region sequence as listed in any one of Tables B1-1 to B1-12, or a corresponding sequence in another ORF1 molecule.

[0966] As used herein, the term "structural jellycoil region" refers to a domain of an anellovirus ORF1 molecule having a structural jellycoil region sequence as listed in any one of Tables B1-1 to B1-12, or a corresponding sequence in another ORF1 molecule.

[0967] As used herein, the term "structural N22 domain" refers to a domain of an anellovirus ORF1 molecule having a structural N22 domain sequence as listed in any one of Tables B1-1 to B1-12, or a corresponding sequence in another ORF1 molecule.

[0968] As used herein, the term "structural C-terminal domain region" refers to a domain of an anellovirus ORF1 molecule having a structural C-terminal domain sequence as listed in any one of Tables B1-1 to B1-12, or a corresponding sequence in another ORF1 molecule.

[0969] As used herein, the term "jelly coil BH chain subdomain" refers to a domain of an anellovirus ORF1 molecule having a jelly coil BH chain subdomain sequence as listed in any one of Tables B1-1 to B1-12, or a corresponding sequence in another ORF1 molecule.

[0970] As used herein, the term "P1 domain" generally refers to a non-contiguous domain comprising the P1-1 subdomain and the P1-2 subdomain, such as anellovirus ORF1 molecule.

[0971] As used herein, the term "P1-1 subdomain" refers to a domain of an anellovirus ORF1 molecule having a P1-1 domain sequence as listed in any one of Tables B1-1 to B1-12, or a corresponding sequence in another ORF1 molecule.

[0972] As used herein, the term "P2 domain" refers to a domain of an anellovirus ORF1 molecule having a P2 domain sequence as listed in any one of Tables B1-1 to B1-12, or a corresponding sequence in another ORF1 molecule.

[0973] As used herein, the term "P1-2 subdomain" refers to a domain of an anellovirus ORF1 molecule having a P1-2 subdomain sequence as listed in any one of Tables B1-1 to B1-12, or a corresponding sequence in another ORF1 molecule.

[0974] As used herein, the term "jelly coil I chain subdomain" refers to a domain of an anellovirus ORF1 molecule having a jelly coil I chain subdomain sequence as listed in any one of Tables B1-1 to B1-12, or a corresponding sequence in another ORF1 molecule.

[0975] As used herein, the term "mutant ORF1," as applied to a particular domain or region of an ORF1 molecule, refers to a non-naturally occurring ORF1 domain or region that comprises at least one sequence difference (e.g., addition, deletion, or substitution) relative to the closest naturally occurring ORF1 domain or region sequence. For example, a "mutant ORF1 structural jelly coil region" comprises at least one sequence difference (e.g., addition, deletion, or substitution) relative to the closest naturally occurring anellovirus ORF1 structural jelly coil region.

[0976] As used herein, a "substantially non-pathogenic" organism, particle, or component refers to an organism, particle (e.g., a virus or ring vector, e.g., as described herein) or component thereof that does not cause or induce a detectable disease or pathogenic condition, e.g., in a host organism, e.g., a mammal, such as a human. In some embodiments, administration of the ring vector to a subject may result in mild reactions or side effects that are acceptable as part of standard treatment.

[0977] As used herein, the term "non-pathogenic" refers to an organism or component thereof that does not cause or induce a detectable disease or pathogenic condition, eg, in a host organism, eg, a mammal, eg, a human.

[0978] As used herein, a "substantially non-integrated" genetic element refers to a genetic element, e.g., a genetic element in a viral or ring vector, e.g., as described herein, wherein less than about 0.01%, 0.05%, 0.1%, 0.5%, or 1% of the genetic element that enters a host cell (e.g., a eukaryotic cell) or organism (e.g., a mammal such as a human) is integrated into the genome. In some embodiments, the genetic element is not detectably integrated into, e.g., the genome of the host cell. In some embodiments, integration of the genetic element into the genome can be detected using techniques as described herein, e.g., nucleic acid sequencing, PCR detection, and / or nucleic acid hybridization.

[0979] As used herein, a "substantially non-immunogenic" organism, particle, or component refers to an organism, particle (e.g., a virus or finger ring vector, e.g., as described herein) or a component thereof that does not cause or induce an undesirable or non-targeted immune response, e.g., in a host tissue or organism (e.g., a mammal, such as a human). In some embodiments, a substantially non-immunogenic organism, particle, or component does not produce a detectable immune response. In some embodiments, a substantially non-immunogenic finger ring vector does not produce a detectable immune response to a protein comprising an amino acid sequence as shown in any one of Tables N1-N26, or encoded by a nucleic acid sequence as shown in any of these tables. In some embodiments, an immune response (e.g., an undesired or untargeted immune response) is detected by measuring the presence or level of antibodies in a subject (e.g., the presence or level of anti-ring vector antibodies, e.g., the presence or level of antibodies to a ring vector as described herein), e.g., according to the anti-TTV antibody detection method described in Tsuda et al. (1999; J. Virol. Methods 77: 199-206; incorporated herein by reference) and / or the anti-TTV IgG level determination method described in Kakkola et al. (2008; Virology 382: 182-189; incorporated herein by reference). Antibodies to ring viruses or ring vectors based thereon can also be detected by methods known in the art for detecting antiviral antibodies, e.g., methods for detecting anti-AAV antibodies, e.g., the methods described in Calcedo et al. (2013; Front. Immunol. 4(341): 1-7; incorporated herein by reference).

[0980] As used herein, "subsequence" refers to a nucleic acid sequence or an amino acid sequence that is respectively included in a larger nucleic acid sequence or an amino acid sequence. In some cases, a subsequence can comprise a domain or a functional fragment of a larger sequence. In some cases, a subsequence can comprise a fragment of a larger sequence, and when separated from the larger sequence, the fragment can form a secondary and / or tertiary structure, similar to the secondary and / or tertiary structure formed by the subsequence when existing together with the remainder of the larger sequence. In some cases, a subsequence can be replaced by another sequence (for example, a subsequence comprising an exogenous sequence or a sequence that is heterologous to the remainder of the larger sequence, for example, from the corresponding subsequences of different anelloviruses).

[0981] As used herein, the term "surface moiety" refers to a moiety that is at least partially exposed on the outer surface of a particle (e.g., exposed to the solution surrounding the particle). A surface moiety is typically attached directly or indirectly to a component of the protein coat of the particle (e.g., an ORF1 molecule). In some cases, a surface moiety is covalently attached to a component of the protein coat of the particle (e.g., an ORF1 molecule). In some cases, a surface moiety is non-covalently attached to a component of the protein coat of the particle (e.g., an ORF1 molecule). In some cases, the surface moiety is bound to a binding moiety that is in turn (e.g., covalently or non-covalently) attached to a component of the protein coat of the particle (e.g., the ORF1 molecule). In some cases, the surface moiety is contained within an ORF1 molecule (e.g., is a heterologous domain of an ORF1 molecule). In some cases, the surface moiety is foreign to the anellovirus (e.g., the anellovirus from which the ORF1 molecule is derived and / or an anellovirus to which the ORF1 protein has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity). In some cases, the surface moiety is foreign to the target cell (e.g., a mammalian cell, e.g., a human cell) to be infected by the particle.

[0982] As used herein, "treatment," "treating," and their cognates refer to the medical management of a subject with the intent to ameliorate, improve, stabilize, prevent, or cure a disease, pathological condition, or disorder. The term includes active treatment (treatment intended to ameliorate a disease, pathological condition, or disorder), causal treatment (treatment directed at the cause of the disease, pathological condition, or disorder in question), palliative treatment (treatment designed to relieve symptoms), prophylactic treatment (treatment intended to prevent, minimize, or partially or completely inhibit the progression of the disease, pathological condition, or disorder in question); and supportive care (treatment used to supplement another therapy).

[0983] As used herein, the term "virome" refers to viruses in a particular environment, eg, a part of the body, eg, in an organism, eg, in a cell, eg, in a tissue.

[0984] The present invention generally relates to finger ring vectors, e.g., synthetic finger ring vectors, and uses thereof. The present disclosure provides finger ring vectors, compositions comprising finger ring vectors, and methods of preparing or using finger ring vectors. Finger ring vectors are typically used as delivery vehicles, e.g., for delivering therapeutic agents to eukaryotic cells. Typically, a finger ring vector will include a genetic element comprising a nucleic acid sequence (e.g., encoding an effector, e.g., an exogenous effector or an endogenous effector) enclosed in a protein coat. Relative to anellovirus sequence (e.g., as described herein), a finger ring vector may include one or more deletions of a sequence (e.g., a region or domain as described herein). Finger ring vectors can be used as substantially non-immunogenic vehicles for delivering genetic elements or effectors encoded therein (e.g., polypeptides or nucleic acid effectors, e.g., as described herein) into eukaryotic cells, e.g., to treat a disease or disorder in a subject comprising these cells.

[0985] Table of contents

[0986] I. Ring Vectors and Ring VLPs

[0987] A. Anellovirus

[0988] B. ORF1 molecule

[0989] i. Deletion and truncation of structural arginine-rich regions

[0990] ii. Structural C-terminal domain deletions and truncations

[0991] iii. Chimeric ORF1 molecules

[0992] (a) N-terminal insertions (e.g., structural arginine-rich region exchange)

[0993] (b) P1 / P2 domain exchange

[0994] (c) Other domain exchanges

[0995] C. ORF2 molecule

[0996] D.Genetic elements

[0997] E. Protein Binding Sequence

[0998] F.5'UTR region

[0999] G.GC-rich region

[1000] H. Effector

[1001] I. Protein coat

[1002] J. Surface part

[1003] i. Click Chemistry

[1004] (a) Exemplary click chemistry

[1005] (b) Surface lysine mutation

[1006] (c) Surface cysteine ​​mutations

[1007] ii. Genetic grafting

[1008] iii. X-fold symmetry

[1009] II. Compositions and methods for preparing finger ring vectors and finger ring VLPs

[1010] A. Components and Assembly of Ring Vectors and Ring VLPs

[1011] i. ORF1 molecules for assembling ring vectors and ring VLPs

[1012] ii. ORF2 molecules for assembling ring vectors and ring VLPs

[1013] iii. Production of protein components

[1014] (a) Baculovirus expression system

[1015] (b) Insect cell system

[1016] (c) Mammalian cell systems

[1017] B. Genetic Element Constructs

[1018] i.Plasmid

[1019] ii. Circular Nucleic Acid Constructs

[1020] iii. In vitro cyclization

[1021] iv. Tandem constructs

[1022] v. cis / trans constructs

[1023] vi. Expression cassette

[1024] vii. Design and Generation of Genetic Element Constructs

[1025] C. Effector

[1026] D.Host cells

[1027] i. Introduction of genetic elements into host cells

[1028] ii. Methods for providing one or more proteins in cis or trans

[1029] iii. Exemplary Cell Types

[1030] E. Culture Conditions

[1031] F. Harvest

[1032] G. In vitro assembly method of ring vectors

[1033] H. In vitro assembly of ring VLPs

[1034] I. Enrichment and Purification

[1035] III. Vector

[1036] IV. Composition

[1037] V. Host cells

[1038] VI. Instructions for Use

[1039] VII. Production Method

[1040] VIII. Administration / Delivery

[1041] I. Ring Carrier and Ring VLP

[1042] In some aspects, the invention described herein comprises compositions and methods for using and making finger ring vectors, finger ring vector formulations, finger ring VLPs, finger ring VLP formulations, and therapeutic compositions.

[1043] Ring carrier

[1044] In some embodiments, the anellovirus vector comprises an anellovirus-based sequence, structure, and / or function that is based on an anellovirus (e.g., an anellovirus as described herein, e.g., an anellovirus comprising a nucleic acid or polypeptide comprising a sequence as shown in any one of Tables A1-A26 or N1-N26), or a fragment or portion thereof, or other substantially non-pathogenic virus (e.g., a symbiotic virus, a commensal virus, a naturally occurring virus). In some embodiments, the anellovirus-based anellovirus vector comprises at least one element that is foreign to the anellovirus, e.g., an exogenous effector or a nucleic acid sequence encoding an exogenous effector within a genetic element of the anellovirus vector. In some embodiments, the anellovirus-based anellovirus vector comprises at least one element that is heterologous to another element from the anellovirus, e.g., an effector-encoding nucleic acid sequence that is heterologous to another associated nucleic acid sequence (e.g., a promoter element). In some embodiments, the finger ring vector comprises a genetic element (e.g., a circular DNA, e.g., a single-stranded DNA) comprising at least one element that is heterologous relative to the rest of the genetic element and / or the protein coat (e.g., an exogenous element encoding an effector, e.g., as described herein). The finger ring vector can be a delivery vehicle (e.g., a substantially non-pathogenic delivery vehicle) for delivering a payload into a host, e.g., a human. In some embodiments, the finger ring vector is capable of replicating in eukaryotic cells, e.g., mammalian cells, e.g., human cells. In some embodiments, the finger ring vector is substantially non-pathogenic and / or substantially non-integrating in mammalian (e.g., human) cells. In some embodiments, the finger ring vector is substantially non-immunogenic in mammals, e.g., humans. In some embodiments, the finger ring vector is replication-defective. In some embodiments, the finger ring vector is replicable.

[1045] In some embodiments, the ring vector comprises a curon or a component thereof (e.g., a genetic element, e.g., comprising a sequence encoding an effector and / or a protein coat), e.g., as described in PCT Application No. PCT / US2018 / 037379, which is incorporated herein by reference in its entirety.

[1046] In one aspect, the invention includes a finger ring vector comprising: (i) a genetic element comprising a promoter element, a sequence encoding an effector (e.g., an endogenous effector or an exogenous effector, e.g., a cargo), and a protein binding sequence (e.g., a coat protein binding sequence, e.g., a packaging signal), wherein the genetic element is single-stranded DNA and has one or both of the following properties: is circular and / or integrates into the genome of a eukaryotic cell at a frequency less than about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5% or 2% of the genetic elements that enter the cell; and (ii) a protein coat; wherein the genetic element is enclosed within the protein coat; and wherein the finger ring vector is capable of delivering the genetic element into a eukaryotic cell.

[1047] In some embodiments of the finger ring vectors described herein, the genetic elements integrate at a frequency of less than about 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5% or 2% of the genetic elements that enter the cell. In some embodiments, less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4% or 5% of the genetic elements of a plurality of finger ring vectors administered to a subject will integrate into the genome of one or more host cells of the subject. In some embodiments, the frequency of integration of the genetic elements of a finger ring vector, e.g., a population of finger ring vectors as described herein, into the host cell genome is lower than the frequency of a similar AAV virus population, e.g., the frequency is about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more lower than that of a similar AAV virus population.

[1048] In one aspect, the invention includes a finger ring vector comprising: (i) a genetic element comprising a promoter element and a sequence encoding an effector (e.g., an endogenous effector or an exogenous effector, e.g., a cargo), and a protein binding sequence (e.g., a coat protein binding sequence), wherein the genetic element has at least 75% (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a wild-type anellovirus sequence (e.g., a wild-type picolovirus (TTV), parvovirus (TTMV) or TTMDV sequence, e.g., a wild-type anellovirus sequence listed in any one of Tables N1-N26); and (ii) a protein coat; wherein the genetic element is enclosed within the protein coat; and wherein the finger ring vector is capable of delivering the genetic element into a eukaryotic cell.

[1049] In one aspect, the present invention comprises a finger-ring vector comprising:

[1050] a) a genetic element comprising (i) a sequence encoding a coat protein (e.g., a non-pathogenic coat protein), (ii) a coat protein binding sequence that enables the genetic element to bind to the non-pathogenic coat protein, and (iii) a sequence encoding an effector (e.g., an endogenous effector or an exogenous effector); and

[1051] b) a proteinaceous coat that is associated with, eg, envelopes, or surrounds the genetic element.

[1052] In some embodiments, the ring vector comprises a sequence or expression product from a non-enveloped circular single-stranded DNA virus (or has >70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% homology thereto). Animal circular single-stranded DNA viruses generally refer to a subgroup of single-stranded DNA (ssDNA) viruses that infect eukaryotic non-plant hosts and have a circular genome. Thus, animal circular ssDNA viruses can be distinguished from ssDNA viruses that infect prokaryotes (i.e., Pinothiaceae and Filoviridae) and ssDNA viruses that infect plants (i.e., Geminiviridae and Gnathoviridae). They can also be distinguished from linear ssDNA viruses that infect non-plant eukaryotes (i.e., Parvoviridae).

[1053] In some embodiments, the ring vector modulates host cell function, e.g., transiently or chronically. In certain embodiments, the cellular function is stably altered, e.g., the modulation persists for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days or longer, or any time therebetween. In certain embodiments, the alteration in cellular function is transient, e.g., the modulation persists for no more than about 30 minutes to about 7 days, or no more than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, or any time therebetween.

[1054] In some embodiments, the genetic element comprises a promoter element. In some embodiments, the promoter element is selected from RNA polymerase II dependent promoter, RNA polymerase III dependent promoter, PGK promoter, CMV promoter, EF-1α promoter, SV40 promoter, CAGG promoter or UBC promoter, TTV virus promoter, tissue-specific promoter, U6 (pollIII), a minimal CMV promoter with an upstream DNA binding site of an activating protein (TetR-VP16, Gal4-VP16, dCas9-VP16, etc.). In some embodiments, the promoter element comprises a TATA box. In some embodiments, the promoter element is endogenous to a wild-type anellovirus, for example, a wild-type anellovirus as described herein.

[1055] In certain embodiments, genetic elements comprise one or more of the following features: single strand, ring-type, minus strand and / or DNA.In certain embodiments, genetic elements comprise episome.In certain embodiments, the combined size of the genetic elements part except that effector is about 2.5kb-5kb (for example, about 2.8kb-4kb, about 2.8kb-3.2kb, about 3.6kb-3.9kb or about 2.8kb-2.9kb), less than about 5kb (for example, less than about 2.9kb, 3.2kb, 3.6kb, 3.9kb or 4kb) or at least 100 Nucleotide (for example, at least 1kb).

[1056] In some cases, the finger ring vectors, compositions comprising finger ring vectors, methods of using such finger ring vectors, etc., as described herein, are based in part on examples that illustrate how different effectors (e.g., miRNAs (e.g., for IFN or miR-625), shRNAs, etc.) and protein binding sequences (e.g., DNA sequences that bind to capsid proteins such as Q99153) can be combined with a protein shell (e.g., a capsid disclosed in Arch Virol (2007) 152: 1961-1975) to produce a finger ring vector that can then be used to deliver the effector to a cell (e.g., an animal cell, e.g., a human cell or a non-human animal cell, such as a pig or mouse cell). In embodiments, the effector can silence the expression of a factor, such as interferon. The examples further describe how the finger ring vector can be prepared by inserting the effector into a sequence, e.g., a sequence derived from an anellovirus. It is based on these examples that the description below contemplates various variations of the particular discoveries and combinations contemplated in the examples. For example, the skilled person will understand from the examples that a particular miRNA is merely an example of an effector, while other effectors may be, for example, other regulatory nucleic acids or therapeutic peptides. Similarly, the particular capsids used in the examples may be replaced with substantially non-pathogenic proteins as described below. The particular anellovirus sequences described in the examples may also be replaced with the anellovirus sequences described below. These considerations also apply to protein binding sequences, regulatory sequences such as promoters, etc. Independently of this, those skilled in the art will particularly consider these embodiments that are closely related to the examples.

[1057] In some embodiments, the ring vector or the genetic element contained therein is introduced into a cell (e.g., a human cell). In some embodiments, for example, once the ring vector or the genetic element has been introduced into a cell (e.g., a human cell), the effector (e.g., RNA, e.g., miRNA) (e.g., encoded by the genetic element of the ring vector) is expressed in the cell. In some embodiments, the introduction of the ring vector or the genetic element contained therein into a cell regulates (e.g., increases or decreases) the level of a target molecule (e.g., a target nucleic acid (e.g., RNA) or a target polypeptide) in the cell, e.g., by changing the expression level of the target molecule by the cell. In some embodiments, the introduction of the ring vector or the genetic element contained therein reduces the level of interferon produced by the cell. In some embodiments, the introduction of the ring vector or the genetic element contained therein into a cell regulates (e.g., increases or decreases) the function of the cell. In some embodiments, the introduction of the ring vector or the genetic element contained therein into a cell regulates (e.g., increases or decreases) the viability of the cell. In some embodiments, the introduction of the ring vector or the genetic element contained therein into a cell reduces the survival rate of a cell (e.g., a cancer cell).

[1058] In some embodiments, the finger ring vectors described herein (e.g., synthetic finger ring vectors) elicit an antibody positivity rate of less than 70% (e.g., less than about 60%, 50%, 40%, 30%, 20%, or 10% antibody positivity). In some embodiments, the antibody positivity rate is determined according to methods known in the art. In some embodiments, the antibody positivity rate is determined by detecting antibodies against an anellovirus (e.g., as described herein) or a finger ring vector based thereon in a biological sample, for example, according to the anti-TTV antibody detection method described in Tsuda et al. (1999; J. Virol. Methods 77:199-206; incorporated herein by reference) and / or according to the method for determining anti-TTV IgG seroprevalence described in Kakkola et al. (2008; Virology 382:182-189; incorporated herein by reference). Antibodies against anelloviruses or anellovirus-based anellovirus vector can also be detected by methods used in the art to detect antiviral antibodies, for example, methods for detecting anti-AAV antibodies, for example, the methods described in the following literature: Calcedo et al. (2013; Front. Immunol. [Immunology Frontier] 4(341): 1-7; which is incorporated herein by reference).

[1059] In certain embodiments, replication defect type, replication defect type or non-replication type genetic elements do not encode all necessary mechanisms or components required for genetic elements replication.In certain embodiments, replication defect type genetic elements do not encode replication factor.In certain embodiments, replication defect type genetic elements do not encode one or more ORFs (for example, ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3 and / or ORF2t / 3, for example, as described herein).In certain embodiments, mechanism or component not encoded by genetic elements can be provided in trans (for example, using auxiliary, for example helper virus or helper plasmid, or encoding in the nucleic acid comprised by host cell, for example, being integrated into the genome of host cell), for example, so that genetic elements can be replicated in the presence of the mechanism or component provided in trans.

[1060] In some embodiments, the packaging-defective, packaging-deficient, or non-packaging genetic element is unable to be packaged into a protein coat (e.g., wherein the protein coat comprises a capsid or a portion thereof, e.g., comprising a polypeptide encoded by an ORF1 nucleic acid, e.g., as described herein). In some embodiments, the packaging-defective genetic element is packaged into a protein coat with less than 10% efficiency (e.g., less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001%) compared to a wild-type anellovirus (e.g., as described herein). In some embodiments, the packaging-defective genetic element is unable to be packaged into a protein coat even in the presence of factors (e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, or ORF2t / 3) that permit packaging of a wild-type anellovirus genetic element (e.g., as described herein). In some embodiments, the packaging-defective genetic element is packaged into the protein coat with less than 10% efficiency (e.g., less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001%) compared to a wild-type anellovirus (e.g., as described herein), even in the presence of factors (e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, or ORF2t / 3) that allow packaging of a wild-type anellovirus genetic element (e.g., as described herein).

[1061] In some embodiments, the packageable genetic element can be packaged into a protein coat (e.g., wherein the protein coat comprises a capsid or a portion thereof, e.g., comprising a polypeptide encoded by an ORF1 nucleic acid, e.g., as described herein). In some embodiments, the efficiency of packaging the packageable genetic element into the protein coat is at least 20% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or more) compared to a wild-type anellovirus (e.g., as described herein). In some embodiments, the packageable genetic element can be packaged into a protein coat in the presence of factors (e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, or ORF2t / 3) that allow packaging of a wild-type anellovirus genetic element (e.g., as described herein). In some embodiments, the efficiency of packaging of the packageable genetic elements into the protein coat is at least 20% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or more) compared to a wild-type anellovirus (e.g., as described herein) in the presence of factors (e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, or ORF2t / 3) that allow packaging of wild-type anellovirus genetic elements (e.g., as described herein).

[1062] Ring VLP

[1063] In some embodiments, the anellovirus VLP has a sequence, structure, and / or function based on an anellovirus (e.g., an anellovirus as described herein, e.g., an anellovirus comprising a nucleic acid or polypeptide comprising a sequence as shown in any one of Tables A1-A26) or a fragment or portion thereof, or other substantially non-pathogenic viruses (e.g., symbiotic viruses, commensal viruses, natural viruses). In some embodiments, the anellovirus-based anellovirus VLP comprises at least one element that is exogenous to the anellovirus, e.g., an exogenous effector or a nucleic acid sequence encoding an exogenous effector. In some embodiments, the anellovirus-based anellovirus VLP comprises a surface portion comprising an exogenous effector. In some embodiments, the anellovirus-based anellovirus VLP comprises at least one element that is heterologous to another element from the anellovirus, e.g., an effector encoding nucleic acid sequence that is heterologous to another connected nucleic acid sequence (e.g., a promoter element). The anellovirus VLP can be a delivery vehicle (e.g., a substantially non-pathogenic delivery vehicle) for delivering a payload into a host, e.g., a human. In some embodiments, the anellovirus vector is not capable of replicating in eukaryotic cells, e.g., mammalian cells, e.g., human cells. In some embodiments, the Finger Ring VLP is substantially non-pathogenic and / or substantially non-integrating in mammalian (eg, human) cells. In some embodiments, the Finger Ring VLP is substantially non-immunogenic in mammals, eg, humans.

[1064] In one aspect, the present invention includes a ring VLP comprising a protein shell and an effector (e.g., an exogenous effector); wherein the ring VLP is capable of delivering the exogenous effector into a eukaryotic cell. In some embodiments, the exogenous effector is enclosed within the protein shell. In some embodiments, the exogenous effector is contained in a surface portion on the surface of the ring VLP (e.g., as described herein). In some embodiments, the protein shell comprises one or more ORF1 molecules (e.g., an anellovirus ORF1 protein, e.g., as described herein, or a polypeptide having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto).

[1065] In some embodiments, the ring VLP comprises a sequence or expression product from a non-enveloped circular single-stranded DNA virus (or has >70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% homology thereto). Animal circular single-stranded DNA viruses generally refer to a subgroup of single-stranded DNA (ssDNA) viruses that infect eukaryotic non-plant hosts and have a circular genome. Thus, animal circular ssDNA viruses can be distinguished from ssDNA viruses that infect prokaryotes (i.e., Microviridae and Filoviridae) and ssDNA viruses that infect plants (i.e., Geminiviridae and Gnathoviridae). They can also be distinguished from linear ssDNA viruses that infect non-plant eukaryotes (i.e., Parvoviridae).

[1066] In some embodiments, the ring VLP modulates host cell function, e.g., transiently or chronically. In certain embodiments, the cellular function is stably altered, e.g., the modulation persists for at least about 1 hour to about 30 days, or at least about 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 60 days or longer, or any time therebetween. In certain embodiments, the alteration in cellular function is transient, e.g., the modulation persists for no more than about 30 minutes to about 7 days, or no more than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, or any time therebetween.

[1067] In some cases, the finger ring VLPs, compositions comprising the finger ring VLPs, methods of using such finger ring VLPs, etc., as described herein, are based in part on examples that illustrate how different effectors (e.g., miRNAs (e.g., for IFN or miR-625), shRNAs, etc.) and protein binding sequences (e.g., DNA sequences that bind to capsid proteins such as Q99153) can be combined with a protein shell (e.g., a capsid disclosed in Arch Virol (2007) 152: 1961-1975) to produce finger ring VLPs that can then be used to deliver the effector to a cell (e.g., an animal cell, e.g., a human cell or a non-human animal cell, such as a pig or mouse cell). In embodiments, the effector can silence the expression of a factor such as interferon. The examples further describe how to prepare finger ring VLPs by inserting the effector into a sequence, e.g., a sequence derived from an anellovirus. It is based on these examples that the description below contemplates various variations of the particular discoveries and combinations contemplated in the examples. For example, the skilled person will understand from the examples that a particular miRNA is merely an example of an effector, while other effectors may be, for example, other regulatory nucleic acids or therapeutic peptides. Similarly, the particular capsids used in the examples may be replaced with substantially non-pathogenic proteins as described below. The particular anellovirus sequences described in the examples may also be replaced with the anellovirus sequences described below. These considerations also apply to protein binding sequences, regulatory sequences such as promoters, etc. Independently of this, those skilled in the art will particularly consider these embodiments that are closely related to the examples.

[1068] In some embodiments, the ring VLP is introduced into a cell (e.g., a human cell). In some embodiments, the exogenous effector is delivered to the cell. In some embodiments, the delivery of an exogenous effector to a cell regulates (e.g., increases or decreases) the level of a target molecule (e.g., a target nucleic acid (e.g., RNA) or a target polypeptide) in the cell, for example, by changing the expression level of the target molecule by the cell. In some embodiments, the delivery of an exogenous effector to a cell regulates (e.g., increases or decreases) the function of the cell. In some embodiments, the delivery of an exogenous effector to a cell regulates (e.g., increases or decreases) the viability of the cell. In some embodiments, the delivery of an exogenous effector to a cell reduces the viability of a cell (e.g., a cancer cell).

[1069] In some embodiments, the antibody positivity elicited by the finger ring VLPs described herein (e.g., synthetic finger ring VLPs) is less than 70% (e.g., less than about 60%, 50%, 40%, 30%, 20%, or 10%). In some embodiments, the antibody positivity is determined according to methods known in the art. In some embodiments, the antibody positivity is determined by detecting antibodies against an anellovirus (e.g., as described herein) or a finger ring VLP based thereon in a biological sample, for example, according to the anti-TTV antibody detection method described in Tsuda et al. (1999; J. Virol. Methods 77:199-206; incorporated herein by reference) and / or according to the method for determining anti-TTV IgG seroprevalence described in Kakkola et al. (2008; Virology 382:182-189; incorporated herein by reference). Antibodies against ring viruses or ring VLPs based thereon can also be detected by methods used in the art to detect antiviral antibodies, for example, methods for detecting anti-AAV antibodies, for example, the methods described in the following literature: Calcedo et al. (2013; Front. Immunol. [Immunology Frontier] 4(341): 1-7; which is incorporated herein by reference).

[1070] Anellovirus

[1071] In some embodiments, for example, as described herein, the ring vectors or ring VLPs comprise sequences or expression products derived from an anellovirus. In some embodiments, the ring vectors or ring VLPs comprise one or more sequences or expression products that are exogenous to the anellovirus. In some embodiments, the ring vectors or ring VLPs comprise one or more sequences or expression products that are endogenous to the anellovirus. In some embodiments, the ring vectors or ring VLPs comprise one or more sequences or expression products that are heterologous to one or more other sequences or expression products in the ring vector. Anelloviruses typically have a single-stranded circular DNA genome with negative polarity. Anelloviruses are generally not associated with any human disease. However, attempts to link anellovirus infection to human disease have been hampered by the high incidence of asymptomatic anellovirus viremia in one or more control cohorts, the significant genomic diversity within the anellovirus family, the historical inability to propagate the pathogens in vitro, and the lack of one or more animal models of anellovirus disease (Yzebe et al., Panminerva Med. [Italian Medical Association Journal] (2002) 44:167-177; Biagini, P., Vet. Microbiol. [Veterinary Microbiology] (2004) 98:95-101).

[1072] Anelloviruses are typically transmitted through oral or fecal-oral infection, mother-to-child transmission, and / or intrauterine transmission (Gerner et al., Ped. Infect. Dis. J. [Pediatric Infectious Diseases Journal] (2000) 19:1074-1077). In some cases, infected individuals may be characterized by prolonged (months to years) anellovirus viremia. A person can be co-infected with more than one genogroup or strain (Saback et al., Scad. J. Infect. Dis. [Scandinavian Journal of Infectious Diseases] (2001) 33:121-125). It has been suggested that these genogroups can recombine within an infected person (Rey et al., Infect. [Infection] (2003) 31:226-233). Double-chain isoform (replicative) intermediates have been found in several tissues such as liver, peripheral blood mononuclear cells, and bone marrow (Kikuchi et al., J. Med. Virol. (2000) 61:165-170; Okamoto et al., Biochem. Biophys. Res. Commun. (2002) 270:657-662; Rodriguez-lígo et al., Am. J. Pathol. (2000) 156:1227-1234).

[1073] In some embodiments, the ring vector or ring VLP as described herein comprises one or more polypeptides (e.g., ORF1 molecules) comprising an amino acid sequence having at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to, for example, an anellovirus sequence as described herein, or a fragment thereof. In embodiments, the polypeptide comprises an amino acid sequence encoded by a nucleic acid sequence selected from any one of the sequences shown in Tables N1-N26, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto. In embodiments, the polypeptide comprises a sequence as shown in any one of Tables A1-A26, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[1074] In some embodiments, the genetic element comprises a nucleotide sequence encoding an amino acid sequence or a functional fragment thereof, or a sequence having at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the amino acid sequences described herein (e.g., an anellovirus amino acid sequence).

[1075] In some embodiments, the finger ring vectors as described herein comprise one or more nucleic acid molecules (e.g., genetic elements as described herein) comprising a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an anellovirus sequence, e.g., an anellovirus sequence as described herein, or a fragment thereof. In embodiments, the finger ring vector comprises a nucleic acid sequence selected from any one of Tables N1-N26, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto. In an embodiment, the ring vector comprises a polypeptide comprising a sequence as shown in any one of Tables A1-A26, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[1076] In some embodiments, the anellovirus vectors described herein comprise one or more nucleic acid molecules (e.g., genetic elements as described herein) comprising a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more of the TATA box, capping site, initiation element, transcription start site, 5'UTR conserved domain, ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, three open reading frame regions, poly(A) signal, GC-rich region, or any combination thereof of any of the anelloviruses described herein (e.g., an anellovirus sequence annotated in any of Tables N1-N26 or an anellovirus sequence encoded by a sequence listed in any of Tables N1-N26). In some embodiments, the nucleic acid molecule comprises a sequence encoding a capsid protein, e.g., an ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, or ORF2t / 3 sequence of any of the anelloviruses described herein (e.g., an anellovirus sequence annotated in any of Tables N1-N26 or an anellovirus sequence encoded by a sequence listed in any of Tables N1-N26). In some embodiments, the nucleic acid molecule comprises a sequence encoding a capsid protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an anellovirus ORF1 or ORF2 protein (e.g., an ORF1 or ORF2 amino acid sequence set forth in any of Tables A1-A26, or an ORF1 or ORF2 amino acid sequence encoded by a nucleic acid sequence set forth in any of Tables N1-N26). In embodiments, the nucleic acid molecule comprises a sequence encoding a capsid protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an Anellovirus ORF1 protein (e.g., an ORF1 amino acid sequence set forth in any one of Tables A1-A26, or an ORF1 amino acid sequence encoded by a nucleic acid sequence set forth in any one of Tables N1-N26).

[1077] Nucleic acid sequence

[1078] In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus ORF1 nucleotide sequence of any one of Tables N1-N26. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus ORF2 nucleotide sequence of any one of Tables N1-N26. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus ORF3 nucleotide sequence of any one of Tables N1-N26. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus GC-rich region nucleotide sequence of any one of Tables N1-N26. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus 5'UTR conserved domain nucleotide sequence of any one of Tables N1-N26.

[1079] Amino acid sequence encoded by the nucleic acid sequence

[1080] In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the Anellovirus ORF1 amino acid sequences in Tables A1-A26. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the Anellovirus ORF2 amino acid sequences in Tables A1-A26. In embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the Anellovirus ORF3 amino acid sequence of any one of Tables A1-A26.

[1081] Protein containing amino acid sequence

[1082] In embodiments, the finger ring vectors described herein comprise a protein having an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the anellovirus ORF1 amino acid sequence of any one of Tables A1-A26. In embodiments, the finger ring vectors described herein comprise a protein having an amino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the anellovirus ORF2 amino acid sequence of any one of Tables A1-A26. In embodiments, the finger ring vectors described herein comprise a protein having an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the anellovirus ORF3 amino acid sequences in Tables A1-A26. In some embodiments, the ORF1 molecule (e.g., contained in the finger ring vector) comprises any one of the anellovirus ORF1 proteins in Tables A1-A26, or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof. In some embodiments, the ORF2 molecule (e.g., contained in the finger ring vector) comprises any one of the anellovirus ORF2 proteins in Tables A1-A26, or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof. In some embodiments, the ORF3 molecule (e.g., contained in the finger ring vector) comprises any one of the anellovirus ORF3 proteins in Tables A1-A26, or a splice variant or post-translational processing (e.g., proteolytic processing) variant thereof.

[1083] A polypeptide comprising an amino acid sequence

[1084] In some embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus ORF1 amino acid sequence described herein. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus ORF1 amino acid sequence of any one of Tables A1-A26.

[1085] In some embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an anellovirus ORF1 nucleic acid described herein. In some embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF1 molecule encoded by an anellovirus ORF1 nucleic acid listed in any one of Tables A1-A26.

[1086] In some embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an Anellovirus ORF2 amino acid sequence described herein. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an Anellovirus ORF2 amino acid sequence of any one of Tables A1-A26.

[1087] In some embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF2 molecule encoded by an anellovirus ORF2 nucleic acid described herein. In some embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF2 molecule encoded by an anellovirus ORF2 nucleic acid listed in any one of Tables A1-A26.

[1088] In some embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus ORF3 amino acid sequence described herein. In embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus ORF3 amino acid sequence of any one of Tables A1-A26.

[1089] In some embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF3 molecule encoded by an anellovirus ORF3 nucleic acid described herein. In some embodiments, the polypeptides described herein comprise an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an ORF3 molecule encoded by an anellovirus ORF3 nucleic acid listed in any one of Tables A1-A26.

[1090] In some embodiments, the polypeptide comprises an amino acid sequence as shown in any one of Tables A1-A26 (e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, or ORF2t / 3 sequence), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[1091] Table N1. Novel anellovirus nucleic acid sequences (alpha-type anellovirus)

[1092] NameTTV-RTx1

[1093] Genus / clade Alpha-Perivirus, clade 6

[1094] Accession number SRR2167793

[1095] Full sequence: 3648 bp

[1096]

[1097]

[1098]

[1099] Notes:

[1100]

[1101] Table A1. Novel anellovirus amino acid sequences (Alpha-type anellovirus, clade 6)

[1102]

[1103]

[1104] Table N2. Novel anellovirus nucleic acid sequences (Alpha-type anellovirus) NameTTV-RTx2 Genus / clade Alpha-Perivirus, clade 6 Accession number SRR3479021

[1105] Full sequence: 3704bp

[1106]

[1107]

[1108] Notes:

[1109]

[1110]

[1111] Table A2. Novel anellovirus amino acid sequences (Alpha-type anellovirus, clade 6)

[1112]

[1113]

[1114] Table N3. Novel anellovirus nucleic acid sequences (Alpha-type anellovirus)

[1115] NameTTV-RTx3

[1116] Genus / clade Alpha-Perivirus, clade 4 Accession number SRR3479781

[1117] Full sequence: 3653bp

[1118]

[1119]

[1120]

[1121]

[1122] Table A3. Novel anellovirus amino acid sequences (Alpha-type anellovirus, clade 4)

[1123]

[1124]

[1125]

[1126] Table N4. Novel Anellovirus Nucleic Acid Sequences (Alpha-type Anellovirus)

[1127] NameTTV-RTx4

[1128] Genus / clade Alpha-Perivirus, clade 4 Accession number SRR3481579 Full sequence: 3742 bp

[1129]

[1130]

[1131] Notes:

[1132]

[1133] Table A4. Novel anellovirus amino acid sequences (Al...

Claims

1. A polypeptide comprising (e.g., in the N-terminal to C-terminal direction): (i) Structural jelly-coil region of anellovirus ORF1 molecule; (ii) the structural N22 domain of the anellovirus ORF1 molecule; and (iii) a portion of a structural C-terminal domain (CTD) of an anellovirus ORF1 molecule, the portion comprising a deletion of about 20-30, 30-40 (e.g., about 37), 40-50 (e.g., about 55), 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130 (e.g., about 129), 130-140 (e.g., about 131), 140-150 (e.g., about 148), or 150-160 (e.g., about 155) amino acids at the C-terminus of the structural CTD relative to the corresponding wild-type structural CTD of the anellovirus ORF1 molecule.

2. A polypeptide comprising (e.g., in the N-terminal to C-terminal direction): (i) optionally a first portion of the structural jelly coil region of an Orovirus ORF1 molecule (eg, beta strand BH comprising the structural jelly coil region); (ii) a first portion of the P1 domain of an Anellovirus ORF1 molecule (e.g., a P1-1 domain as described herein); (iii) P2 domain of anellovirus ORF1 molecule; (iv) a second portion of the P1 domain of an Anellovirus ORF1 molecule (e.g., a P1-2 domain as described herein); (v) optionally a second portion of the structural jelly coil region of the Anellovirus ORF1 molecule (eg, beta strand I comprising the structural jelly coil region); and (vi) a portion of a structural C-terminal domain (CTD) of an anellovirus ORF1 molecule, the portion comprising a deletion of about 20-30, 30-40 (e.g., about 37), 40-50 (e.g., about 55), 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 110-120, 120-130 (e.g., about 129), 130-140 (e.g., about 131), 140-150 (e.g., about 148), or 150-160 (e.g., about 155) amino acids at the C-terminus of the structural CTD relative to the corresponding wild-type structural CTD of the anellovirus ORF1 molecule.

3. A polypeptide comprising (e.g., in the N-terminal to C-terminal direction): (i) a structural jelly coil region of an Anellovirus ORF1 molecule, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto; and (ii) the structural N22 domain of anellovirus ORF1 molecule, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto; and The polypeptide lacks the structural arginine-rich region of the anellovirus ORF1 molecule.

4. A polypeptide comprising (e.g., in the N-terminal to C-terminal direction): (i) optionally a first portion of the structural jelly coil region of an Orovirus ORF1 molecule (eg, beta strand BH comprising the structural jelly coil region); (ii) a first portion of the P1 domain of an Anellovirus ORF1 molecule (e.g., a P1-1 domain as described herein); (iii) P2 domain of anellovirus ORF1 molecule; (iv) a second portion of the P1 domain of an Anellovirus ORF1 molecule (e.g., a P1-2 domain as described herein); (v) optionally a second portion of the structural jelly coil region of the Anellovirus ORF1 molecule (eg, beta strand I comprising the structural jelly coil region); and The polypeptide lacks the structural arginine-rich region of the anellovirus ORF1 molecule.

5. A polypeptide comprising (e.g., in the N-terminal to C-terminal direction): (i) a structural arginine-rich region of a first anellovirus ORF1 molecule, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto; (ii) a structural jelly-coil region of a second anellovirus ORF1 molecule, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto; as well as (iii) the structural N22 domain of the second anellovirus ORF1 molecule, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto; The sequence of (i) comprises at least one amino acid sequence difference relative to the structural arginine-rich region of the second anellovirus ORF1 molecule.

6. A polypeptide comprising (e.g., in the N-terminal to C-terminal direction): (i) a structural arginine-rich region of a first anellovirus ORF1 molecule, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto; (ii) optionally a first portion of the structural jelly coil region of a second anellovirus ORF1 molecule (e.g., beta strand BH comprising the structural jelly coil region); (iii) a first portion of the P1 domain of the second anellovirus ORF1 molecule (e.g., a P1-1 domain as described herein); (iv) the P2 domain of the second anellovirus ORF1 molecule; (v) a second portion of the P1 domain of the second anellovirus ORF1 molecule (e.g., a P1-2 domain as described herein); (vi) optionally a second portion of the structural jelly coil region of the second anellovirus ORF1 molecule (eg, beta strand I comprising the structural jelly coil region); and The sequence of (i) comprises at least one amino acid sequence difference relative to the structural arginine-rich region of the second anellovirus ORF1 molecule.

7. A polypeptide comprising (e.g., in the N-terminal to C-terminal direction): (i) the N-terminal portion of a first Anellovirus ORF1 molecule, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, wherein the N-terminal portion of the first Anellovirus ORF1 molecule has a length of 30-40, 40-50, 50-60, 60-70, 70-80 or 80-90 amino acids; and (ii) the C-terminal portion of a second Anellovirus ORF1 molecule, or an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto, wherein the C-terminal portion of the second Anellovirus ORF1 molecule has a length of 590-600, 600-610, 610-620, 620-630, 630-640, 640-650, 650-660, 660-670, 670-680, 680-690, 690-700, 700-710, 710-720 or 720-730 amino acids; and The sequence of (i) comprises at least one amino acid sequence difference relative to the structural arginine-rich region of the second anellovirus ORF1 molecule.

8. A polypeptide comprising (e.g., in the N-terminal to C-terminal direction): (i) the structural jelly-coil region of the anellovirus ORF1 molecule; and (ii) the structural N22 domain of the anellovirus ORF1 molecule; and wherein the polypeptide lacks the structural arginine-rich region of the anellovirus ORF1 molecule; and The polypeptide lacks the structural C-terminal domain of the anellovirus ORF1 molecule.

9. A polypeptide comprising (e.g., in the N-terminal to C-terminal direction): (i) optionally a structural arginine-rich region of an Anellovirus ORF1 (e.g., the full-length arginine-rich region or a portion of the structural arginine-rich region), (ii) Structural jelly-coil region of anellovirus ORF1 molecule, (iii) the P1 domain of the anellovirus ORF1 molecule, (iv) the P2 domain of the anellovirus ORF1 molecule, and (v) optionally a structural C-terminal domain (CTD) of an Angiovirus ORF1 molecule (e.g., a full-length structural CTD or a portion of a structural CTD); in: (a) the P1 domain is from an anellovirus that is different from one or more (e.g., 1, 2, 3, or 4) of the structural arginine-rich region, the structural jellycoil region, the P2 domain, and / or the structural CTD; (b) the P2 domain is from an anellovirus that is different from one or more (e.g., 1, 2, 3, or 4) of the structural arginine-rich region, the structural jelly-coil region, the P1 domain, and / or the structural CTD; or (c) the P1 domain and the P2 domain are from an anellovirus different from one or more (e.g., 1, 2 or 3) of the structural arginine-rich region, the structural jelly coil region and / or the structural CTD, optionally wherein the P1 domain and the P2 domain are from the same anellovirus.

10. A finger-ring carrier, comprising: (i) a protein coat comprising: an Anellovirus ORF1 protein listed in Table A26 or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, and (ii) a genetic element surrounded by the protein coat, wherein the genetic element comprises a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector); wherein the protein coat and / or the genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type anellovirus ORF1 protein and / or a wild-type anellovirus genome (e.g., as described herein), such as an insertion, substitution, chemical or enzymatic modification, and / or deletion, such as a deletion of a domain (e.g., one or more of a structural arginine-rich region, a structural jelly coil domain, a structural HVR, a structural N22, a structural CTD, a P1 domain, or a P2 domain, such as described herein) or a genomic region (e.g., one or more of a TATA box, a capping site, a transcription start site, a 5'UTR, an open reading frame (ORF), a poly(A) signal, or a GC-rich region, such as described herein).

11. A finger-ring carrier, comprising: (i) a protein coat comprising a polypeptide encoded by an Anellovirus ORF1 nucleic acid sequence listed in Table N24, or a polypeptide encoded by a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the Anellovirus ORF1 nucleic acid sequence, and (ii) a genetic element surrounded by the protein coat, wherein the genetic element comprises a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector); wherein the protein coat and / or the genetic element comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) relative to a wild-type anellovirus ORF1 protein and / or a wild-type anellovirus genome (e.g., as described herein), such as an insertion, substitution, chemical or enzymatic modification, and / or deletion, such as a deletion of a domain (e.g., one or more of a structural arginine-rich region, a structural jelly coil domain, a structural HVR, a structural N22, a structural CTD, a P1 domain, or a P2 domain, such as described herein) or a genomic region (e.g., one or more of a TATA box, a capping site, a transcription start site, a 5'UTR, an open reading frame (ORF), a poly(A) signal, or a GC-rich region, such as described herein).

12. An ORF1 molecule comprising an exogenous surface moiety, wherein the exogenous surface moiety is attached to (e.g., conjugated to) loop 10 of ORF1, e.g., at positions 254, 263, 264, 265, 272, 273, 274, 276, 283, 284, 285, 287, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331 374, 376, 378, 380 or 381 corresponding to an amino acid residue (e.g., a cysteine ​​residue).

13. A protein complex comprising five ORF1 molecules, wherein each of the ORF1 molecules comprises: (i) ORF1 domain, and (ii) exogenous surface portion; The exogenous surface portions of the five ORF1 molecules form a pentamer.

14. A protein complex comprising three ORF1 molecules, wherein each of the ORF1 molecules comprises: (i) ORF1 domain, and (ii) exogenous surface portion; The exogenous surface parts of the three ORF1 molecules form a trimer.

15. A protein complex comprising two ORF1 molecules, wherein each of the ORF1 molecules comprises: (i) ORF1 domain, and (ii) exogenous surface portion; The exogenous surface parts of the two ORF1 molecules form a dimer.

16. A particle comprising: (a) a protein coat comprising an ORF1 molecule; and (b) a genetic element comprising a heterologous nucleic acid sequence encoding an exogenous effector; wherein the genetic element is enclosed within the protein coat; and The particles have one or more of the following characteristics: (i) the genetic element (eg, a DNA genetic element) does not comprise anellovirus 5'UTR or an origin of replication; (ii) the sequence encoding the exogenous effector occupies at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the genetic element (e.g., DNA genetic element); (iii) the heterologous nucleic acid sequence occupies at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the genetic element (e.g., DNA genetic element); (iv) the particle does not contain a detectable amount of (e.g., any) a polypeptide from a host cell, or contains fewer than 5, 10, 15, 20, 25, 30, 40, or 50 copies of a polypeptide from a host cell; (v) the particle contains no detectable amount (e.g., any) of a nucleic acid molecule from a host cell, or contains fewer than 2, 3, 4, or 5 copies of a nucleic acid molecule from a host cell; (vi) the particles comprise a denaturant at a concentration of less than about 0.01 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1 M, 1.1 M, 1.2 M, 1.3 M, 1.5 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, or 2 M; (vii) does not substantially replicate when introduced into a cell (e.g., a human cell); and / or (viii) It has a symmetrical shape.

17. A particle comprising: a protein coat comprising about 40-80 (e.g., about 60) copies of the ORF1 molecule; and The particles: (i) does not contain (e.g., does not surround) a polynucleotide, (ii) does not contain (e.g., does not surround) a detectable level of a polynucleotide, (iii) does not comprise (e.g., does not surround) a polynucleotide greater than 1000, 500, 200 or 100 nucleotides in length, (iv) does not comprise (e.g., does not encompass) a polynucleotide comprising any contiguous nucleic acid sequence of at least 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides in length that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a contiguous sequence in a wild-type anellovirus genome (e.g., as described herein), and / or (v) does not contain a polynucleotide containing anellovirus 5'UTR or origin of replication.

18. A composition comprising a plurality of particles comprising a protein shell comprising about 40-80 (e.g., about 60) copies of an ORF1 molecule; wherein at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% of the particles do not contain (e.g., do not surround): (i) a polynucleotide, (ii) a nucleic acid molecule of greater than 1000, 500, 200 or 100 nucleotides in length, (iii) a plurality of polynucleotides, (iv) a circular nucleic acid molecule, (v) single-stranded nucleic acid molecules, and / or (vi) a genetic element (e.g., a genetic element of a ring vector), e.g., as described herein; or The composition comprises less than 10 10 -10 14 (For example, less than 10 10 -10 11 , 10 11 -10 12 , 10 12 -10 13 or 10 13 -10 14 ) viral genome equivalent of nucleic acid molecules (e.g., genetic elements, e.g., genetic elements of a ring vector as described herein) per kilogram of the subject to be administered the composition (e.g., as determined by qPCR or by measuring optical density).

19. A method for decomposing particles, the method comprising: (a) providing a mixture comprising particles and a denaturing agent, wherein the particles comprise: (i) a protein coat comprising a plurality of anellovirus ORF1 molecules, and (ii) a nucleic acid molecule (e.g., a nucleic acid endogenous to the host cell or a nucleic acid exogenous to the host cell, such as an anellovirus genome); and (b) incubating the mixture under conditions suitable for: The protein coat breaks down, and The nucleic acid molecule dissociates from the protein coat.

20. A method for preparing a finger ring carrier, the method comprising: (a) providing a mixture comprising a plurality of anellovirus ORF1 molecules, wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the plurality of ORF1 molecules are not contained in particles comprising about 40-80 (e.g., about 60) copies of the ORF1 molecule; (b) subjecting the mixture to conditions suitable for in vitro assembly of the anellovirus ORF1 molecules; as well as (c) incubating the anellovirus ORF1 molecules with a plurality of genetic elements under conditions suitable for assembling the anellovirus ORF1 molecules into one or more finger ring vectors each enclosing one or more of the genetic elements.

21. A method for preparing a finger ring carrier, the method comprising: (a) providing a mixture comprising a plurality of anellovirus ORF1 molecules, and subjecting the mixture to denaturing conditions (e.g., providing a denaturing agent as part of the mixture, e.g., contacting the mixture with a denaturing agent), wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the plurality of ORF1 molecules are not contained in particles comprising about 40-80 (e.g., about 60) copies of the ORF1 molecule; (b) subjecting the mixture to non-denaturing conditions (e.g., reducing the concentration of the denaturing agent to a level) suitable for in vitro assembly of the Anellovirus ORF1 molecules (e.g., by dialysis); and (c) incubating the anellovirus ORF1 molecules with a plurality of genetic elements under conditions suitable for assembling the anellovirus ORF1 molecules into one or more finger ring vectors each enclosing one or more of the genetic elements.

22. A method for preparing a ring VLP, the method comprising: (a) providing a mixture comprising a plurality of anellovirus ORF1 molecules, wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the plurality of ORF1 molecules are not contained in particles comprising about 40-80 (e.g., about 60) copies of the ORF1 molecule; (b) subjecting the mixture to conditions suitable for in vitro assembly of the anellovirus ORF1 molecules; as well as (c) incubating the Anellovirus ORF1 molecules with multiple effectors (eg, exogenous effectors) under conditions suitable for assembling the Anellovirus ORF1 molecules into one or more Anellovirus VLPs that each surround one or more of the effectors.

23. A method for preparing a ring VLP, the method comprising: (a) providing a mixture comprising a plurality of anellovirus ORF1 molecules and a denaturing agent, wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the plurality of ORF1 molecules are not contained in particles comprising about 40-80 (e.g., about 60) copies of the ORF1 molecule; (b) reducing the concentration of the denaturing agent to a level suitable for the in vitro assembly of the anellovirus ORF1 molecules; and (c) incubating the anellovirus ORF1 molecules with multiple effectors (eg, exogenous effectors) under conditions suitable for: assembling the anellovirus ORF1 molecules in vitro into one or more anellovirus VLPs that each enclose one or more of the effectors.

24. A method for preparing a ring VLP, the method comprising: (a) providing a mixture comprising particles and a denaturing agent, wherein the particles comprise: (i) a protein coat comprising a plurality of anellovirus ORF1 molecules, and (ii) a nucleic acid molecule (e.g., a host cell nucleic acid molecule); and (b) incubating the mixture under conditions suitable for: The protein coat breaks down, and The nucleic acid molecule dissociates from the protein coat; (c) providing a mixture comprising a plurality of Anellovirus ORF1 molecules and a denaturing agent, wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the plurality of ORF1 molecules are not contained in particles comprising about 40-80 (e.g., about 60) copies of the ORF1 molecule.

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