Surface-modified and modular virus particles

Abstract

The present invention relates generally to annelovectors, annelovLPs, and compositions and uses thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 289,967, filed December 15, 2021, U.S. Provisional Application No. 63 / 289,975, filed December 15, 2021, and U.S. Provisional Application No. 63 / 344,029, filed May 19, 2022, the contents of which are hereby incorporated by reference in their entireties.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is hereby incorporated by reference in its entirety. Said XML copy, created on December 14, 2022, is named V2057-7020WO_SL.txt and is 1,080,558 bytes in size. [Background technology]

[0003] There is a continuing need to develop vectors suitable for delivering therapeutic agents to patients. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure provides anaerovectors, e.g., synthetic anaerovectors, that can be used, for example, as a delivery vehicle to deliver genetic material, to deliver an effector, e.g., a payload, or to deliver a therapeutic agent or effector to a eukaryotic cell (e.g., a human cell or tissue). The anaerovector generally comprises a surface moiety as described herein on its outer surface (e.g., associated with a proteinaceous outer portion). In some embodiments, the anaerovector (e.g., a particle, e.g., a viral particle, e.g., an Anellovirus particle) comprises a genetic element (e.g., a genetic element comprising a therapeutic DNA sequence) encapsulated in a proteinaceous outer portion (e.g., a proteinaceous outer portion comprising a polypeptide encoded by an Anellovirus capsid protein, e.g., an Anellovirus ORF1 molecule or an Anellovirus ORF1 nucleic acid, e.g., as described herein), and the proteinaceous outer portion is capable of introducing the genetic element into a cell (e.g., a mammalian cell, e.g., a human cell). In some embodiments, the anaerovector is a particle that includes a proteinaceous outer part that includes a polypeptide encoded by an Anellovirus ORF1 nucleic acid (e.g., an ORF1 nucleic acid of Betatorquevirus, e.g., as described herein). The genetic elements of the anaerovectors of the present disclosure are typically circular and / or single-stranded DNA molecules (e.g., circular and single-stranded) and generally include a protein binding sequence that may bind to the proteinaceous outer part that encapsulates it, or to a polypeptide associated with it, thereby facilitating encapsulation of the genetic element within the proteinaceous outer part and / or enrichment of the genetic element relative to other nucleic acids within the proteinaceous outer part. In some examples, the genetic element is circular or linear. In some examples, the genetic element includes or encodes an effector (e.g., a nucleic acid effector such as a non-coding RNA, or a polypeptide effector, e.g., a protein), e.g., the effector can be expressed in the cell.In some embodiments, the effector is a therapeutic agent or therapeutic effector, e.g., as described herein. In some embodiments, the effector is an endogenous or exogenous effector, e.g., to the wild-type Anellovirus or the target cell. In some examples, the effector is an exogenous effector to the wild-type Anellovirus or the target cell. In some embodiments, the Anellovector can deliver an effector into a cell by contacting the cell and introducing a genetic element encoding the effector into the cell such that the effector is produced or expressed by the cell. In certain cases, the effector is an endogenous effector (e.g., endogenous to the target cell but provided in increased amounts, e.g., by the Anellovector). In other cases, the effector is an exogenous effector. The effector can, in some cases, modulate the function of a cell or modulate the activity or level of a target molecule in a cell. For example, the effector can reduce the level of a target protein in a cell. In another example, anaerovectors can deliver and express effectors, e.g., exogenous proteins, in vivo. Anellovectors can be used, for example, to deliver genetic material to a target cell, tissue, or subject; to deliver effectors to a target cell, tissue, or subject; or for treating a disease or disorder, e.g., by delivering an effector that can act as a therapeutic agent to a desired cell, tissue, or subject. In some examples, anaerovectors are made by in vitro assembly. In vitro assembly of anaerovectors generally involves the formation of a proteinaceous exterior that encapsulates the genetic elements, which is performed outside the host cell (e.g., in a cell-free suspension, lysate, or supernatant). In vitro assembly utilizes components produced in the host cell in some examples, but generally does not require a host cell for particle assembly.

[0005] The present disclosure provides anaeroVLPs, e.g., synthetic anaeroVLPs, that can be used, for example, as a delivery vehicle for delivering genetic material, for delivering an effector, e.g., a payload, or for delivering a therapeutic agent or therapeutic effector to a eukaryotic cell (e.g., a human cell or human tissue). An anaeroVLP generally comprises a surface moiety as described herein on its outer surface (e.g., associated with a proteinaceous outer moiety). In some embodiments, the surface moiety comprises an effector. In some embodiments, the surface moiety comprises a targeting agent (e.g., an agent that targets the anaeroVLP to a target cell or tissue). In some embodiments, an anaeroVLP (e.g., a particle, e.g., a viral particle, e.g., an Anellovirus particle) comprises a proteinaceous outer moiety (e.g., a proteinaceous outer moiety comprising an Anellovirus capsid protein, e.g., a polypeptide encoded by an Anellovirus ORF1 molecule or an Anellovirus ORF1 nucleic acid, e.g., as described herein). In some embodiments, anelloVLPs are particles that include a proteinaceous outer part that includes a polypeptide encoded by an Anellovirus ORF1 nucleic acid (e.g., an ORF1 nucleic acid of Betatorquevirus, e.g., as described herein). In some embodiments, the proteinaceous outer part encapsulates an effector. In some embodiments, the effector is a therapeutic agent or a therapeutic effector, e.g., as described herein. In some examples, the effector is an endogenous effector or an exogenous effector, e.g., to a wild-type Anellovirus or a target cell. In some embodiments, the effector is exogenous to a wild-type Anellovirus or a target cell. In some embodiments, anelloVLPs can deliver an effector to a cell by contacting the cell and introducing the effector into the cell.In certain instances, the effector is an endogenous effector (e.g., endogenous to the target cell but provided in increased amounts, e.g., by anelloVLP). In other instances, the effector is an exogenous effector. The effector can, in some instances, modulate the function of a cell or modulate the activity or level of a target molecule in a cell. For example, the effector can reduce target protein levels in a cell. In another example, anelloVLP can deliver an effector, e.g., an exogenous protein, in vivo. AnelloVLPs can be used, for example, to deliver an effector to a target cell, tissue, or subject; or can be used for the treatment of diseases and disorders, for example, by delivering an effector that can act as a therapeutic agent to a desired cell, tissue, or subject. In some instances, anelloVLPs are created by in vitro assembly. In vitro assembly of anelloVLPs generally involves the formation of a proteinaceous outer portion (e.g., a proteinaceous outer portion that encapsulates an effector) that is linked to an effector, and is performed outside of a host cell (e.g., in a cell-free suspension, lysate, or supernatant). In vitro assembly of anelloVLPs utilizes components produced in the host cell in some instances, but generally does not require a host cell for particle assembly.

[0006] The present invention further provides synthetic anaerovectors and synthetic anaeroVLPs. The synthetic anaerovectors or synthetic anaeroVLPs have at least one structural difference, such as a deletion, insertion, substitution, modification (e.g., enzymatic modification) compared to a wild-type virus (e.g., a wild-type Anellovirus, e.g., as described herein), compared to the wild-type virus. Generally, the synthetic anaerovectors and synthetic anaeroVLPs include a proteinaceous outer part, which can be used to deliver an effector (e.g., an exogenous effector or an endogenous effector) to a eukaryotic (e.g., human) cell. In some embodiments, the anaerovectors or anaeroVLPs do not elicit a detectable and / or undesirable immune or inflammatory response, e.g., do not elicit greater than a 1%, 5%, 10%, 15% increase in one or more inflammatory molecular markers, e.g., TNF-α, IL-6, IL-12, IFN, and B cell responses, e.g., reactive or neutralizing antibodies, e.g., the anaerovectors or anaeroVLPs may be substantially non-immunogenic to a target cell, tissue, or subject.

[0007] In one aspect, the invention features an anaerovector that includes: (i) a genetic element that includes a promoter element and a sequence encoding an effector (e.g., an endogenous or exogenous effector), and a protein binding sequence (e.g., an exterior portion protein binding sequence, e.g., a packaging signal); and (ii) a proteinaceous exterior portion; the genetic element is encapsulated within the proteinaceous exterior portion (e.g., a capsid); and the anaerovector is capable of delivering the genetic element to a eukaryotic (e.g., mammalian, e.g., human) cell. In some embodiments, the anaerovector includes a surface moiety (e.g., a surface moiety having an effector and / or targeting functionality), e.g., displayed on the exterior portion surface of the anaerovector (e.g., as described herein). In some embodiments, the surface moiety includes an effector.

[0008] 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 properties: circular, single-stranded, integrates into the genome of the cell 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 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 per genome. In some embodiments, the integration frequency is determined, for example, as described in Wang et al. (2004, Gene Therapy 11:711-721, incorporated herein by reference in its entirety). In some embodiments, the genetic element is encapsulated within a proteinaceous exterior. In some embodiments, the annelovector is capable of delivering genetic elements into eukaryotic cells. In some embodiments, the genetic element comprises a nucleic acid sequence (e.g., a nucleic acid sequence of between 300 and 4000 nucleotides, e.g., between 300 and 3500 nucleotides, between 300 and 3000 nucleotides, between 300 and 2500 nucleotides, between 300 and 2000 nucleotides, between 300 and 1500 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 (e.g., a wild-type Torque Teno virus (TTV), Torque Teno mini virus (TTMV), or TTMDV sequence, e.g., a wild-type Anellovirus sequence as listed in any one of Tables A1-A25 or N1-N25).In some embodiments, the genetic element comprises a nucleic acid sequence (e.g. a nucleic acid sequence of at least 300 nucleotides, 500 nucleotides, 1000 nucleotides, 1500 nucleotides, 2000 nucleotides, 2500 nucleotides, 3000 nucleotides or more) 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 (e.g. a wild-type Anellovirus sequence as listed in any one of Tables A1-A25 or N1-N25 described herein). In some embodiments, the nucleic acid sequence is codon optimized, for example for expression in a mammalian (e.g. human) cell. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the codons in a nucleic acid sequence are codon optimized, e.g., for expression in a mammalian (e.g., human) cell.

[0009] In one aspect, the invention features an annellovLP comprising a proteinaceous outer part (e.g., a capsid) and an effector; the annellovLP having the ability to deliver the effector to a eukaryotic (e.g., mammalian, e.g., human) cell. In some embodiments, the effector is included in a surface moiety, e.g., displayed on the outer part surface of the annellovLP (e.g., as described herein).

[0010] In some aspects, the invention features an infectious (to a human cell) particle that includes an anellovirus capsid (e.g., a capsid that includes an anellovirus ORF, e.g., ORF1, polypeptide). In some embodiments, the infectious particle encapsulates a genetic element that includes a protein binding sequence that binds to the capsid and a heterologous (to the anellovirus) sequence that encodes a therapeutic effector. In some embodiments, the particle is capable of delivering the genetic element to a mammalian cell, e.g., a human cell. In some embodiments, the genetic element has less than about 6% identity (e.g., less than 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, or less) with 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 with a wild-type anellovirus. In some embodiments, the genetic element has at least about 2% to at least about 5.5% (e.g., 2-5%, 3%-5%, 4%-5%) identity to a wild-type Anellovirus. In some embodiments, the genetic element has about 2000, 3000, 4000, 4500, or more than 5000 nucleotides of non-viral sequence (e.g., non-Anellovirus genomic sequence). In some embodiments, the genetic element has about 2000-5000, 2500-4500, 3000-4500, 2500-4500, 3500, or more than 4000, 4500 (e.g., about 3000-4500) nucleotides of non-viral sequence (e.g., non-Anellovirus genomic sequence). In some embodiments, the genetic element is single-stranded, circular DNA.Alternatively, or in combination, the genetic element has one, two or three of the following properties: it is circular, it 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 it 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, the integration frequency is determined, for example, as described in Wang et al. (2004, Gene Therapy 11:711-721, incorporated herein by reference in its entirety).

[0011] Also described herein are anellovirus-based viral vectors and viral particles that can be used to deliver agents (e.g., exogenous or endogenous effectors, e.g., therapeutic effectors) to cells (e.g., cells of a subject undergoing a therapeutic treatment). In some embodiments, anelloviruses can be used as an effective delivery vehicle to introduce agents, such as the effectors described herein, into target cells, e.g., target cells of a subject undergoing a therapeutic or prophylactic treatment.

[0012] In one aspect, the present invention provides a method for (e.g., in order) (i) a first region comprising an arginine-rich region, e.g., 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, or a sequence of at least about 40 amino acids in which at least 60%, 70%, or 80% are basic residues (e.g., arginine, lysine, or a combination thereof); (ii) a second region comprising a jellyroll domain, e.g., 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 jellyroll domain sequence described herein or a sequence comprising at least six β-strands; (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 an N22 domain sequence described herein; (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 C-terminal domain (CTD) sequence described herein. and (v) optionally, a polypeptide having an amino acid sequence having less than 100%, 99%, 98%, 95%, 90%, 85%, 80% sequence identity to a wild-type anellovirus ORF1 protein described herein.

[0013] In some embodiments, the polypeptide comprises 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-A25). In some embodiments, the polypeptide comprises at least about 70, 80, 90, 95, 96, 97, 98, 99, or 100% sequence identity to a subsequence (e.g., an arginine (Arg)-rich domain, a jelly-roll domain, a hypervariable region (HVR), an N22 domain, or a C-terminal domain (CTD)) of an anellovirus ORF1 molecule as described herein (e.g., as listed in any one of Tables A1-A25). In one embodiment, the amino acid sequences of regions (i), (ii), (iii), and (iv) have at least 90% sequence identity to their respective references, wherein the polypeptide has an amino acid sequence having less than 100%, 99%, 98%, 95%, 90%, 85%, 80% sequence identity to a wild-type anellovirus ORF1 protein described herein.

[0014] In one aspect, the invention features a complex that includes a polypeptide as described herein (e.g., an anellovirus ORF1 molecule as described herein) and a genetic element that includes 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.

[0015] The disclosure further provides nucleic acid molecules, such as nucleic acid molecules comprising genetic elements as described herein, or nucleic acid molecules comprising sequences encoding proteinaceous exterior proteins as described herein. The nucleic acid molecules of the invention may comprise one or both of (a) genetic elements as described herein, and (b) nucleic acid sequences encoding proteinaceous exterior proteins as described herein.

[0016] In one aspect, the invention features an isolated nucleic acid that includes a genetic element that includes a promoter element operably linked to a sequence encoding an effector, e.g., a payload, and an exogenous protein binding sequence. In some embodiments, the exogenous protein binding sequence includes a sequence that is at least 75% (at least 80%, 85%, 90%, 95%, 97%, 100%) identical to a 5'UTR sequence of an Anellovirus disclosed herein. In some embodiments, the genetic element is single stranded DNA, is circular, is integrated 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, and / or is integrated 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 is integrated 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, 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 some embodiments, the effector is not derived from TTV and is not SV40-miR-S1. In some embodiments, the nucleic acid molecule does not include the polynucleotide sequence of TTMV-LY2. In some embodiments, the promoter element can direct the expression of the effector in eukaryotic (e.g., mammalian, e.g., human) cells.

[0017] In some embodiments, the nucleic acid molecule is circular. In some embodiments, the nucleic acid molecule is linear. In some embodiments, the nucleic acid molecule described herein comprises one or more modified nucleotides (e.g., base, sugar, or backbone modifications).

[0018] 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 that includes one, two, or three of the following: (i) a promoter element and a sequence encoding an effector, e.g., an exogenous or endogenous effector; (ii) at least 72 contiguous nucleic acids (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%) having sequence identity to a wild-type Anellovirus sequence; or at least 72 contiguous nucleic acids (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, 100, or 150%) sequence identity to a wild-type Anellovirus sequence; 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, e.g., an exogenous protein binding sequence, wherein the nucleic acid construct comprises a single and the nucleic acid construct is circular and integrates at a frequency of 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 / 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.In some embodiments, a genetic element encoding an effector (e.g., an exogenous or endogenous effector) 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 anellovector, e.g., as described herein. In some embodiments, the genetic element described herein comprises one or more modified nucleotides (e.g., a base modification, a sugar modification, or a backbone modification). 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 (e.g., an Anellovirus ORF3 protein, e.g., as described herein).

[0019] In some aspects, the invention features a host or helper cell that includes: (a) a nucleic acid that includes a sequence encoding one or more of an ORF1, ORF2, or ORF3 molecule (e.g., a sequence encoding an anellovirus ORF1 polypeptide described herein), the nucleic acid being a plasmid, a viral nucleic acid, or integrated into a helper cell chromosome; and (b) a genetic element that includes (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 genetic element that includes a protein binding sequence that binds the polypeptide of (a), where optionally, the genetic element does not encode the ORF1 polypeptide (e.g., an ORF1 protein). For example, the host or helper cell includes (a) and (b) in either cis (both portions of the same nucleic acid molecule) or in trans (each portion of a different nucleic acid molecule). In some embodiments, the genetic element of (b) is circular, single-stranded DNA. In some embodiments, the host cell is a manufacturing cell line. In some embodiments, the host or helper cells are adherent or in suspension, or both. In some embodiments, the host or helper cells are grown in microcarriers. In some embodiments, the host or helper cells comply with cGMP manufacturing practices. In some embodiments, the host or helper cells are grown in a medium suitable for promoting cell growth. In certain embodiments, once the host or helper cells have grown sufficiently (e.g., to an appropriate cell density), the medium may be replaced with a medium suitable for production of the anaerovector by the host or helper cells.

[0020] In some aspects, the invention features a pharmaceutical composition that includes an anelovector (e.g., a synthetic anelovector) described herein. In embodiments, the pharmaceutical composition further includes a pharma- ceutical acceptable carrier or excipient. In some embodiments, the pharmaceutical composition further includes a pharma- ceutical acceptable carrier or excipient. In some embodiments, the pharmaceutical composition is administered at about 10 per kilogram of the subject. 5~10 14 In some embodiments, the pharmaceutical composition comprising the preparation is stable for an acceptable period of time and temperature, and / or is compatible with the desired route of administration and / or any device required by this route of administration, such as 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, e.g., by a medical professional. In some embodiments, the pharmaceutical composition comprises a desired concentration of anellovector genome or genome equivalent (e.g., defined by the number of genomes per volume).

[0021] In one aspect, the invention features a method of treating a disease or disorder in a subject, the method includes administering to the subject an annelovector, e.g., a synthetic annelovector as described herein.

[0022] In one aspect, the invention features a method of delivering an effector or payload (e.g., an endogenous effector or an exogenous effector) to a cell, tissue, or subject, the method includes administering to a subject an anaerovector, e.g., a synthetic anaerovector as described herein, where the anaerovector includes a nucleic acid encoding the effector. In some embodiments, the payload is a nucleic acid. In some embodiments, the payload is a polypeptide.

[0023] In one aspect, the invention features a method of delivering an annelovector to a cell, including contacting an annelovector, e.g., a synthetic annelovector as described herein, with a cell, e.g., a eukaryotic cell, e.g., a mammalian cell, e.g., in vivo or ex vivo.

[0024] In one aspect, the invention features a method of treating a disease or disorder in a subject, the method including administering to the subject an anaeroVLP, e.g., a synthetic anaeroVLP, e.g., as described herein.

[0025] 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, comprising administering to a subject an anaeroVLP, e.g., a synthetic anaeroVLP, e.g., as described herein, where the anaeroVLP comprises an effector (e.g., where the proteinaceous outer portion of the anaeroVLP encapsulates the effector). In an embodiment, the payload is a nucleic acid. In an embodiment, the payload is a polypeptide (e.g., a protein).

[0026] In one aspect, the invention features a method of delivering anelloVLP to a cell, the method including contacting an anneloVLP, e.g., as described herein, e.g., a synthetic anneloVLP, with a cell, e.g., a eukaryotic cell, e.g., a mammalian cell, e.g., in vivo or ex vivo.

[0027] In one aspect, the invention features a method of making an annellvector, e.g., a synthetic annellvector, comprising: a) providing a host cell, the host cell comprising: (i) a first nucleic acid molecule comprising a nucleic acid sequence of a genetic element of an anaerovector, e.g., a synthetic anaerovector, as described herein; and (ii) a first nucleic acid or a second nucleic acid molecule encoding one or more of the amino acid sequences selected from ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, or ORF1 / 2, e.g., as set forth in any one of Tables A1 to A25, 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; Contains; and b) incubating the host cells under conditions suitable for producing the anellovector; Includes.

[0028] In some embodiments, the method further comprises introducing the first and / or second nucleic acid molecule into the host cell prior to step (a). In some embodiments, the second nucleic acid molecule is introduced into the host cell prior to, 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 a helper (e.g., a helper plasmid or the genome of a helper virus).

[0029] In another aspect, the present invention provides a method for producing an anelovectin composition, comprising the steps of: a) providing a host cell that contains, e.g., expresses, one or more components (e.g., all components) of an anaerovector, e.g., a synthetic anaerovector, e.g., as described herein. For example, the host cell contains (a) a nucleic acid that contains a sequence encoding an anellovirus ORF1 polypeptide described herein, which is a plasmid, a viral nucleic acid, or is integrated into a helper cell chromosome; and (b) a genetic element that contains (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 the polypeptide of (a), wherein the host cell or helper cell contains (a) and (b) either 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 manufacturing cell line; b) culturing the host cells under conditions suitable for producing an anaerovector preparation from the host cells, wherein the anaerovector in the preparation comprises a proteinaceous outer portion (e.g., comprising an ORF1 molecule) that encapsulates a genetic element (e.g., as described herein), thereby creating an anaerovector preparation; and Optionally, c) formulating the anellovector preparation, e.g., as a pharmaceutical composition suitable for administration to a subject. The present invention is characterized by a method comprising the steps of:

[0030] In some embodiments, at the time of production, the anaerovector construct is introduced into a host cell (e.g., by transient transfection). In some embodiments, the host cell stably expresses the anaerovector construct (e.g., where one or more nucleic acids encoding the anaerovector construct are introduced into the host cell, or a progenitor cell thereof, e.g., by stable transfection).

[0031] In some embodiments, the method further comprises one or more purification steps (e.g., purification by precipitation, 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 preparation. In some embodiments, the resulting preparation or pharmaceutical composition comprising the preparation will be stable for an acceptable period of time and temperature, and / or compatible with the desired route of administration and / or any equipment that may be required for that route of administration, e.g., a needle or syringe.

[0032] In one aspect, the invention features a method of producing an anaerovector composition, comprising: a) providing a plurality of anaerovectors described herein, or a preparation of an anaerovector described herein; and b) formulating the anaerovector or the preparation thereof, e.g., as a pharmaceutical composition suitable for administration to a subject.

[0033] In one aspect, the invention features a method of producing an anelloVLP composition, the method including: a) providing a plurality of anelloVLPs described herein, or a preparation of anelloVLPs described herein; and b) formulating the anelloVLPs or the preparation thereof, e.g., as a pharmaceutical composition suitable for administration to a subject.

[0034] In one aspect, the invention features a method of producing a population of host cells, e.g., a first host cell or producer cell (e.g., such as those depicted in FIG. 20), e.g., a first host cell comprising an anaerovector, the method including introducing a genetic element (e.g., such as those described herein) into the host cell and culturing the host cell under conditions suitable for production of the anaerovector. In some embodiments, the method further includes introducing a helper, e.g., a helper virus, into the host cell. In some embodiments, the introducing includes transfection (e.g., chemical transfection) or electroporation of the host cell with the anaerovector.

[0035] In one aspect, the invention features a method of making an anaerovector, including providing a host cell, e.g., a first host cell or producer cell (e.g., such as that shown in FIG. 20), including an anaerovector, e.g., one described herein, and purifying the anaerovector from the host cell. In some embodiments, the method further includes, prior to the providing step, contacting the host cell with an anaerovector, e.g., one described herein, and incubating the host cell under conditions suitable for production of the anaerovector. In some embodiments, the host cell is a first host cell or producer cell described in the above method of making a host cell. In some embodiments, purifying the anaerovector from the host cell includes lysing the host cell.

[0036] In some embodiments, the method further comprises a second step of contacting the anaerovector produced by the first host cell or producer cell with a second host cell, e.g., a permissive cell (e.g., as shown in FIG. 20), e.g., a population of second host cells. In some embodiments, the method further comprises incubating the second host cell under conditions suitable for production of the anaerovector. In some embodiments, the method further comprises purifying the anaerovector from the second host cell, e.g., thereby producing an anaerovector seed population. In some embodiments, at least about 2-100 times more anaerovector is produced from the population of second host cells compared to that from the population of first host cells. In some embodiments, purifying the anaerovector from the second host cell comprises lysing the second host cell. In some embodiments, the method further comprises contacting the anaerovector produced by the second host cell with a third host cell, e.g., a permissive cell (e.g., as shown in FIG. 20), e.g., a population of third host cells. In some embodiments, the method further comprises incubating a third host cell under conditions suitable for production of the anaerovector. In some embodiments, the method comprises purifying the anaerovector from the third host cell, e.g., thereby producing an anaerovector stock population. In some embodiments, purifying the anaerovector from the third host cell comprises lysing the third host cell. In some embodiments, at least about 2-100 fold more anaerovector is produced from the population of third host cells compared to that from the population of second host cells.

[0037] In some embodiments, the host cells are grown in a medium suitable for promoting cell growth. In certain embodiments, once the host cells or helper cells have grown sufficiently (e.g., to a suitable cell density), the medium may be replaced with a medium suitable for production of the anaerovector by the host cells or helper cells. In some embodiments, the anaerovector produced by the host cells is separated from the host cells (e.g., by lysing the host cells) prior to contacting with a second host cell. In some embodiments, the anaerovector produced by the host cells is contacted with a second host cell without an intervening purification step.

[0038] In one aspect, the invention features a method of making a pharmaceutical anaerovector preparation, the method including the steps of: (a) making an anaerovector preparation as described herein; (b) evaluating the preparation (e.g., a pharmaceutical anaerovector preparation, an anaerovector seed population, or an anaerovector stock population) for one or more pharmaceutical quality control parameters, e.g., identity, purity, titer, potency (e.g., in genome equivalents per anaerovector particle), and / or nucleic acid sequences from genetic elements contained in the anaerovector; and (c) formulating the preparation for pharmaceutical use for evaluation that it meets predetermined criteria, e.g., meets pharmaceutical specifications. In some embodiments, the step of evaluating the identity includes evaluating (e.g., confirming) sequences of genetic elements of the anaerovector, e.g., sequences encoding effectors. In some embodiments, the step of assessing purity includes assessing the amount of impurities, such as mycoplasma, endotoxins, host cell nucleic acids (e.g., host cell DNA and / or host cell RNA), animal-derived impurities (e.g., serum albumin or trypsin), replicative causative agents (RCA), such as replicative viruses or unwanted anaerovectors (e.g., anaerovectors other than the desired anaerovector, e.g., synthetic anaerovectors described herein), free viral capsid proteins, adventitious materials, and aggregates. In some embodiments, the step of assessing potency includes assessing the ratio of functional and non-functional (e.g., infectious and non-infectious) anaerovectors in the preparation (e.g., by HPLC, etc.). In some embodiments, the step of assessing potency includes assessing the level of anaerovector function detectable in the preparation (e.g., expression and / or function or genomic equivalent of effectors encoded therein). In some embodiments, the impurities include residual denaturants (e.g., urea) or cellular substituents (e.g., proteasomes or ferritin).

[0039] In some embodiments, the formulated preparation 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 anaerovectors can be produced in a single batch. In some embodiments, the levels of anaerovectors produced in a batch can be assessed (e.g., individually or together).

[0040] In one aspect, the invention features a method of making a pharmaceutical anaero-VLP preparation, the method including (a) making an anaero-VLP preparation as described herein, (b) evaluating the preparation (e.g., a pharmaceutical anaero-VLP preparation, an anaero-VLP seed population, or an anaero-VLP stock population) for one or more pharmaceutical quality control parameters, e.g., identity, purity, potency, efficacy, and (c) formulating the preparation for pharmaceutical use if the evaluation meets predetermined criteria, e.g., meets pharmaceutical specifications. In some embodiments, evaluating purity includes evaluating the amount of impurities, e.g., mycoplasma, 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 agents (RCA), e.g., replication-competent viruses or undesired VLPs (e.g., anaeroVLPs other than desired anaeroVLPs, e.g., synthetic anaeroVLPs as described herein), free viral capsid proteins, adventitious agents, and aggregates. In some embodiments, evaluating potency includes evaluating the ratio of functional to non-functional (e.g., infectious to non-infectious) anaeroVLPs in the preparation (e.g., as assessed by HPLC). In some embodiments, evaluating potency includes evaluating the level of detectable anaeroVLP function (e.g., expression and / or function or genomic equivalent of an effector encoded therein) in the preparation. In some embodiments, impurities include residual denaturants (eg, urea) or cellular substitutes (eg, proteasomes or ferritin).

[0041] In some embodiments, the formulated preparation is substantially free of pathogens, host cell contaminants or impurities; has a predetermined level of non-infectious particles or a predetermined particle:infectious unit ratio (e.g., <300:1, <200:1, <100:1, or <50:1). In some embodiments, multiple anelloVLPs can be produced in a single batch. In some embodiments, the level of anelloVLPs produced in a batch can be assessed (e.g., individually or together).

[0042] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a first nucleic acid molecule comprising a nucleic acid sequence of a genetic element of an anaerovector as described herein; and (ii) optionally, a second nucleic acid molecule encoding one or more of the amino acid sequences selected from ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, or ORF1 / 2, as listed in any one of Tables A1 to A25, 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. The present invention features a host cell comprising:

[0043] In one aspect, the invention features a reaction mixture including an annelovector described herein and a helper virus, where the helper virus includes a polynucleotide, e.g., a polynucleotide encoding an outer part protein (e.g., an outer part protein having the ability to bind to an outer part protein binding sequence and, optionally, a lipid envelope), a polynucleotide encoding a replication protein (e.g., a polymerase), or any combination thereof.

[0044] In some embodiments, the anaerovector (e.g., a synthetic anaerovector) is isolated, e.g., isolated from the host cell and / or isolated from other components in the solution (e.g., the supernatant). In some embodiments, the anaerovector (e.g., a synthetic anaerovector) is purified, e.g., from the solution (e.g., the supernatant). In some embodiments, the anaerovector is concentrated in the solution relative to other components in the solution.

[0045] In some embodiments of any of the foregoing anaerovectors, compositions, or methods, providing the anaerovector comprises isolating (e.g., harvesting) the anaerovector from a composition comprising an anaerovector-producing cell, such as those described herein. In other embodiments, providing the anaerovector comprises obtaining the anaerovector or a preparation thereof, e.g., from a third party.

[0046] In some embodiments of any of the above anaerovectors, anaerovectors, compositions or methods, the genetic element comprises an anaerovector genome, e.g., as identified by the methods described in Example 9. In embodiments, the anaerovector genome is an anaerovector genome capable of self-replication and / or self-amplification. In some embodiments, the anaerovector genome does not have the ability to self-replicate and / or self-amplify. In some embodiments, the anaerovector genome is capable of replicating and / or undergoing amplification in trans, e.g., in the presence of a helper, e.g., a helper virus.

[0047] Additional features of any of the annelovectors, annelovLPs, compositions or methods described above include one or more of the embodiments listed below.

[0048] 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 which are intended to be encompassed by the embodiments recited below.

[0049] Enumerated Embodiments 1. A proteinaceous outer portion comprising about 40 to 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. A particle comprising: (i) does not contain (e.g., does not encapsulate) polynucleotides (e.g., as determined using a nuclease protection assay as described herein); (ii) does not contain (e.g., does not encapsulate) polynucleotides greater than 1000, 500, 200, or 100 nucleotides in length; or (iii) contains 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. particle.

[0050] 2. The particle of embodiment 1, wherein the anellovirus ORF1 molecule comprises an ORF1 domain and an exogenous surface moiety.

[0051] 3. The particle of embodiment 1, wherein the anellovirus ORF1 molecule is attached to the exogenous surface moiety by non-covalent incorporation or by a covalent bond other than a peptide bond.

[0052] 4. The particle of embodiment 1, wherein the anellovirus ORF1 molecule does not contain an arginine-rich domain.

[0053] 5. The particle of embodiment 1, which is a virus-like particle (VLP).

[0054] 6. (a) a proteinaceous outer portion comprising 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 and an exogenous surface portion; and (b) a genetic element comprising a heterologous nucleic acid sequence encoding an exogenous effector. Particles containing.

[0055] 7. A proteinaceous outer portion comprising an anellovirus ORF1 molecule, the ORF1 molecule comprising an ORF1 domain and an exogenous surface portion. A particle comprising: One or more of the following: a) the exogenous surface moiety is selected from a receptor, a ligand, an antibody molecule (e.g., an scFv), an antigen (e.g., a viral, bacterial, fungal, or parasitic antigen) an adjuvant (e.g., a TLR agonist, e.g., bacterial flagellin); b) the ORF1 molecule contains a hypervariable region (HVR); c) the particle contains a genetic element encoding a peptide or polypeptide that boosts the immune response (e.g., an adjuvant, a TCR agonist (e.g., bacterial flagellin)); d) 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; e) a polypeptide linker region is located between the exogenous surface portion and the ORF1 molecule; f) 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 moiety; g) the proteinaceous exo-portion comprises (i) a plurality of ORF1 molecules lacking exogenous surface moieties (e.g., wild-type ORF1 molecules) and (ii) a plurality of ORF1 molecules comprising exogenous surface moieties, optionally with a ratio of (i):(ii) of 10:1 to 5:1, 5:1 to 2:1, 2:1 to 1:2, 1:2 to 1:5, or 1:5 to 1:10; and / or h) the particle further comprises a second exogenous surface moiety; particle.

[0056] 8. The particle of embodiment 7, wherein the exogenous surface portion is located at an insertion point between the N-terminal portion of the ORF1 domain and the C-terminal portion of the ORF1 domain.

[0057] 9. The particle of embodiment 8, wherein the insertion point is in the HVR.

[0058] 10. A particle according to any one of embodiments 7 to 9, further comprising a genetic element comprising a heterologous nucleic acid sequence encoding an exogenous effector.

[0059] 11. A particle according to any one of embodiments 7 to 10, which does not contain (e.g., is not encapsulated in) a polynucleotide, or does not contain (e.g., is not encapsulated in) a polynucleotide that is more than 1000, 500, 200, or 100 nucleotides in length, or contains 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 in length.

[0060] 12. A proteinaceous outer portion comprising an anellovirus ORF1 molecule and an exogenous surface moiety that is covalently attached to the ORF1 molecule by a bond other than a peptide bond. Particles containing.

[0061] 13. The particle of embodiment 12, wherein the exogenous surface moiety is linked to an NHS moiety and the exogenous surface moiety is bound to the ORF1 molecule via the NHS moiety.

[0062] 14. The particle according to embodiment 12 or 13, wherein a non-polypeptide linker is located between the exogenous surface moiety and the ORF1 molecule.

[0063] 15. The particle of embodiment 14, wherein the non-polypeptide linker comprises a click bond.

[0064] 16. The particle according to embodiment 14, wherein the non-polypeptide linker is produced by a click reaction between a DBCO moiety and an azide moiety.

[0065] 17. The particle of embodiment 16, wherein the DBCO moiety is linked to the anellovirus ORF1 molecule via the NHS moiety prior to the click reaction.

[0066] 18. A particle according to embodiment 16 or 17, wherein prior to the Click reaction, the azide moiety is linked to the exogenous surface moiety via an NHS moiety.

[0067] 19. The particle of embodiment 17 or 18, wherein the NHS moiety of the DBCO moiety is linked to a lysine residue on the surface of the anellovirus ORF1 molecule.

[0068] 20. A particle according to any one of embodiments 17 to 19, wherein the NHS portion of the azide moiety is attached to a lysine residue on the surface of the exogenous surface moiety.

[0069] 21. The particle of embodiment 16, wherein the DBCO moiety is linked to the exogenous surface moiety via the NHS moiety prior to the click reaction.

[0070] 22. The particle according to embodiment 16 or 21, wherein prior to the click reaction, the azide moiety is linked to the anellovirus ORF1 molecule via the NHS moiety.

[0071] 23. The particle of embodiment 21 or 22, wherein the NHS moiety of the DBCO moiety is linked to a lysine residue on the surface of the exogenous surface moiety.

[0072] 24. A particle according to any one of embodiments 21 to 23, wherein the NHS portion of the azide moiety is attached to a lysine residue on the surface of the anellovirus ORF1 molecule.

[0073] 25. A proteinaceous outer portion comprising an anellovirus ORF1 molecule and an exogenous surface portion that is non-covalently bound to the ORF1 molecule. Particles containing.

[0074] 26. The particle of embodiment 25, wherein the ORF1 molecule comprises an exogenous binding domain (e.g., MS2 coat protein or avidin) and the exogenous surface moiety comprises a cognate binding moiety that binds the exogenous binding domain (e.g., MS2 hairpin or biotin).

[0075] 27. A particle according to any one of embodiments 12 to 26, wherein the exogenous surface moiety comprises a polypeptide.

[0076] 28. A particle according to any one of embodiments 12 to 27, wherein the exogenous surface moiety comprises a small molecule or a nucleic acid molecule (e.g., a polynucleotide).

[0077] 29. A particle according to any one of the preceding claims, wherein the ratio of ORF1 molecules to exogenous surface moieties is about 60:1 to 30:1, 30:1 to 20:1, 20:1 to 10:1, or 10:1 to 1:1.

[0078] 30. A particle according to any one of embodiments 1 to 29, wherein the antibody molecule is a bispecific antibody molecule.

[0079] 31. A bispecific antibody molecule comprising: a first antigen-binding domain that binds to a first antigen on a first type of host cell; and a second antigen-binding domain that binds to a second antigen on a second type of host cell. 31. The particle of embodiment 30, comprising:

[0080] 32. A particle according to any one of the preceding embodiments, which has the ability to enter a target cell, for example by endocytosis.

[0081] 33. The particle of embodiment 32, wherein the exogenous surface moiety binds to a cognate moiety on a target cell.

[0082] 34. The particle according to embodiment 32, wherein the particle comprises a genetic element encoding an exogenous effector to be delivered inside the target cell.

[0083] 35. A particle according to any one of the preceding embodiments, wherein the genetic element is encapsulated within a proteinaceous outer part.

[0084] 36. A particle according to any one of the preceding embodiments, which does not contain a polynucleotide or does not contain a polynucleotide greater than 1000, 500, 200, or 100 nucleotides in length.

[0085] 37. The anellovirus ORF1 molecule is (b) a first region comprising an anellovirus ORF1 jellyroll region, e.g., an anellovirus ORF1 jellyroll region sequence described herein, 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 thereto, or a sequence comprising at least six β strands; (c) a second 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 N22 domain, e.g., an anellovirus ORF1 N22 domain sequence described herein; and (d) 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 anellovirus ORF1 C-terminal domain (CTD), e.g., an anellovirus ORF1 CTD sequence described herein. including; and The anellovirus ORF1 molecule comprises an anellovirus ORF1 arginine-rich region, e.g., a sequence of at least about 40 amino acids in which at least 60%, 70%, or 80% are basic residues (e.g., arginine, lysine, or a combination thereof); A particle according to any one of embodiments 1 to 36.

[0086] 38. A preparation comprising particles according to any one of embodiments 1 to 37.

[0087] 39. 10 mg / kg of subject to which the composition is to be administered 10 ~10 14Less than (e.g., 10 10 ~10 11 , 10 11 ~10 12 , 10 12 ~10 13 , or 10 13 ~10 14 39. The preparation of embodiment 38, comprising a nucleic acid molecule (e.g., a genetic element, e.g., of an anaerovector as described herein) equivalent to less than 1000000000 viral genome (e.g., as determined by qPCR or by measuring optical density).

[0088] 40. (b) a first region comprising an anellovirus ORF1 jellyroll region, e.g., an anellovirus ORF1 jellyroll region sequence described herein 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 thereto, or a sequence comprising at least six β strands; (c) a second 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 N22 domain, e.g., an anellovirus ORF1 N22 domain sequence described herein; and (d) 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 anellovirus ORF1 C-terminal domain (CTD), e.g., an anellovirus ORF1 CTD sequence described herein. and A polypeptide that does not include an anellovirus ORF1 arginine-rich region, e.g., a sequence of at least about 40 amino acids in which at least 60%, 70%, or 80% are basic residues (e.g., arginine, lysine, or a combination thereof).

[0089] 41. A nucleic acid molecule encoding a polypeptide according to embodiment 40.

[0090] 42. A particle comprising a proteinaceous outer part comprising an anellovirus ORF1 molecule, the ORF1 molecule comprising a proteinaceous outer part comprising an ORF1 domain and an exogenous surface domain, A particle produced by contacting a plurality of anellovirus ORF1 molecules in a cell-free solution under conditions suitable for the formation of a proteinaceous outer portion comprising a plurality of anellovirus ORF1 molecules.

[0091] 43. The particle of embodiment 42, which does not contain (e.g., is not encapsulated in) a polynucleotide or does not contain (e.g., is not encapsulated in) a polynucleotide that is more than 1000, 500, 200, or 100 nucleotides in length.

[0092] 44. A method for making particles, comprising: contacting a plurality of anellovirus ORF1 molecules in a cell-free solution under conditions suitable for the formation of a proteinaceous outer portion comprising the plurality of anellovirus ORF1 molecules. Includes; How to create particles with it.

[0093] 45. A method for regulating the biological activity of a cell, comprising: Contacting a cell with a particle according to any one of embodiments 1 to 44. Includes; A method wherein the cell comprises a moiety on its surface that binds to the exogenous surface moiety of the particle.

[0094] 46. ​​A method for targeting a particle to a cell, comprising: Contacting a cell with a particle according to any one of embodiments 1 to 45. Includes; A method wherein the cell comprises a moiety on its surface that binds to the exogenous surface moiety of the particle.

[0095] 47. The polypeptide, particle, nucleic acid molecule, or method of any one of embodiments 1 to 46, wherein the exogenous surface moiety is fused to the N-terminus of the anellovirus ORF1 molecule.

[0096] 48. The polypeptide, particle, nucleic acid molecule, or method of any one of embodiments 1 to 47, wherein the exogenous surface moiety is fused to the C-terminus of the anellovirus ORF1 molecule.

[0097] 49. The polypeptide, particle, nucleic acid molecule, or method of any one of embodiments 1 to 48, wherein the exogenous surface moiety is inserted within the amino acid sequence of the anellovirus ORF1 molecule.

[0098] 50. An ORF1 molecule comprising an exogenous surface moiety, wherein the exogenous surface moiety is fused within, replaces, and / or is located at an insertion point of the ORF1 domain (e.g., within HVR or P2).

[0099] 51. 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 284-285 of Ring10 ORF1, e.g., in the ORF1 domain (e.g., within an HVR).

[0100] 52. 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 328-329 in the ORF1 domain (e.g., within an HVR) of Ring10 ORF1.

[0101] 53. 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 256-383 in the ORF1 domain (e.g., within an HVR) of Ring10 ORF1.

[0102] 54. 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 251-383 in the ORF1 domain (e.g., within an HVR) of Ring10 ORF1.

[0103] 55. 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 251-384 in the ORF1 domain (e.g., within an HVR) of Ring10 ORF1.

[0104] 56. An ORF1 molecule comprising an exogenous surface moiety, wherein the exogenous surface moiety is Ring10. ORF1 molecule that is linked to (e.g., conjugated to) an amino acid residue (e.g., a cysteine ​​residue) corresponding to position 254, 263, 264, 265, 272, 273, 274, 276, 283, 284, 285, 287, 288, 290, 291, 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 of ORF1, for example, in the ORF1 domain (e.g., within an HVR).

[0105] 57. The ORF1 molecule of any one of embodiments 51 to 56, wherein the exogenous surface portion forms a pentamer when the ORF1 molecule is complexed with four other ORF1 molecules (e.g., four other copies of the ORF1 molecule).

[0106] 58. The ORF1 molecule of any one of embodiments 51 to 56, wherein the exogenous surface portion forms a trimer when the ORF1 molecule is complexed with four other ORF1 molecules (e.g., four other copies of the ORF1 molecule).

[0107] 59. The ORF1 molecule of any one of embodiments 51 to 56, wherein the exogenous surface portion forms a dimer when the ORF1 molecule is complexed with four other ORF1 molecules (e.g., four other copies of the ORF1 molecule).

[0108] 60. A protein complex comprising five ORF1 molecules, each of which is (i) the ORF1 domain, and (ii) extrinsic surface moieties; Includes; A protein complex in which the exogenous surface portions of five ORF1 molecules form a pentamer.

[0109] 61. A protein complex according to embodiment 60, wherein each of the exogenous surface moieties is fused within, replaces and / or is located at an insertion point (e.g., within an HVR) of the ORF1 domain of the corresponding ORF1 molecule.

[0110] 62. The protein complex of embodiment 60, wherein each of the exogenous surface moieties is fused within, replaces, and / or is located at an insertion point between residues corresponding to positions 284-285 in the ORF1 domain of Ring10 ORF1 (e.g., within an HVR).

[0111] 63. The protein complex of embodiment 60, wherein each of the exogenous surface moieties is fused within, replaces, and / or is located at an insertion point between residues corresponding to positions 328-329 in the ORF1 domain of Ring10 ORF1 (e.g., within an HVR).

[0112] 64. The protein complex of embodiment 60, wherein each of the exogenous surface moieties is fused within, replaces, and / or is located at an insertion point between residues corresponding to positions 256 to 383 in the ORF1 domain of Ring10 ORF1 (e.g., within an HVR).

[0113] 65. The protein complex of embodiment 60, wherein each of the exogenous surface moieties is fused within, replaces, and / or is located at an insertion point between residues corresponding to positions 251 to 383 in the ORF1 domain of Ring10 ORF1 (e.g., within an HVR).

[0114] 66. The protein complex of embodiment 60, wherein each of the exogenous surface moieties is fused within, replaces, and / or is located at an insertion point between residues corresponding to positions 251 to 384 in the ORF1 domain of Ring10 ORF1 (e.g., within an HVR).

[0115] 67. Each of the exogenous surface portions is a Ring10 61. The protein complex of embodiment 60, wherein the protein complex is linked to (e.g., conjugated to) an amino acid residue (e.g., a cysteine ​​residue) corresponding to position 254, 263, 264, 265, 272, 273, 274, 276, 283, 284, 285, 287, 288, 290, 291, 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 of ORF1, e.g., in the ORF1 domain (e.g., within an HVR).

[0116] 68. A protein complex according to any one of embodiments 60 to 67, in which the exogenous surface portions of the ORF1 molecules have the same amino acid sequence.

[0117] 69. A protein complex according to any one of embodiments 60 to 67, wherein at least two (e.g., at least 2, 3, 4, or 5) of the exogenous surface portions of the ORF1 molecule have different amino acid sequences.

[0118] 70. A protein complex comprising three ORF1 molecules, each of which is (i) the ORF1 domain, and (ii) extrinsic surface moieties; Includes; A protein complex in which the exogenous surface portions of three ORF1 molecules form a trimer.

[0119] 71. A protein complex according to embodiment 70, wherein each of the exogenous surface moieties is fused within, replaces and / or is located at an insertion point (e.g., within an HVR) of the ORF1 domain of the corresponding ORF1 molecule.

[0120] 72. A protein complex according to embodiment 70, wherein each of the exogenous surface moieties is fused within, replaces and / or is located at an insertion point at positions 284-285 in the ORF1 domain of the corresponding ORF1 molecule (e.g., within an HVR).

[0121] 73. A protein complex according to embodiment 70, wherein each of the exogenous surface moieties is fused within, replaces and / or is located at an insertion point at positions 328 to 329 in the ORF1 domain of the corresponding ORF1 molecule (e.g., within an HVR).

[0122] 74. A protein complex according to embodiment 70, wherein each of the exogenous surface moieties is fused to, replaces and / or is located at an insertion point between positions 256 and 383 in the ORF1 domain of the corresponding ORF1 molecule (e.g., within an HVR).

[0123] 75. A protein complex according to embodiment 70, wherein each of the exogenous surface moieties is fused to, replaces and / or is located at an insertion point at positions 251 to 383 in the ORF1 domain of the corresponding ORF1 molecule (e.g., within an HVR).

[0124] 76. A protein complex according to embodiment 70, wherein each of the exogenous surface moieties is fused to, replaces and / or is located at an insertion point at positions 251 to 384 in the ORF1 domain of the corresponding ORF1 molecule (e.g., within an HVR).

[0125] 77. Each of the exogenous surface moieties is a 254, 263, 264, 265, 272, 273, 274, 276, 283, 284, 285, 287, 288, 290, 291, 308, 311, 312, 313, 314, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 71. The protein complex of embodiment 70, wherein the protein complex is linked to (e.g., conjugated to) an amino acid residue (e.g., a cysteine ​​residue) at position 21, 324, 328, 329, 341, 343, 354, 358, 361, 362, 363, 364, 365, 368, 369, 371, 374, 376, 378, 380, or 381.

[0126] 78. A protein complex according to any one of embodiments 70 to 77, wherein the exogenous surface portions of the ORF1 molecules have the same amino acid sequence.

[0127] 79. A protein complex according to any one of embodiments 70 to 78, wherein at least two (e.g., at least two or three) of the exogenous surface portions of the ORF1 molecule have different amino acid sequences.

[0128] 80. A protein complex comprising two ORF1 molecules, each of which is (i) the ORF1 domain, and (ii) extrinsic surface moieties; Includes; A protein complex in which the exogenous surface portions of two ORF1 molecules form a dimer.

[0129] 81. A protein complex according to embodiment 80, wherein each of the exogenous surface moieties is fused within, replaces and / or is located at an insertion point (e.g., within an HVR) of the ORF1 domain of the corresponding ORF1 molecule.

[0130] 82. The protein complex of embodiment 80, wherein each of the exogenous surface moieties is fused within, replaces, and / or is located at an insertion point between residues corresponding to positions 284-285 in the ORF1 domain of Ring10 ORF1 (e.g., within an HVR).

[0131] 83. The protein complex of embodiment 80, wherein each of the exogenous surface moieties is fused within, replaces, and / or is located at an insertion point between residues corresponding to positions 328-329 in the ORF1 domain of Ring10 ORF1 (e.g., within an HVR).

[0132] 84. The protein complex of embodiment 80, wherein each of the exogenous surface moieties is fused within, replaces, and / or is located at an insertion point between residues corresponding to positions 256 to 383 in the ORF1 domain of Ring10 ORF1 (e.g., within an HVR).

[0133] 85. The protein complex of embodiment 80, wherein each of the exogenous surface moieties is fused within, replaces, and / or is located at an insertion point between residues corresponding to positions 251 to 383 in the ORF1 domain of Ring10 ORF1 (e.g., within an HVR).

[0134] 86. The protein complex of embodiment 80, wherein each of the exogenous surface moieties is fused within, replaces, and / or is located at an insertion point between residues corresponding to positions 251 to 384 in the ORF1 domain of Ring10 ORF1 (e.g., within an HVR).

[0135] 87. Each of the exogenous surface portions is a Ring10 81. The protein complex of embodiment 80, wherein the protein complex is linked to (e.g., conjugated to) an amino acid residue (e.g., a cysteine ​​residue) corresponding to position 254, 263, 264, 265, 272, 273, 274, 276, 283, 284, 285, 287, 288, 290, 291, 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 of ORF1, e.g., in the ORF1 domain (e.g., within an HVR).

[0136] 88. A protein complex according to any one of embodiments 80 to 87, wherein the exogenous surface portions of the two ORF1 molecules have the same amino acid sequence.

[0137] 89. A protein complex according to any one of embodiments 80 to 87, wherein the exogenous surface portions of the two ORF1 molecules have different amino acid sequences.

[0138] 90. A polypeptide, particle, nucleic acid molecule, method, or protein complex according to any one of embodiments 1 to 89, wherein the polypeptide or ORF1 molecule comprises one or more substitutions of cysteine ​​residues (e.g., one or more cysteine ​​to alanine substitutions or one or more cysteine ​​to serine substitutions).

[0139] 91. The polypeptide, particle, nucleic acid molecule, method, or protein complex of any one of embodiments 1 to 90, 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 the Ring10 ORF1 protein (e.g., as described herein).

[0140] 92. The polypeptide, particle, nucleic acid molecule, method, or protein complex of any one of embodiments 1 to 91, 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 the Ring10 ORF1 protein (e.g., as described herein).

[0141] 93. The polypeptide, particle, nucleic acid molecule, method, or protein complex of any one of embodiments 1 to 92, wherein the polypeptide or ORF1 molecule comprises a cysteine ​​to serine mutation at a residue corresponding to position 70 of the Ring10 ORF1 protein (e.g., as described herein).

[0142] 94. A polypeptide, particle, nucleic acid molecule, method, or protein complex according to any one of embodiments 1 to 93, 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) with a cysteine.

[0143] 95. The polypeptide, particle, nucleic acid molecule, method, or protein complex of embodiment 94, wherein the polypeptide or ORF1 molecule comprises a threonine to cysteine ​​substitution, e.g., at a position corresponding to position 365 of the Ring10 ORF1 protein (e.g., as described herein).

[0144] 96. The polypeptide, particle, nucleic acid molecule, method, or protein complex of embodiment 94, wherein the polypeptide or ORF1 molecule comprises a serine to cysteine ​​substitution, for example, at a position corresponding to position 284 of the Ring10 ORF1 protein (e.g., as described herein).

[0145] 97. The polypeptide, particle, nucleic acid molecule, method, or protein complex of embodiment 94, wherein the polypeptide or ORF1 molecule comprises an asparagine to cysteine ​​substitution, e.g., at a position corresponding to position 290 of the Ring10 ORF1 protein (e.g., as described herein).

[0146] 98. The polypeptide, particle, nucleic acid molecule, method, or protein complex of embodiment 94, wherein the polypeptide or ORF1 molecule comprises a lysine to cysteine ​​substitution, e.g., at a position corresponding to position 317 of the Ring10 ORF1 protein (e.g., as described herein).

[0147] 99. The polypeptide, particle, nucleic acid molecule, method, or protein complex of embodiment 94, wherein the polypeptide or ORF1 molecule comprises a lysine to cysteine ​​substitution, e.g., at a position corresponding to position 324 of the Ring10 ORF1 protein (e.g., as described herein).

[0148] 100. The polypeptide, particle, nucleic acid molecule, method, or protein complex of embodiment 94, wherein the polypeptide or ORF1 molecule comprises an alanine to cysteine ​​substitution, for example, at a position corresponding to position 362 of the Ring10 ORF1 protein (e.g., as described herein).

[0149] 101. The polypeptide, particle, nucleic acid molecule, method, or protein complex of embodiment 94, wherein the polypeptide or ORF1 molecule comprises a serine to cysteine ​​substitution, e.g., at a position corresponding to position 363 of the Ring10 ORF1 protein (e.g., as described herein).

[0150] 102. The polypeptide, particle, nucleic acid molecule, method, or protein complex of embodiment 94, wherein the polypeptide or ORF1 molecule comprises an asparagine to cysteine ​​substitution, e.g., at a position corresponding to position 369 of the Ring10 ORF1 protein (e.g., as described herein).

[0151] 103. The polypeptide, particle, nucleic acid molecule, method, or protein complex of embodiment 94, wherein the polypeptide or ORF1 molecule comprises a lysine to cysteine ​​substitution, e.g., at a position corresponding to position 371 of the Ring10 ORF1 protein (e.g., as described herein).

[0152] 104. The polypeptide, particle, nucleic acid molecule, method, or protein complex of embodiment 94, wherein the polypeptide or ORF1 molecule comprises a glutamine to cysteine ​​substitution, e.g., at a position corresponding to position 287 of the Ring10 ORF1 protein (e.g., as described herein).

[0153] 105. The polypeptide, particle, nucleic acid molecule, method, or protein complex of embodiment 94, wherein the polypeptide or ORF1 molecule 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 I381 of the Ring10 ORF1 protein (e.g., as described herein).

[0154] 106. (a) a proteinaceous outer portion that contains the ORF1 molecule; and (b) a genetic element comprising a heterologous nucleic acid sequence encoding an exogenous effector. A particle comprising: the genetic element is encapsulated within a proteinaceous outer part; and The particles have one or more of the following properties: (i) the genetic element (e.g., a DNA genetic element) does not contain an anellovirus 5' UTR or origin of replication; (ii) the sequence encoding the exogenous effector occupies at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the genetic elements (e.g., DNA genetic elements); (iii) the heterologous nucleic acid sequences account for at least 90%, 95%, 96%, 97%, 98%, 99% or 100% of the genetic elements (e.g., DNA genetic elements); (iv) the particles do not contain a detectable amount (e.g., any amount) of a polypeptide from the host cell, or contain less than 5, 10, 15, 20, 25, 30, 40, or 50 copies of a polypeptide from the host cell; (v) the particles do not contain detectable amounts (e.g., any amounts) of nucleic acid molecules from the host cell, or contain less than 2, 3, 4, or 5 copies of nucleic acid molecules from the host cell; (vi) the particles comprise a denaturing agent at a concentration of less than about 0.01M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.5M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, or 2M; (vii) does not substantially replicate when introduced into a cell (e.g., a human cell); and / or (viii) Particles having a symmetrical morphology.

[0155] 107. The particle according to embodiment 106, 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.

[0156] 108. A population of particles according to embodiment 106, wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the particles in the population contain at least 50, 55, or 60 copies of anellovirus ORF1 molecules.

[0157] 109. A population of particles according to embodiment 106, wherein at least 90% of the particles in the population have a diameter of at least 30, 31, 32, 33, 34, or 35 nm.

[0158] 110. The population of particles of embodiment 106, wherein at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the particles have a symmetrical morphology.

[0159] 111. A population of particles according to embodiment 106, wherein the population does not contain detectable amounts of a polypeptide from the host cell, or contains less than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, or 50 copies of a polypeptide from the host cell per particle.

[0160] 112. A population of particles according to embodiment 106, wherein the population does not contain detectable amounts of nucleic acid molecules from the host cell, or contains less than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, or 50 copies of nucleic acid molecules from the host cell per particle.

[0161] 113. The population of particles according to embodiment 106, wherein the population comprises less than 10 ng of nucleic acid.

[0162] 114. A population of particles according to embodiment 106, wherein the population does not contain detectable amounts of nucleic acid molecules from the host cell, or contains less than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, or 50 copies of nucleic acid molecules of 200 bp or less in length from the host cell per particle.

[0163] 115. A proteinaceous outer region comprising about 40 to 80 (e.g., about 60) copies of ORF1 molecules. A particle comprising: (i) does not contain (e.g., is not encapsulated in) a polynucleotide; (ii) does not contain detectable levels of polynucleotides (e.g., is not encapsulated); (iii) does not contain (e.g., is not encapsulated in) a polynucleotide greater than 1000, 500, 200, or 100 nucleotides in length; (iv) does not contain (e.g., does not encapsulate) 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) The particle does not contain a polynucleotide comprising an anellovirus 5'UTR or replication origin.

[0164] 116. The particle according to embodiment 115, further comprising an exogenous effector.

[0165] 117. The particle according to embodiment 116, wherein the exogenous effector is encapsulated within the proteinaceous outer part.

[0166] 118. The particle according to embodiment 115 or 116, wherein the exogenous effector is a polypeptide.

[0167] 119. A particle according to any one of embodiments 115 to 118, wherein the exogenous effector is a small molecule.

[0168] 120. A composition comprising a plurality of particles comprising a proteinaceous outer portion comprising about 40-80 (e.g., about 60) copies of an ORF1 molecule; At least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of the particles are (i) a polynucleotide; (ii) a nucleic acid molecule that is more than 1000, 500, 200, or 100 nucleotides in length; (iii) a plurality of polynucleotides; (iv) a circular nucleic acid molecule; (v) a single-stranded nucleic acid molecule, and / or (vi) a genetic element (e.g., a genetic element of an anaerovector), e.g., as described herein. does not contain (e.g., is not encapsulated in); or 10 per kilogram of subject to which the composition is to be administered 10 ~10 14 Less than (e.g., 10 10 ~10 11 , 10 11 ~10 12 , 10 12 ~10 13 , or 10 13 ~10 14 A composition comprising a nucleic acid molecule (e.g., a genetic element, e.g., of an anaerovector as described herein) equivalent to a viral genome (e.g., less than 1000 ng / ml) (e.g., as determined by qPCR or by measuring optical density).

[0169] 121. The composition of embodiment 120, further comprising a denaturing agent (e.g., urea); proteasome; or ferritin, for example, at a concentration of less than about 0.01M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.5M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, or 2M.

[0170] 122. The composition of embodiment 120 or 121, comprising 0.01 to 100 mg of particles (e.g., 0.01 to 1, 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, or 90 to 100 mg of particles).

[0171] 123. A method for disintegrating particles, comprising: (a) providing a mixture comprising particles and a modifier, the particles comprising: (i) a proteinaceous outer portion comprising a plurality of anellovirus ORF1 molecules; and (ii) a nucleic acid molecule (e.g., a nucleic acid endogenous to a host cell or a nucleic acid exogenous to a host cell, e.g., an anellovirus genome) Contains; and (b) subjecting the mixture to Degradation of the proteinaceous outer part, and Dissociation of nucleic acid molecules from their proteinaceous exterior Incubate under conditions suitable for The method includes:

[0172] 124. The method of embodiment 123, wherein the anellovirus ORF1 molecule is produced in a mammalian cell.

[0173] 125. The method of embodiment 123 or 124, wherein the conditions suitable for decomposing the proteinaceous outer part include one or more of the following conditions: 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, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, or 5 M), or a predetermined temperature.

[0174] 126. The method of any one of embodiments 123-125, wherein the mixture comprises a population of particles.

[0175] 127. The method of any one of embodiments 123-126, wherein the incubation in (b) results in degradation of at least 50%, 60%...95%, or 100% of the population of particles.

[0176] 128. The method of any one of embodiments 123 to 127, further comprising the step of (c) removing (partially or completely) the nucleic acid molecules from the mixture, for example by washing.

[0177] 129. The method of any one of embodiments 123 to 128, wherein the host cell is a human cell.

[0178] 130. A method for producing an anellovector, 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 to 80 (e.g., about 60) copies of ORF1 molecules; (b) subjecting the mixture to conditions suitable for the in vitro assembly of anellovirus ORF1 molecules; and (c) incubating the anellovirus ORF1 molecule with the plurality of genetic elements under conditions suitable for assembling the anellovirus ORF1 molecules into one or more anellovectors, each encapsulating one or more of the genetic elements. The method includes:

[0179] 131. The method of embodiment 130, wherein the mixture provided in (a) is under denaturing conditions, e.g., the mixture contains a denaturant at a level sufficient to degrade complexes (e.g., proteinaceous outer portions) comprising at least about 20, 30, 40, 50, or 60 copies, or 20-30, 30-40, 40-50, or 50-60 copies of anellovirus ORF1 molecules.

[0180] 132. The method of embodiment 130 or 131, wherein the conditions suitable for in vitro assembly comprise reducing the concentration of a denaturing agent or removing the mixture from denaturing conditions.

[0181] 133. A method for producing an anellovector, 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 the 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 to 80 (e.g., about 60) copies of ORF1 molecules; (b) subjecting the mixture to non-denaturing conditions suitable for in vitro assembly of anellovirus ORF1 molecules (e.g., by dialysis) (e.g., reducing the concentration of denaturing agents to a certain level); and (c) incubating the anellovirus ORF1 molecule with the plurality of genetic elements under conditions suitable for assembling the anellovirus ORF1 molecules into one or more anellovectors, each encapsulating one or more of the genetic elements. The method includes:

[0182] 134. The method of embodiment 133, wherein (b) and (c) are performed simultaneously.

[0183] 135. The method of embodiment 133, wherein (b) is performed before (c).

[0184] 136. The method according to any one of embodiments 133 to 135, wherein the genetic element is introduced into the mixture comprising the anellovirus ORF1 molecule prior to, simultaneously with, or subsequent to (b).

[0185] 137. The method of any one of embodiments 133-136, wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the ORF1 molecules in the mixture of (a) are contained in capsomers (e.g., decamers or particles having a diameter of 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).

[0186] 138. The method of any one of embodiments 133 to 137, wherein the ratio of ORF1 molecules in the mixture (a) contained in the capsomers (e.g., decamers) compared to ORF1 molecules in the mixture (a) contained in the particles 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.

[0187] 139. The method of any one of embodiments 133 to 138, wherein after incubation of (c), at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the plurality of ORF1 molecules are contained in an anellovector (e.g., a 60-mer or a particle having a diameter of at least 30, 31, 32, 33, 34, or 35 nm).

[0188] 140. The method of any one of embodiments 133 to 139, wherein the genetic element encodes an exogenous effector.

[0189] 141. The method according to any one of embodiments 133 to 140, wherein the genetic element is an oligonucleotide.

[0190] 142. The method according to any one of embodiments 133 to 141, wherein the genetic element does not encode a polypeptide or a functional nucleic acid.

[0191] 143. The method of any one of embodiments 133 to 142, wherein the concentration of the denaturing agent after step (b) does not exceed about 0.01M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.5M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, or 2M.

[0192] 144. The method of any one of embodiments 133 to 143, wherein after incubation of (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 anellovirus ORF1 molecules.

[0193] 145. The method of any one of embodiments 133 to 144, wherein after incubation of (c), at least 90% of the particles in the mixture have a diameter of at least 30, 31, 32, 33, 34, or 35 nm.

[0194] 146. The method of any one of embodiments 133 to 145, wherein after incubation (c), at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the particles in the mixture have a symmetrical morphology.

[0195] 147. The method of any one of embodiments 133 to 146, wherein the denaturing agent is selected from a chaotropic agent (e.g., urea), heat (e.g., a temperature greater than about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C.), or pH (e.g., an acidic or basic pH).

[0196] 148. A method for producing anelloVLPs, 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 to 80 (e.g., about 60) copies of ORF1 molecules; (b) subjecting the mixture to conditions suitable for the in vitro assembly of anellovirus ORF1 molecules; and (c) incubating the anellovirus ORF1 molecule with a plurality of effectors (e.g., exogenous effectors) under conditions suitable for assembling the anellovirus ORF1 molecule into one or more anelloVLPs, each of which encapsulates one or more of the effectors. The method includes:

[0197] 149. The method of embodiment 148, wherein the mixture provided in (a) is under denaturing conditions, e.g., the mixture contains a denaturant at a level sufficient to degrade complexes (e.g., proteinaceous outer portions) comprising at least about 20, 30, 40, 50, or 60 copies, or 20-30, 30-40, 40-50, or 50-60 copies of anellovirus ORF1 molecules.

[0198] 150. The method of embodiment 148 or 149, wherein the conditions suitable for in vitro assembly comprise reducing the concentration of a denaturing agent or removing the mixture from denaturing conditions.

[0199] 151. A method for producing anelloVLPs, 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 to 80 (e.g., about 60) copies of the ORF1 molecule; (b) reducing the concentration of the denaturing agent to a level suitable for in vitro assembly of anellovirus ORF1 molecules; and (c) incubating the anellovirus ORF1 molecule with a plurality of effectors (e.g., exogenous effectors) under conditions suitable for in vitro assembly of the anellovirus ORF1 molecule into one or more anelloVLPs, each of which encapsulates one or more of the effectors. The method includes:

[0200] 152. The method of embodiment 151, wherein an effector is introduced into the mixture containing the anellovirus ORF1 molecules prior to, simultaneously with, or after (b).

[0201] 153. The method of embodiment 151 or 152, wherein at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the ORF1 molecules in the mixture of (a) are contained in capsomers (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).

[0202] 154. The method of any one of embodiments 151 to 153, wherein the ratio of ORF1 molecules in the mixture (a) contained in the capsomers (e.g., decamers) compared to ORF1 molecules in the mixture (a) contained in the particles 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.

[0203] 155. The method of any one of embodiments 151 to 154, wherein after incubation of (c), at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the plurality of ORF1 molecules are contained in anelloVLPs (e.g., 60-mers or particles having a diameter of at least 30, 31, 32, 33, 34, or 35 nm).

[0204] 156. The anelloVLP has one or more of the following characteristics: (i) does not contain (e.g., is not encapsulated in) a polynucleotide; (ii) does not contain detectable levels of polynucleotides (e.g., is not encapsulated); (iii) does not contain (e.g., is not encapsulated in) a polynucleotide greater than 1000, 500, 200, or 100 nucleotides in length; (iv) does not contain (e.g., does not encapsulate) 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 an anellovirus 5' UTR or replication origin; The method according to any one of embodiments 151 to 155.

[0205] 157. The method of any one of embodiments 151 to 156, wherein the concentration of the denaturing agent after step (b) does not exceed about 0.01M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.5M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, or 2M.

[0206] 158. The method of any one of embodiments 151 to 157, wherein after incubation of (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 anellovirus ORF1 molecules.

[0207] 159. The method of any one of embodiments 151 to 158, wherein after incubation of (c), at least 90% of the particles in the mixture have a diameter of at least 30, 31, 32, 33, 34, or 35 nm.

[0208] 160. The method of any one of embodiments 151 to 159, wherein after incubation (c), at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the particles in the mixture have a symmetrical morphology.

[0209] 161. The method of any one of embodiments 151 to 160, wherein the denaturing agent is selected from a chaotropic agent (e.g., urea), heat (e.g., a temperature greater than about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C.), or pH (e.g., an acidic or basic pH).

[0210] 162. A method for producing anelloVLPs, comprising: (a) providing a mixture comprising particles and a modifier, the particles comprising: (i) a proteinaceous outer portion comprising a plurality of anellovirus ORF1 molecules; and (ii) a nucleic acid molecule (e.g., a host cell nucleic acid molecule) Contains; and (b) subjecting the mixture to Degradation of the proteinaceous outer part, and Dissociation of nucleic acid molecules from their proteinaceous exterior incubating under conditions suitable for (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 to 80 (e.g., about 60) copies of the ORF1 molecule; (d) reducing the concentration of the denaturing agent to a level suitable for in vitro assembly of anellovirus ORF1 molecules; and (e) incubating the anellovirus ORF1 molecule with a plurality of effectors (e.g., exogenous effectors) under conditions suitable for assembling the anellovirus ORF1 molecule into one or more anelloVLPs, each of which encapsulates one or more of the effectors. The method includes:

[0211] 163. (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 (e.g., MPYYYRRRRYNYRRPRWYGRGWIRRPFRRRFRRKRRVR (SEQ ID NO: 216) or MAWGWWKRRRRWWFRKRWTRGRLRRRWPRSARRRPRRRRVRRRRRWRRGRRKTRTYRRRRRFRRRGRK (SEQ ID NO: 186), or as set forth in any one of Tables A1-A25), or a sequence of at least about 40 amino acids in which at least 60%, 70%, or 80% are basic residues (e.g., arginine, lysine, or a combination thereof); (b) a jelly-roll region sequence described herein (e.g., [ka] or as set forth in any one of Tables A1 through A25), or a sequence that includes at least six (e.g., at least six, seven, eight, nine, ten, eleven, or twelve) beta strands; (c) an N22 domain sequence described herein (e.g., [ka] or any one of Tables A1-A25); and (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., WGGSPPKAINVENPAHQIQYPIPRNEHETTSLQSPGEAPESILYSFDYRHGNYTTTALSRISQDWALKDTVSKITEPDRQQLLKQALECLQISEETQEKKEKEVQQLISNLRQQQQLYRERIISLLKDQ (SEQ ID NO: 220), or as set forth in any one of Tables A1-A25). A polypeptide, e.g., an ORF1 molecule, comprising one or more of: A polypeptide, wherein the ORF1 molecule includes at least one difference (e.g., a mutation, a chemical modification, or an epigenetic change) compared to a wild-type ORF1 protein (e.g., as described herein), such as an insertion, substitution, chemical or enzymatic modification, and / or a deletion, such as a deletion of a domain (e.g., one or more of the arginine-rich region, the jelly-roll domain, the HVR, N22, or the CTD, e.g., as described herein).

[0212] 164. The polypeptide of embodiment 163, wherein the amino acid sequences of regions (a), (b), (c), and (d) have at least 90% sequence identity with their respective references.

[0213] 165.(i) The first and second areas; (ii) the first region and the third region; (iii) the first region and the fourth region; (iv) the second and third regions; (v) the second and fourth regions; (vi) the third and fourth regions; (vii) a first region, a second region, and a third region; (viii) a first region, a second region, and a fourth region; (ix) the first region, the third region, and the fourth region; or (x) The second area, the third area, and the fourth area 164. The polypeptide of embodiment 163, comprising:

[0214] 166. 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 to A25; 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 jellyroll region sequence as listed in any one of Tables A1-A25; the third 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 N22 domain sequence as listed in any one of Tables A1 to A25; and / or The fourth 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 CTD sequence as set forth in any one of Tables A1 to A25; 166. A polypeptide according to any one of embodiments 163 to 165.

[0215] 167. The polypeptide of embodiment 166, wherein the amino acid sequences of the first, second, third and fourth regions have at least 90% sequence identity with their respective references.

[0216] 168. The polypeptide according to any one of embodiments 1 to 167, comprising, in order from N-terminus to C-terminus, a first domain, a second domain, a third domain, and a fourth domain.

[0217] 169. The polypeptide according to any one of the preceding embodiments, wherein the at least one difference comprises at least one difference in the first region compared to the arginine-rich region of the wild-type ORF1 protein.

[0218] 170. The polypeptide according to any one of embodiments 1 to 169, wherein the first region comprises an arginine-rich region from an ORF1 protein of an anellovirus other than a wild-type anellovirus with which the polypeptide, or a portion thereof excluding the first region, exhibits the highest sequence identity.

[0219] 171. The polypeptide of any one of embodiments 1 to 170, wherein the first region comprises an amino acid sequence having at least 70% sequence identity to an arginine-rich region from an anellovirus other than a wild-type anellovirus with which the polypeptide exhibits the highest sequence identity.

[0220] 172. The polypeptide of any one of embodiments 1 to 171, wherein the second region comprises a jelly-roll region from an ORF1 protein of an anellovirus other than a wild-type anellovirus with which the polypeptide, or a portion thereof excluding the second region, exhibits the highest sequence identity.

[0221] 173. The polypeptide of any one of embodiments 1 to 172, wherein the second region comprises an amino acid sequence having at least 70% sequence identity to a jellyroll region from an anellovirus other than a wild-type anellovirus with which the polypeptide exhibits the highest sequence identity.

[0222] 174. The polypeptide of any one of embodiments 1 to 173, wherein the third region comprises an N22 domain from an ORF1 protein of an anellovirus other than a wild-type anellovirus with which the polypeptide, or a portion thereof excluding the third region, exhibits the highest sequence identity.

[0223] 175. The polypeptide of any one of embodiments 1 to 174, wherein the third region comprises an amino acid sequence having at least 70% sequence identity to an N22 region from an anellovirus other than the wild-type anellovirus with which the polypeptide exhibits the highest sequence identity.

[0224] 176. The polypeptide of any one of embodiments 1 to 175, wherein the fourth region comprises a CTD domain from an ORF1 protein of an anellovirus other than a wild-type anellovirus with which the polypeptide, or a portion thereof excluding the fourth region, exhibits the highest sequence identity.

[0225] 177. The polypeptide of any one of embodiments 1 to 176, wherein the fourth region comprises an amino acid sequence having at least 70% sequence identity to a CTD region from an anellovirus other than the wild-type anellovirus with which the polypeptide exhibits the highest sequence identity.

[0226] 178. The polypeptide according to any one of embodiments 163-177, wherein the HVR sequence is located between the second and third regions.

[0227] 179. The polypeptide of embodiment 178, wherein the HVR sequence 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 with an HVR from an anellovirus other than the wild-type anellovirus with which the ORF1 protein shows the highest sequence identity.

[0228] 180. The polypeptide of embodiment 178 or 179, wherein the HVR sequences are heterologous compared to one or more of the first region, the second region, the third region, and / or the fourth region.

[0229] 181. The polypeptide according to any one of embodiments 178 to 180, wherein the HVR sequence comprises an HVR from an ORF1 protein of an anellovirus other than a wild-type anellovirus with which the polypeptide, or a portion thereof excluding the HVR sequence, exhibits the highest sequence identity.

[0230] 182. The polypeptide according to any one of embodiments 178 to 181, wherein the HVR sequence comprises an amino acid sequence having at least 70% sequence identity with an HVR from an anellovirus other than the wild-type anellovirus with which the polypeptide exhibits the highest sequence identity.

[0231] 183. The anaerovector according to any one of the preceding embodiments, wherein the proteinaceous outer part comprises a polypeptide according to any one of the preceding embodiments 58 to 77.

[0232] 184. A particle according to any one of the preceding embodiments, wherein the proteinaceous outer part comprises a polypeptide according to any one of the preceding embodiments 58 to 77.

[0233] 185. The anelloVLP according to any one of embodiments 1 to 184, wherein the proteinaceous outer part comprises a polypeptide according to any one of embodiments 58 to 77.

[0234] 186. A method for making two or more different anellovirus ORF molecules, comprising: (i) providing an insect cell containing 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); (ii) incubating the insect cells under conditions suitable for expression of two or more different anellovirus ORF molecules. The method includes:

[0235] 187. The method of embodiment 186, wherein the nucleic acid construct comprises sequences encoding all of the ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, and / or ORF1 / 2 molecules.

[0236] 188. The method of embodiment 186, further comprising incubating the insect cells under conditions suitable for secretion of the anellovirus ORF molecule.

[0237] 189. The method of embodiment 186, further isolating the anellovirus ORF molecule from the insect cell.

[0238] 190. The method of embodiment 189, wherein the isolating step comprises lysing the insect cells.

[0239] 191. The method of any one of embodiments 186 to 190, wherein the anellovirus ORF comprises an anellovirus ORF1 molecule.

[0240] 192. A method for producing an anellovirus ORF1 molecule, comprising: (i) providing an insect cell comprising a nucleic acid construct encoding an anellovirus ORF1 molecule, (a) the anellovirus ORF1 molecule has a molecular weight of at least 101 kDa; (b) whether the anellovirus ORF1 molecule is a full-length anellovirus ORF1 protein; (c) a plurality of anellovirus ORF1 molecules, when in the presence of anellovirus genetic elements, encapsulates the anellovirus genetic element; (d) whether the anellovirus ORF1 molecule is the TTV ORF1 protein; (e) the anellovirus ORF1 molecule is a Betatorquevirus or a Gammatorquevirus ORF1 molecule; or (f) the anellovirus ORF1 molecule comprises an anellovirus ORF1 arginine-rich region and an anellovirus C-terminal domain; (ii) incubating the insect cells under conditions suitable for expression of the anellovirus ORF1 molecule; The method includes:

[0241] 193. The method of embodiment 192, further comprising incubating the insect cells under conditions suitable for secretion of the anellovirus ORF1 molecule.

[0242] 194. The method of embodiment 192, further isolating the anellovirus ORF1 molecule from the insect cell.

[0243] 195. The method of embodiment 194, wherein the isolating step comprises lysing the insect cells.

[0244] 196. The method of any one of embodiments 1-195, wherein the incubation step produces a detectable amount of anellovirus ORF1 molecules, e.g., by Western blot as described herein.

[0245] 197. A method of making an anellovirus ORF molecule (e.g., an ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, and / or ORF1 / 2 molecule), comprising: (i) providing an insect cell (e.g., an Sf9 cell) containing a nucleic acid construct encoding an anellovirus ORF molecule; (ii) incubating the insect cells under conditions suitable for expression of the plurality of anellovirus ORF molecules; and (iii) optionally isolating, purifying, and / or concentrating the plurality of anellovirus ORF molecules from the insect cells or other components or components. Includes; thereby generating an anellovirus ORF molecule.

[0246] 198. The method of embodiment 197, wherein the anellovirus ORF molecule is fused, for example, at its N-terminal end or at its C-terminal end to a marker (e.g., a His tag) (e.g., as described in Table E1 and / or Example 9).

[0247] 199. 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 the method comprises: For example, prior to, simultaneously with, or subsequent to step (ii), incubating the insect cells under conditions suitable for expression of a plurality of the one or more additional anellovirus ORF molecules; and Optionally, for example, prior to, simultaneously with, or subsequent to step (iii), isolating, purifying, and / or concentrating the plurality of one or more additional anellovirus ORF molecules from the insect cells or other components or components. 199. The method of embodiment 197 or 198, further comprising:

[0248] 200. The method of embodiment 199, wherein the nucleic acid construct encoding the one or more additional anellovirus ORF molecules is the same as the nucleic acid construct of (i).

[0249] 201. The method of embodiment 200, wherein the nucleic acid construct of (i) comprises a sequence encoding two, three, four, five, or all six of the anellovirus ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, and / or ORF1 / 2 molecules.

[0250] 202. The method of embodiment 200, wherein the nucleic acid construct of (i) encodes anellovirus ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, and ORF1 / 2 molecules.

[0251] 203. The method of embodiment 200, wherein the nucleic acid construct of (i) comprises a complete open reading frame region of an anellovirus genome.

[0252] 204. The method of embodiment 199, wherein the nucleic acid construct encoding the one or more additional anellovirus ORF molecules is different from the nucleic acid construct of (i).

[0253] 205. The method of any one of embodiments 199 to 204, wherein the anellovirus ORF molecules are from the same anellovirus genome.

[0254] 206. The method of any one of embodiments 199 to 204, wherein the anellovirus ORF molecules are from multiple anellovirus genomes (e.g., the ORF1 molecule is from one anellovirus genome and the ORF2 molecule is from a different anellovirus genome).

[0255] 207. The method of any one of embodiments 199-206, wherein one or more of the anellovirus ORF molecules are from an alphatorque virus (e.g., as listed in Table E2).

[0256] 208. The method of any one of embodiments 199-207, wherein one or more of the anellovirus ORF molecules is from a Betatorque virus (e.g., as listed in Table E2).

[0257] 209. The method of any one of embodiments 199-208, wherein one or more of the anellovirus ORF molecules is from a Gamma Torque virus (e.g., as listed in Table E2).

[0258] 210. The method of any one of embodiments 199 to 209, wherein 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 polyhedrin promoter).

[0259] 211. The method according to any one of embodiments 199 to 210, further comprising incubating the insect cells under conditions suitable for secretion of the anellovirus ORF molecule.

[0260] 212. The method of any one of embodiments 199-211, wherein the isolating step comprises lysing the insect cells.

[0261] 213. The method of any one of embodiments 199-212, wherein the incubation step produces a detectable amount of anellovirus ORF molecule (e.g., ORF1 molecule), e.g., by Western blot as described herein.

[0262] 214. The method of any one of emcodiments 199 to 213, wherein the incubation step produces at least 1, 2, 3, 4, 5, or 6 mg of anellovirus ORF1 molecules per L of cell culture (e.g., Sf9 culture).

[0263] 215. The method of any one of embodiments 1 to 214, wherein the anellovirus ORF molecules are isolated, purified or concentrated by isopycnic centrifugation.

[0264] 216. The anellovirus ORF molecule is an anellovirus ORF1 molecule, and the method comprises: contacting an isolated, purified or concentrated anellovirus ORF1 molecule with a genetic element in vitro under conditions suitable for encapsulation of the genetic element with a proteinaceous outer part comprising an anellovirus ORF1 molecule, e.g. as described herein; 216. The method of any one of embodiments 1 to 215, further comprising:

[0265] Other features, objects, and advantages of the invention will become apparent from the specification and drawings, and from the claims.

[0266] 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 this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0267] The following detailed description of the embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the drawings show embodiments which are given as examples herein. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication in color will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0268] [Figure 1] FIG. 1 is a series of diagrams showing the generation of exemplary malaria peptide conjugation constructs comprising the C-terminal region of the CS protein. [Diagram 2] FIG. 1 is a series of diagrams showing the structures of exemplary malaria peptide conjugation constructs on the surface of anellovector capsids. [Diagram 3]FIG. 1 shows the separation of in vitro circularized DNA into positive- and negative-sense circularized single-stranded DNA (ssDNA) after denaturation. [Figure 4A]

[0023] Figure 1 is a series of figures showing successful expression of anellovirus ORF1 molecules in cells. (A) Anellovirus strains for which ORF1 expression was detected in Sf9 cells included Ring2, Ring3, Ring4, Ring5, Ring6, Ring9, and Ring10 (e.g., as described herein). (B) Virus-like particles (VLPs) were observed by electron microscopy following production of anellovirus ORF1 protein for Ring2 and Ring10, as well as for Chicken Anemia Virus (CAV) VP1. [Figure 4B] Same as above [Diagram 5] Figure 1 is a series of figures showing successful expression and purification of Ring2 ORF1 and ORF2 proteins from Sf9 cells. (A) An exemplary workflow for purification of ORF1 and ORF2 proteins from Sf9 cells. (B) Western blot using anti-ORF1 antibody showing detection of Ring2 ORF1 protein produced in Sf9 cells as indicated. (C) Electron microscopy images showing formation of virus-like particles (VLPs) from Ring2 ORF1 protein produced in Sf9 cells as indicated. [Figure 6] A series of figures showing the disassembly of Ring2 VLPs using urea as a chaotropic denaturant. (A) Ring2 ORF1 protein was produced and purified from Sf9 cells as described, and VLPs were detected by electron microscopy. (B) After treatment with urea, VLPs are no longer observed, but instead the solution contains small ORF1 capsomers. (C) Enlargement of the boxed area shown in Figure 6B, showing details of the capsomers. [Figure 7AB] 1 is a series of electron microscope images showing exemplary symmetric (A) and asymmetric (B) particle morphologies. [Figure 8] Illustrates expression of C-terminally His-tagged Ring2 ORF1 in insect cells. [Figure 9]Illustrates expression of C-terminally His-tagged Ring1 ORF1 and ORF1 / 1 in insect cells. [Figure 10] 1 illustrates the expression of N-terminally His-tagged Ring2 ORF1 in insect cells with or without the PreScission cleavage sequence. [Figure 11] 1 illustrates the expression of Ring1 ORF1 / 1, 1 / 2, 2, 2 / 2, and 2 / 3 as C-terminally His-tagged recombinant proteins in insect cells. [Figure 12] Figure 1 illustrates the expression of individual Ring2 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 Ring2 constructs tested as indicated. [Figure 13] 1 illustrates baculovirus-mediated co-expression of Ring2 ORF1+"FullORF", ORF1+ORF2, ORF1+ORF2 / 2, and ORF1+ORF2 / 3 in insect cells. [Figure 14] FIG. 1 illustrates simultaneous co-expression of multiple Ring2 proteins in insect cells using baculovirus. [Figure 15] Illustrates expression of ORFs from anellovirus genomes delivered to insect cells by baculovirus and by transfection. [Figure 16] 1 shows that expression of Ring1 ORF2 in Sf9 cells is independent of the polyhedrin promoter (arrow labeled pH). [Figure 17] Figure 1 illustrates the co-delivery of Ring2 ORF1-His and Ring2 genomic DNA into Sf9 cells, followed by incubation and fractionation on a linear CsCl gradient. The top of the figure shows an anti-His tag Western blot of the fractions, as well as a qPCR assay for each fraction. The bottom panel shows transmission electron microscopy images of two individual fractions and the fraction pool, as indicated by boxes on the Western blot. The inset in the middle panel is a magnified image showing the proteasome-like structures. [Figure 18] Illustrated is the characterization of Sf9 isopycnic fractions by immunogold electron microscopy. [Figure 19] 1 illustrates expression of ORF1 from additional anellovirus strains. [Figure 20] FIG. 1 is a schematic diagram showing an exemplary workflow for the generation of anaerovectors (e.g., replication-competent or replication-deficient anaerovectors as described herein). [Figure 21] Graph showing primer specificity of primer sets designed for quantification of TTV and TTMV genome equivalents. Quantitative PCR based on SYBR green chemistry shows one characteristic peak for each amplification product using TTMV or TTV specific primer sets as indicated on plasmids encoding the respective genomes. [Figure 22] Graph showing exemplary amplification plots for linear amplification of TTMV (target 1) or TTV (target 2) across seven log10 genome equivalent concentrations. Genome equivalents were quantified across seven 10-fold dilutions, resulting in high PCR efficiency and linearity (R2 TTMV: 0.996; R2 TTV: 0.997). [Diagram 23AB]

[0023] Figure 1 is a series of figures showing that tandem anelloviral plasmids can increase anellovirus or anaerovector production. (A) Plasmid maps of exemplary tandem anelloviral plasmids. (B) Transfection of HEK293T cells with tandem anelloviral plasmids resulted in production of 4-fold higher viral genome numbers compared to plasmids harboring a single copy. [Figure 23C] 1 is a gel electrophoresis image showing circularization of TTMV-LY2 plasmids pVL46-063 and pVL46-240. [Figure 23D] Chromatograms showing copy numbers of linear and circular TTMV-LY2 constructs as determined by size exclusion chromatography (SEC). [Figure 24] FIG. 1 is a schematic showing the domains of anellovirus ORF1 molecule and the hypervariable regions that will be replaced with hypervariable domains from different anelloviruses. [Diagram 25]FIG. 1 is a schematic diagram showing the domains of ORF1 and the hypervariable regions that will be replaced with a protein or peptide of interest (POI) from a non-anellovirus source. [Figure 26] FIG. 1 is a series of diagrams showing the design of exemplary anaerovector genetic elements based on the anellovirus genome. Protein coding regions were deleted from the anellovirus genome (left), leaving an anellovirus non-coding region (NCR) that includes the viral promoter, the 5'UTR conserved domain (5CD), and a GC-rich region. Payload DNA was inserted into the non-coding region located at the protein-coding locus (right). The resulting anellovector harbored payload DNA (including open reading frames, genes, non-coding RNA, etc.) and essential anaerovirus cis-replication and packaging elements, but lacked the protein elements essential for replication and packaging. [Figure 27] FIG. 1 shows an alignment of 36-nucleotide GC-rich regions from nine anellovirus genome sequences and consensus sequences based thereon (SEQ ID NOs: 818 to 827, respectively, in order of appearance). [Figure 28] FIG. 1 is a series of diagrams showing the ORF1 structures from anellovirus strains LY2 and CBD203. Putative domains are labeled as indicated: arginine-rich region (arg-rich), core region including the jelly-roll domain, hypervariable region (HVR), N22 region, and C-terminal domain (CTD). [Figure 29] FIG. 1 is a graph showing the ability of in vitro circularized (IVC) TTV-tth8 genome (IVC TTV-tth8) to generate TTV-tth8 genome copies at the expected density in HEK293T cells compared to the TTV-tth8 genome in a plasmid. [Diagram 30] 1 is a series of graphs showing the ability of in vitro circularized (IVC) LY2 genome (WT LY2 IVC) and wild-type LY2 genome in a plasmid (WT LY2 plasmid) to generate LY2 genome copies at the expected density in Jurkat cells. [Figure 31-1](FIG. 31A) Schematic representation of full-length Ring10 ORF1 (also referred to herein as Ly1) with color-coded domains with label names. The structural arginine-rich motif (ARM) is shown in purple, the structural jelly-roll (JR) domain in red, the spike P1 domain in blue, the spike P2 domain in green, and the C-terminal domain in cyan. Residue numbers starting at each domain and the structural C-terminal domain (also referred to as C-terminus) are indicated above. (FIG. 31B) Sequence of full-length Ring10 ORF1, color-coded as in FIG. 31A, with residue numbers indicated above. In bold are residues included in the Ring10 delARM construct, with the leading residue K46 indicated. Dashed lines above the sequence indicate residues not observed in density. Secondary structure elements are indicated above with β-strands as arrows and α-helices as zigzag lines. JR β-strands are conventionally labeled B through I, while additional secondary structures are numbered according to their domains. The three peptides used to generate polyclonal antibodies are underlined. [Figure 31-2](Fig. 31C) Western blot analysis of Ring10 delARM after expression (express) and after purification and storage (purify). Molecular weight markers are indicated to the left of the gel, while arrows on the right indicate the Ring10 delARM bands before (Ring10 delARM) and after proteolysis (Ring10 delARM fragment). The polyclonal antibodies used to probe the Western blot are indicated below and color-coded by the peptide used to generate them. (Fig. 31D) Overlay of Ring10 delARM VLP electron density and 3D reconstruction of the 60mer VLP molecular structure color-coded as in Fig. 31A. Spike P1 and P2 domains are labeled. (Fig. 31E) One ORF1 protomer shown in its electron density, domains labeled and color-coded as in Fig. 31A. (Fig. 31F) Electron density of Ring10 delARM VLP color-coded by its local resolution. The bar (left) indicates a color scale for resolution (in Angstroms). The particles (right) are oriented as in Figure 31D. [Figure 32A] Sixty Ring10 structural jelly-roll (JR) domains are illustrated, with one uniquely colored red. The sixty Ring10 structural jelly-roll (JR) domains form the core of the anellovirus particle. [Figure 32B] The two JR domains are shown diagrammatically (in red) with the observed C-terminal domain backbone coloured in cyan. The JR domains are arbitrarily labelled JR1 and JR2 and, for clarity, the first (K48) and last (V562) residues observed in each protomer are labelled with the corresponding numbers. [Figure 32C] A single JR domain is shown oriented to reveal the β-sheets within the particle core, with the side chains of basic residues appropriately positioned to make contact with the viral genome indicated and labeled. [Fig. 32D]The structural arginine-rich region, JR, and structural C-terminal domains of Ring10 aligned with the corresponding ORF1 sequences from various different Anellovirus genera (indicated in brackets). Ring10 residues are colored as in Figures 31A-D. Basic residues in Ring10 that are in potential contact with the viral genome are indicated with an asterisk. [Figure 33A] Illustrates the anellovirus particle structure as shown as a surface rendering. The particle is shown in grey with five spikes forming a crown structure, numbered for clarity and colour coded as in Figures 31A-31D. The spike domains extend from the core on a five-fold axis. [Figure 33B] The outer portion of the crown structure is illustrated in side view. The five spike domains are colored as in Figure 33A. Conserved hydrophobic and hydrophilic residues are colored light blue and magenta, respectively. [Figure 33C] The same spike domain from FIG. 33B is shown rotated to reveal residues within the crown structure. [Figure 33D] Illustrated is the Ring10 spike domain (color coded as in Figures 31A-31D) aligned with ORF1 sequences representing a variety of different anellovirus genera (indicated in brackets). Magenta and black asterisks indicate surface exposed residues in the P1 and P2 domains, respectively. Below the alignment are >30% consensus residues, or φ or γ, which indicate that the residue is >70% hydrophobic or >60% hydrophilic, respectively. [Diagram 34](FIG. 34A) Schematic representation of full-length Ring10 (top), which is shown by negative stain electron microscopy to be a highly heterogeneous particle (bottom). Scale bar=100 nm. (FIG. 34B) Schematic representation of Ring10 delARM (arginine-rich motif; top), which demonstrates a structurally homologous virus-like particle (VLP) as shown by negative stain electron microscopy (bottom). Scale bar=100 nm. (FIG. 34C) Schematic representation of Ring10 delARM delCTD (top), in which further truncation of the structural C-terminal domain (Δ552-672) maintains a structured VLP as shown by negative stain electron microscopy (bottom). Scale bar=100 nm. [Diagram 35] The data processing steps of the Ring10 delARM cryo-electron microscopy (cryo-EM) reconstruction are illustrated. Briefly, 58,391 particles were selected from 11,083 micrographs by crYOLO. Several 2D classification rounds resulted in 11,185 particles. After de novo reconstruction of the initial model with Relion, Relion 3D refinement was performed to obtain orientation parameters. All particles with parameters were fed into 3D classification. The most abundant particle population class resulted in a resolution of 3.98 Å. [Figure 36A] Representative negative staining micrograph of Rig 10 delARM. This micrograph was captured at NanoImaging Service. [Figure 36B] Representative cryo-EM micrograph of Ring10 delARM. The micrograph was captured at NanoImaging Service. [Fig. 37AB]Circular dichroism (CD) results of the TTMV-Ring10 C-terminal peptide (CSEEEEESNLFERLLRQRTKQLQLKRRIIQTLKDLQKLE) are illustrated. Figure 37A is a table showing the average secondary structure percentages predicted by different packages in CDPro. α-helices predominate in the secondary structure assignments from CD spectra. Figure 37B shows an overlay of the experimental spectrum (shown in red) of the C-terminal peptide from three different packages (SELCON3, CDSSTR, and CONTINLL) and the reference ensemble spectra calculated and averaged (shown in blue). [Figure 38-1] Shown is a sequence alignment of 15 known anelloviruses within various different genera indicated in brackets. Conserved amino acids are shown in the first blue line beneath the sequences. The top blue line beneath the sequence alignment indicates homologous sequences where conservation is greater than 30%. The bottom blue line indicates whether the amino acid is hydrophobic (φ, within 70% similarity) or 60% positively charged (γ, within 60% similarity), respectively. Alignment was performed with Clustal Omega as implemented in Geneious. [Figure 38-2] Same as above [Figure 38-3] Same as above [Figure 38-4] Same as above [Figure 38-5] Same as above [Figure 38-6] Same as above [Figure 38-7] Same as above [Figure 39] JA20 and MN779270.1 spikes predicted by Ring10 or alpha-fold are illustrated. Figure 39A illustrates the globular representation of the Ring10 spike (same as Figure 33C). Figures 39B and 39C are the alpha-fold predicted spike representations of JA20 and MN779270.1, respectively. P1 and P2 domains are demonstrated in blue and green. Light blue and magenta are conserved hydrophobic and basic residues. Figure 39D shows the sequence alignment between Ring10, JA20, and MN779210.1. [Fig. 40AB] FIG. 1 is a series of figures showing Coomassie staining (FIG. A1) and Western blot (FIG. A2) of Ring2 virus-like particles (VLPs). [Diagram 41] Electron microscopy images showing Ring2 VLPs obtained after Capto400 purification. [Fig. 42AB] A series of figures showing Coomassie staining (Fig. A4) and Western blot (Fig. A5) of Ring19 VLPs. [Diagram 43] Electron microscopy images showing Ring19 VLPs obtained after Capto400 purification. [Diagram 44] FIG. 1 shows an exemplary workflow for conjugating NHS ester moieties to surface lysines of anelloVLPs using click chemistry. [Fig. 45AB] FIG. 2 is a series of figures showing Coomassie staining (FIG. B2) and Western blot (FIG. B3) of Ring2 VLPs conjugated with NHS ester 647. [Fig. 46AB] FIG. 2 is a series of figures showing Western blots of Ring2 anelloVLPs conjugated with NHS-ester biotin, labeled using streptavidin CV 800 antibody (FIG. B4) or Ring19 HVR3 primary antibody and goat anti-rabbit secondary antibody (FIG. B5). [Figure 47] FIG. 1 shows an exemplary two-step process for conjugating surface effector moieties to the surface of anelloVLPs. [Figure 48] FIG. 1 shows Coomassie staining, Western blot, and UV labeling of SARS-CoV-2 receptor binding domains (RBDs) attached to DBCO, CalFluor 488, azide, or Alexa488, as indicated. [Figure 49] FIG. 13 shows Coomassie staining and Western blot of pRTx-2652 (Ring2 ORF1 mutant) prepared using SE-FPLC. [Figure 50]FIG. 13 shows Coomassie staining and Western blot showing conjugation of RBD linked to an azide moiety with pRTx-2652 ORF1 polypeptide linked to DBCO to generate RBD-pRTx-2652 conjugate anelloVLP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0269] definition The present invention will be described with respect to specific embodiments and with reference to certain figures but the invention is not limited thereto except as claimed. The terms set forth below should generally be understood in their ordinary meaning unless otherwise indicated.

[0270] When the term "comprising" is used in the present specification and claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". Hereinafter, when a group is defined to include at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists only of these embodiments.

[0271] Where an indefinite or definite article is used to indicate a singular noun, e.g. "a", "an" or "the", this includes a plural of that noun, unless specifically stated otherwise.

[0272] The phrase "compounds, compositions, products, etc. for the purpose of treatment, regulation, etc." should be understood to refer, by itself, to compounds, compositions, products, etc. that are suitable for the stated purpose, such as treatment, regulation, etc. The phrase "compounds, compositions, products, etc. for the purpose of treatment, regulation, etc." also discloses, as an embodiment, that such compounds, compositions, products, etc. are used for treatment, regulation, etc.

[0273] The phrases "compounds, compositions, products, etc. for use in...", "use of compounds, compositions, products, etc. in the manufacture of a medicament, pharmaceutical composition, veterinary composition, diagnostic composition, etc. for...", or "compounds, compositions, products, etc. for use as a medicament..." indicate that such compounds, compositions, products, etc. are intended to be used in a method of treatment that may be performed on the human or animal body. They are considered as equivalent disclosures of embodiments and claims relating to methods of treatment, etc. If an embodiment or claim thus refers to a "compound for use in the treatment of a human or animal suspected of suffering from a disease", this is also considered to be a disclosure of "use of a compound in the manufacture of a medicament for the treatment of a human or animal suspected of suffering from a disease" or "method of treatment by administering a compound to a human or animal suspected of suffering from a disease". The phrase "compounds, compositions, products, etc. for treatment, modulation, etc." should be understood to refer to a compound, composition, product, etc. suitable for the stated purpose of treatment, modulation, etc. as such.

[0274] Hereinafter, when an example of a term, value, number, etc. is given in parentheses, this should be understood as indicating that the example described in parentheses may constitute an embodiment. For example, when it is described that "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 the Anellovirus ORF1 encoding the nucleotide sequence of Table 1 (e.g., nucleotides 571-2613 of the nucleic acid sequence of Table 1)," some embodiments relate to a nucleic acid molecule comprising a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotides 571-2613 of the nucleic acid sequence of Table 1.

[0275] As used herein, the term "anellovector" refers to a vehicle that includes a genetic element, e.g., an episome, e.g., circular DNA, enclosed in a proteinaceous outer part. A "synthetic anaellovector" as used herein generally refers to an anaellovector that does not occur in nature, e.g., an anaellovector that has a different sequence compared to a wild-type virus (e.g., a wild-type Anellovirus as described herein). In some embodiments, the proteinaceous outer part includes an ORF1 molecule (e.g., anellovirus ORF1 protein), e.g., as described herein. In some embodiments, the proteinaceous outer part includes a plurality of ORF1 molecules (e.g., anellovirus ORF1 proteins), e.g., at least about 40, 45, 50, 55, 60, 65, or 70 ORF1 molecules. In some embodiments, a synthetic anaellovector is engineered or recombinant, e.g., includes a genetic element that includes a difference or modification relative to a wild-type virus genome (e.g., a wild-type Anellovirus genome as described herein). In some embodiments, enclosed within the proteinaceous exterior includes 100% coverage by the proteinaceous exterior, as well as less than 100% coverage, e.g., 95%, 90%, 85%, 80%, 70%, 60%, 50% or less coverage. For example, so long as the genetic elements are retained within the proteinaceous exterior, e.g., prior to entry into the host cell, gaps or discontinuities (e.g., that render the proteinaceous exterior permeable to water, ions, peptides, or small molecules) may be present in the proteinaceous exterior. In some embodiments, the anaerovector is purified, e.g., it is separated from the original source and / or is substantially free (>50%, >60%, >70%, >80%, >90%) of other constituents.

[0276] An anaerovector, in some embodiments, comprises a nucleic acid vector that comprises a nucleic acid sequence derived from or highly similar to (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to) an Anellovirus genomic sequence, or a contiguous portion thereof, sufficient to permit packaging into a proteinaceous outer portion (e.g., capsid), and further comprises a heterologous sequence. In some embodiments, an anaerovector is a viral vector or a naked nucleic acid. In some embodiments, the anaerovector 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 anaerovector 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 anaerovector comprises genetic elements as described herein, e.g., a genetic element comprising a promoter, a sequence encoding a therapeutic effector, and a capsid binding sequence.

[0277] As used herein, the term "anelloVLP" refers to a vehicle (e.g., a virus-like particle) that comprises a proteinaceous outer portion and an effector (e.g., an exogenous effector). In some examples, an anaeroVLP does not comprise a substantial amount of nucleic acid. In some embodiments, the proteinaceous outer portion comprises an ORF1 molecule (e.g., anellovirus ORF1 protein), e.g., as described herein. In some embodiments, the proteinaceous outer portion comprises a plurality of ORF1 molecules (e.g., anellovirus ORF1 protein), e.g., at least about 40, 45, 50, 55, 60, 65, or 70 ORF1 molecules. In some embodiments, the effector is encapsulated in the proteinaceous outer portion. In some embodiments, the effector is on the surface of the proteinaceous outer portion (e.g., included in a surface portion as described herein). In some embodiments, the anaeroVLP 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 anaeroVLP does not comprise a polynucleotide comprising an anaerovirus 5'UTR or an anaerovirus origin of replication. In some embodiments, anelloVLPs do not include 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).

[0278] As used herein, the term "antibody molecule" refers to a protein, e.g., an immunoglobulin chain or fragment thereof, that comprises at least one immunoglobulin variable domain sequence. The term "antibody molecule" encompasses full-length antibodies and antibody fragments (e.g., scFv). In some embodiments, an antibody molecule is a multispecific antibody molecule, e.g., an antibody molecule comprises a plurality of immunoglobulin variable domain sequences, where a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In some embodiments, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody molecule is generally characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[0279] The term "disassembly", as used herein in relation to a particle, such as a virus-like particle (VLP), or a proteinaceous outer portion, refers to the dissociation of one or more components of the particle (e.g., capsid proteins, e.g., ORF1 molecules as described herein) from the remainder of the particle. In some examples, disassembly of a particle (e.g., a VLP) includes ORF1 molecules moving far enough apart that they no longer form a proteinaceous outer portion. In some examples, ORF1 molecules that are separated by the disassembly of the particle form capsomers (e.g., decameric capsomers), e.g., as described herein. In some embodiments, disassembly reduces the particle to individual monomers. In some embodiments, multimers, e.g., decamers, monomers, and / or pentamers, remain after disassembly. In some examples, disassembly includes denaturation of protein complexes of the particle (e.g., breaking of non-covalent bonds between ORF1 molecules in the proteinaceous outer portion). In some examples, disassembly is driven by a denaturing agent, as described herein.

[0280] The term "in vitro assembly," as used herein in reference to an anaerovector or anaeroVLP, refers to the formation of a proteinaceous outer part comprising an ORF1 molecule, where 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 examples, in vitro assembly of an anaerovector involves the encapsulation of a genetic element (e.g., as described herein) within the proteinaceous outer part outside a cell. In some examples, in vitro assembly of an anaeroVLP involves the association of an effector (e.g., an exogenous effector, e.g., as described herein) with the proteinaceous outer part (e.g., encapsulated within the proteinaceous outer part) outside a cell. In vitro assembly of the proteinaceous exterior portion may in some instances be performed under conditions suitable for multimerization of multiple ORF1 molecules (e.g., non-denaturing conditions), e.g., multimers of more than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ORF1 molecules are formed. In some instances, the in vitro assembly results in the formation of a proteinaceous exterior portion comprising at least about 20, 30, 40, 50, or 60 ORF1 molecules, or about 20-30, 30-40, 40-50, 50-60, or 60-70 ORF1 molecules). In some instances, the proteinaceous exterior portion is formed from ORF1 molecules produced in and subsequently purified from cells. In some instances, the in vitro assembly is performed in a solution that does not contain cells or components thereof. In other instances, the in vitro assembly is performed in a solution that includes cell debris (e.g., from lysed cells). In some examples, in vitro assembly is performed 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, "encoding" nucleic acid 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).

[0281] As used herein, an "exogenous" agent (e.g., effector, nucleic acid (e.g., RNA), gene, payload, protein) refers to an agent not contained in or encoded by a corresponding wild-type virus, e.g., an Anellovirus described herein. In some embodiments, an exogenous agent is not naturally occurring, such as a protein or nucleic acid that has a sequence that is altered (e.g., by insertion, deletion, or substitution) relative to a naturally occurring protein or nucleic acid. In some embodiments, an exogenous agent is not naturally occurring in a host cell. In some embodiments, an exogenous agent is naturally occurring in a host cell but is exogenous to the virus. In some embodiments, an exogenous agent is naturally occurring in a host cell but not at a desired level or at a desired time.

[0282] A "heterologous" substance or element (e.g., effector, nucleic acid sequence, amino acid sequence), as used herein with respect to another substance or element (e.g., effector, nucleic acid sequence, amino acid sequence), refers to a substance or element that is not naturally present together in, for example, a wild-type virus, e.g., 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 that is naturally present in an anellovirus). In some embodiments, a heterologous substance or element is exogenous to the anellovirus that is the substrate for other (remaining) elements of the anellovector.

[0283] As used herein, the term "genetic element" refers to a nucleic acid sequence, generally a nucleic acid sequence in an anaerovector. It is understood that a genetic element can be made as naked DNA and optionally further assembled into a proteinaceous exterior. It is also understood that an anaerovector can insert its genetic element into a cell, and thus a genetic element can be present in a cell and a proteinaceous exterior without necessarily entering the cell.

[0284] As used herein, the term "ORF1 molecule" refers to a polypeptide having an 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-A25), or a functional fragment thereof. An ORF1 molecule, in some cases, comprises 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 N22 domain (e.g., as described herein, e.g., an N22 domain from an anellovirus ORF1 protein described herein), and / or a fourth region comprising the structure or activity of an anellovirus C-terminal domain (CTD) (e.g., as described herein, e.g., a CTD from an anellovirus ORF1 protein described herein). In some cases, an ORF1 molecule comprises, in order from the N-terminus to the C-terminus, the first, second, third, and fourth regions. In some cases, the anaerovector comprises an ORF1 molecule comprising, in order from N-terminus to C-terminus, a first, second, third, and fourth region. The ORF1 molecule may, in some cases, comprise a polypeptide encoded by an anellovirus ORF1 nucleic acid (e.g., as listed in any one of Tables N1-N25). The ORF1 molecule may, in some cases, further comprise a heterologous sequence, e.g., a hypervariable region (HVR), e.g., an HVR from an anellovirus ORF1 protein, e.g., as described herein. "Anellovirus ORF1 protein," as used herein, refers to an ORF1 protein encoded by an anellovirus genome (e.g., a wild-type anellovirus genome, e.g., as described herein), e.g., an ORF1 protein having an amino acid sequence as listed in any one of Tables A1-A25, or as encoded by an ORF1 gene as listed in any one of Tables N1-N25.

[0285] The term "ORF1 domain" as used herein in reference to an ORF1 molecule refers to a portion of an ORF1 molecule having the structure or function of an anellovirus ORF1 protein. The ORF1 domain generally has the ability to form multimers with other copies of the ORF1 domain (e.g., in other ORF1 molecules) or with other ORF1 molecules, e.g., to form a proteinaceous outer portion (e.g., of an anellovector or anelloVLP as described herein). In some examples, an ORF1 molecule may include one or more additional domains other than the ORF1 domain (e.g., a domain that includes or is associated with a surface effector, e.g., as described herein). In some examples, the amino acid sequence of the ORF1 domain includes an insertion (e.g., an insertion that encodes a surface moiety or a domain that has the ability to bind to a surface moiety), for example, between the N-terminal and C-terminal ends of the ORF1 domain. In certain instances, the insertion does not substantially disrupt the structure and / or function of the ORF1 domain, such that, for example, the ORF1 domain remains capable of forming multimers with other ORF1 domains or ORF1 molecules. The location in the ORF1 domain sequence where the insertion is made is referred to herein as the "insertion point." The insertion can be made into the ORF1 domain by any gene 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 an ORF1 domain and a heterologous domain (e.g., a surface portion as described herein). In some embodiments, the ORF1 domain is connected to the surface portion by a polypeptide linker region.

[0286] As used herein, the term "ORF2 molecule" refers to a polypeptide having activity and / or structural characteristics of an anellovirus ORF2 protein (e.g., as described herein, e.g., an anellovirus ORF2 protein as listed in any one of Tables A1-A25), or a functional fragment thereof. "Anellovirus ORF2 protein" as used herein 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-A25, or as encoded by the ORF2 gene as listed in any one of Tables N1-N25.

[0287] As used herein, the term "particle" refers to a medium that includes a proteinaceous exterior that is less than 100 nm in diameter (e.g., about 20-25, 25-30, 30-35, or 35-40 nm). In some examples, the particle includes multiple ORF1 molecules. The proteinaceous exterior of the particle generally forms an inclusion that is capable of restricting or preventing the movement of certain molecules between the inside and outside of the proteinaceous exterior. In some embodiments, the proteinaceous exterior may have gaps or discontinuities (e.g., that render the proteinaceous exterior permeable to water, ions, peptides, or small molecules). In certain embodiments, the gaps or discontinuities are of a size (e.g., diameter) that is small enough that the proteinaceous exterior restricts or prevents the passage of one or more large macromolecules (e.g., peptides, polypeptides, polynucleotides, lipids, or polysaccharides) through the proteinaceous exterior.

[0288] As used herein, the term "proteinaceous exterior" refers to exterior components that are predominantly (e.g., >50%, >60%, >70%, >80%, >90%) protein.

[0289] As used herein, the term "regulatory nucleic acid" refers to a nucleic acid sequence that modulates the expression, e.g., transcription and / or translation, of a DNA sequence encoding an expression product. In embodiments, the expression product comprises an RNA or a protein.

[0290] As used herein, the term "regulatory sequence" refers to a nucleic acid sequence that modulates the transcription of a target gene product. In some embodiments, a regulatory sequence is a promoter or enhancer.

[0291] As used herein, the term "replication protein" refers to a protein, e.g., a viral protein, that is utilized during infection, viral genome replication / expression, viral protein synthesis, and / or assembly of viral components.

[0292] When viewed by electron microscopy, anaerovector or anaeroVLP particles typically adopt one of two conformations: a symmetrical morphology (e.g., as illustrated in FIG. 7A) and an asymmetrical or less symmetrical morphology (e.g., as illustrated in FIG. 7B). Thus, the term "symmetrical morphology," as used herein in connection with anaerovector or anaeroVLP particle morphology, refers to a particle having a predominantly symmetrical shape. A particle having a symmetrical morphology may, in some instances, be approximately circular. A particle having a symmetrical morphology may, in some instances, not be perfectly circular or spherical (e.g., may be ovoid). In some instances, 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 recesses from its surface).

[0293] As used herein, a "substantially non-pathogenic" organism, particle, or construct refers to an organism, particle (e.g., a virus or anellovector, as described herein, for example), or construct thereof, that does not induce or induce a detectable disease or pathology in a host organism, e.g., a mammal, e.g., a human. In some embodiments, administration of an anelovector to a subject may result in some reactions or side effects that are acceptable as part of standard treatment.

[0294] As used herein, "non-pathogenic" refers to an organism or component thereof that does not induce or induce detectable disease or pathology in a host organism, e.g., a mammal, e.g., a human.

[0295] As used herein, a "substantially non-integrated" genetic element refers to a genetic element within a virus or anellovector (e.g., those described herein) where less than about 0.01%, 0.05%, 0.1%, 0.5%, or 1% of the genetic elements that enter a host cell (e.g., a eukaryotic cell) or organism (e.g., a mammal, e.g., a human) are integrated into the genome. In some embodiments, the genetic element is not detectably integrated, for example, into the genome of the host cell. In some embodiments, integration of the genetic element into the genome can be detected using techniques described herein, e.g., nucleic acid sequencing, PCR detection, and / or nucleic acid hybridization.

[0296] As used herein, a "substantially non-immunogenic" organism, particle, or component refers to, for example, an organism, particle (e.g., a virus or anellovector, e.g., as described herein), or component thereof, that does not provoke or elicit an undesirable or off-target immune response in a host tissue or organism (e.g., a mammal, e.g., a human). In some embodiments, a substantially non-immunogenic organism, particle, or component does not elicit a detectable immune response. In some embodiments, a substantially non-immunogenic anaellovector does not elicit a detectable immune response against a protein comprising an amino acid sequence or encoded by a nucleic acid sequence set forth in any one of Tables N1-N25. In some embodiments, an immune response (e.g., an unwanted or non-targeted immune response) is determined by assaying the presence or level of a subject's antibodies (e.g., the presence or level of anti-anellovector antibodies, e.g., the presence or level of antibodies to an anaellovector described herein), for example, according to the anti-TTV antibody detection methods described in Tsuda et al. (1999; J. Virol. Methods 77:199-206; incorporated herein by reference) and / or the method of measuring anti-TTV IgG levels described in Kakkola et al. (2008; Virology 382:182-189; incorporated herein by reference). Antibodies to anelloviruses or anaellovectors based thereon can also be detected by methods in the art for detecting anti-viral antibodies, for example, the methods for detecting anti-AAV antibodies described in Calcedo et al. (2013; Front. Immunol. 4(341):1-7; incorporated herein by reference).

[0297] As used herein, a "subsequence" refers to a nucleic acid sequence or amino acid sequence contained within a larger nucleic acid sequence or amino acid sequence, respectively. In some cases, a subsequence may comprise a domain or functional fragment of the larger sequence. In some cases, a subsequence may comprise a fragment of the larger sequence that, when isolated from the larger sequence, can form a secondary and / or tertiary structure similar to the secondary and / or tertiary structure formed by the subsequence when present with the remainder of the larger sequence. In some cases, a subsequence can be replaced with another sequence (e.g., a subsequence that comprises a foreign or heterologous sequence relative to the remainder of the larger sequence, e.g., a corresponding subsequence from a different Anellovirus).

[0298] As used herein, the term "surface portion" refers to a portion that is at least partially exposed on the outer surface of the particle (e.g., exposed to the solution surrounding the particle). The surface portion is generally directly or indirectly associated with a component of the proteinaceous outer portion of the particle (e.g., an ORF1 molecule). In some cases, the surface portion is covalently associated with a component of the proteinaceous outer portion of the particle (e.g., an ORF1 molecule). In some cases, the surface portion is non-covalently associated with a component of the proteinaceous outer portion of the particle (e.g., an ORF1 molecule). In some cases, the surface portion is bound to a binding portion, which in turn is bound (e.g., covalently or non-covalently) to a component of the proteinaceous outer portion of the particle (e.g., an ORF1 molecule). In some cases, the surface portion is included in the ORF1 molecule (e.g., is a heterologous domain of the ORF1 molecule). In some examples, the surface moiety is exogenous to the anellovirus (e.g., the anellovirus from which the ORF1 molecule is derived and / or the anellovirus whose ORF1 protein has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the ORF1 molecule). In some examples, the surface moiety is exogenous to a target cell (e.g., a mammalian cell, e.g., a human cell) that is to be infected with the particle.

[0299] As used herein, "treatment," "treating," and 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. This term includes active treatment (treatment directed at ameliorating the disease, pathological condition, or disorder), causal treatment (treatment directed at the cause of the associated disease, pathological condition, or disorder), palliative treatment (treatment designed to relieve symptoms), preventative treatment (treatment directed at preventing, minimizing, or partially or completely preventing the occurrence of the associated disease, pathological condition, or disorder); and supportive treatment (treatment used to complement another treatment).

[0300] As used herein, the term "virome" refers to a virus in a particular environment, e.g., in a part of the body, e.g., in an organism, e.g., in a cell, e.g., in a tissue.

[0301] The present invention generally relates to anaerovectors, e.g., synthetic anaerovectors, and uses thereof. The present disclosure provides anaerovectors, compositions comprising anaerovectors, and methods of making or using anaerovectors. An anaerovectors are generally useful as delivery vehicles, e.g., for delivering therapeutic agents to eukaryotic cells. In general, an anaerovector may contain a genetic element that includes a nucleic acid sequence (e.g., encoding an effector, e.g., an exogenous effector or an endogenous effector) encapsulated in a proteinaceous envelope. An anaerovector may include one or more deletions of sequences (e.g., regions or domains described herein) compared to an anaerovirus sequence (e.g., as described herein). An anaerovector can be used as a substantially non-immunogenic vehicle for delivering a genetic element, or an effector encoded therein (e.g., a polypeptide or nucleic acid effector, e.g., as described herein), to a eukaryotic cell, e.g., for the purpose of treating a disease or disorder in a subject, including the cell.

[0302] table of contents I. Anellovectors and AnelloVLPs A. Anellovirus B.ORF1 molecule C.ORF2 molecule D. genetic elements E. Protein Binding Sequences F.5'UTR region G.GC-rich region H. Effector I. Proteinaceous outer part J. Surface part i. Click chemistry (a) Exemplary Click Chemistry (b) Mutation of surface lysines (c) Mutation of surface cysteines ii. Gene grafting iii.X-fold rotational symmetry II. Compositions and Methods for the Generation of Anaerovectors and AnaeroVLPs A. Components and Assembly of Anellovectors and AnelloVLPs i. ORF1 molecule for assembly of anaerovectors and anaeroVLPs ii. ORF2 molecules for the assembly of anaerovectors and anaeroVLPs iii. Preparation of protein components (a) Baculovirus expression system (b) Insect cell system (c) Mammalian cell line B. Genetic Element Constructs i. Plasmids ii. Circular Nucleic Acid Constructs iii. In vitro cyclization iv. Tandem constructs v. Cis / Trans Constructs vi. Expression Cassette vii. Design and construction of genetic element constructs C. Effector D. host cell i. Introduction of genetic elements into a host cell ii. Methods in which one or more proteins are provided in cis or trans iii. Exemplary Cell Types E.Culture conditions F. Recovery G. In Vitro Assembly Methods for Anellovectors In vitro assembly of H. anelloviral VLPs I. Concentration and Purification III. Vector IV. Composition V. Host cells VI.How to use VII. Preparation method VIII. Administration / Delivery

[0303] I. Anellovectors and AnelloVLPs In some aspects, the invention described herein includes compositions and methods for the use and production of anaerovectors, anaerovector preparations, anaeroVLPs, anaeroVLP preparations, and therapeutic compositions.

[0304] Anello Vector In some embodiments, the anaerovector 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 set forth in any one of Tables A1-A25 or N1-N25), or a fragment or portion thereof, or another substantially non-pathogenic virus, e.g., a mutualistic virus, commensal virus, naturally occurring virus. In some embodiments, an Anellovirus-based anaerovector comprises at least one element exogenous to the Anellovirus, e.g., an exogenous effector or a nucleic acid sequence encoding an exogenous effector that is located within a genetic element of the anaerovector. In some embodiments, an Anellovirus-based anaerovector comprises at least one element heterologous to another element derived from the Anellovirus, e.g., an effector-encoding nucleic acid sequence, e.g., a promoter element, that is heterologous to another linked nucleic acid sequence. In some embodiments, an anaerovector comprises a genetic element (e.g., a circular DNA, e.g., a single-stranded DNA) that comprises at least one element heterologous to the remainder of the genetic element and / or a proteinaceous exterior (e.g., an exogenous element encoding an effector, e.g., as described herein). An anaerovector may be a delivery vehicle (e.g., a substantially non-pathogenic delivery vehicle) of a payload to a host, e.g., a human. In some embodiments, an anaerovector is capable of replicating in a eukaryotic cell, e.g., a mammalian cell, e.g., a human cell. In some embodiments, an anaerovector is substantially non-pathogenic and / or substantially non-integrative in mammalian (e.g., human) cells. In some embodiments, the anaerovector is substantially non-immunogenic in a mammal, e.g., a human. In some embodiments, the anaerovector is replication-deficient. In some embodiments, the anaerovector is replication-competent.

[0305] In some embodiments, the anaerovector comprises a clone, or a component thereof (e.g., a genetic element including, e.g., sequences encoding effectors and / or proteinaceous exterior) as described in PCT Application No. PCT / US2018 / 037379, which is incorporated by reference in its entirety.

[0306] In one aspect, the invention includes: (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 payload), and a protein binding sequence (e.g., an exogenous protein binding sequence, e.g., a packaging signal), wherein the genetic element is single stranded DNA and further has one or both of the following properties: it is circular and / or it integrates into the genome of a eukaryotic 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; and (ii) an anaerovector comprising a proteinaceous exterior, wherein the genetic element is enclosed within the proteinaceous exterior; and the anaerovector is capable of delivering the genetic element into a eukaryotic cell.

[0307] In some embodiments of the anaerovectors described herein, the genetic elements are integrated 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 a 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 from a plurality of anaerovectors administered to a subject are integrated into the genome of one or more host cells of the subject. In some embodiments, for example, as described herein, the genetic elements of a population of anaerovectors are integrated into the genome of a host cell at a frequency lower than that of a comparable population of AAV viruses, e.g., about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more lower than that of a comparable population of AAV viruses.

[0308] In one aspect, the invention provides a genetic element comprising: (i) a promoter element, a sequence encoding an effector (e.g., an endogenous effector or an exogenous effector, e.g., a payload), and a protein binding sequence (e.g., an exogenous protein binding sequence), wherein the genetic element is a wild-type Anellovirus sequence (e.g., wild-type Torque Teno virus (TTV), Torque Teno minivirus (TTV), or a wild-type Torque Teno minivirus (TTV)). a genetic element 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 (TTMV) or TTMDV sequence, e.g., a wild-type Anellovirus sequence as listed in any one of Tables N1 through N25; and (ii) an anaellovector comprising a proteinaceous exterior, where the genetic element is enclosed within the proteinaceous exterior; and the anaellovector is capable of delivering the genetic element into a eukaryotic cell.

[0309] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: a) a genetic element comprising: (i) a sequence encoding a non-pathogenic foreign protein; (ii) an foreign protein binding sequence that binds the genetic element to the non-pathogenic foreign protein; and (iii) a sequence encoding an effector (e.g., an endogenous or exogenous effector); and b) a proteinaceous exterior that is associated with, e.g., encases or confines, the genetic element; The anellovector comprises:

[0310] In some embodiments, the anellovector comprises sequences or expression products derived from (or with >70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% homology to) non-enveloped, circular, single-stranded DNA viruses. 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 are distinguishable from ssDNA viruses that infect prokaryotes (i.e., Microviridae and Inoviridae) and ssDNA viruses that infect plants (i.e., Geminiviridae and Nanoviridae). They are also distinguishable from linear ssDNA viruses that infect non-plant eukaryotic cells (i.e., Parvoviridae).

[0311] In some embodiments, the anellovector modulates a host cell function, e.g., transiently or long term. In some embodiments, the cell function is stably altered, e.g., modulation lasts 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 more, or any time in between. In some embodiments, the function of the cell is transiently altered, for example, modulation lasts for about 30 minutes to about 7 days or less, or for 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 less, or any time in between.

[0312] In some embodiments, the genetic element comprises a promoter element. In some embodiments, the promoter element is selected from an RNA polymerase II-dependent promoter, an RNA polymerase III-dependent promoter, a PGK promoter, a CMV promoter, an EF-1α promoter, an SV40 promoter, a CAGG promoter, or a UBC promoter, a TTV viral promoter, a tissue-specific, U6 (pollIII), minimal CMV promoter with an upstream DNA binding site for an activator protein (such as 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, for example, a wild-type Anellovirus described herein.

[0313] In some embodiments, the genetic element comprises one or more of the following characteristics: single stranded, circular, negative stranded, and / or DNA. In some embodiments, the genetic element comprises an episome. In some embodiments, the portion of the genetic element excluding the effector has a total size of about 2.5-5 kb (e.g. about 2.8-4 kb, about 2.8-3.2 kb, about 3.6-3.9 kb, or about 2.8-2.9 kb), less than about 5 kb (e.g. less than about 2.9 kb, 3.2 kb, 3.6 kb, 3.9 kb, or 4 kb), or at least 100 nucleotides (e.g. at least 1 kb).

[0314] The anaerovectors, compositions comprising anaerovectors, methods of using such anaerovectors, etc. described herein are based in part on examples that describe how, in some cases, different effectors, e.g., miRNAs (e.g., against IFN or miR-625), shRNAs, etc., can be combined with protein binding sequences, e.g., DNA sequences that bind to capsid proteins, such as Q99153, to produce anaerovectors (which can then be used to deliver the effectors to cells (e.g., animal cells, e.g., human cells or non-human cells, such as porcine or mouse cells). In embodiments, the effectors can suppress expression of factors such as interferons. The examples also describe how anaerovectors can be produced, e.g., by inserting effectors into sequences derived from an Anellovirus. Based on these examples, the following description considers various variations of the specific findings and combinations discussed in the examples. For example, one skilled in the art will understand from the examples that the specific miRNA is used as just one example of an effector, and that other effectors may be, for example, other regulatory nucleic acids or therapeutic peptides. Similarly, the specific capsid used in this example may be replaced by a substantially non-pathogenic protein as described later in this specification. Also, the specific Anellovirus sequence described in this example may be replaced by an Anellovirus sequence as described later in this specification. These considerations apply equally to protein binding sequences, regulatory sequences such as promoters, and the like. Independently of them, one skilled in the art will particularly consider such embodiments as are closely related to this example.

[0315] In some embodiments, the anaerovector, or a genetic element contained therein, is introduced into a cell (e.g., a human cell). In some embodiments, for example, once the anaerovector or genetic element is introduced into a cell, an effector (e.g., an RNA, e.g., miRNA) encoded by the genetic element of the anaerovector is expressed in the cell (e.g., a human cell). In some embodiments, introduction of the anaerovector, or a genetic element contained therein, into a cell modulates (e.g., increases or decreases) the level of a target molecule (e.g., a target nucleic acid, e.g., an RNA, or a target polypeptide) in the cell, for example, by modifying the level of expression of the target molecule by the cell. In some embodiments, introduction of the anaerovector, or a genetic element contained therein, into a cell modulates (e.g., increases or decreases) the function of the cell. In some embodiments, introduction of the anaerovector, or a genetic element contained therein, into a cell modulates (e.g., increases or decreases) the viability of the cell. In some embodiments, introduction of the anellovector, or the genetic elements contained therein, into a cell reduces the viability of the cell (e.g., a cancer cell).

[0316] In some embodiments, the anaerovectors described herein (e.g., synthetic anaerovectors) induce an antibody positivity rate of less than 70% (e.g., an antibody positivity rate of less than about 60%, 50%, 40%, 30%, 20%, or 10%). In some embodiments, the antibody positivity rate is measured according to methods known in the art. In some embodiments, the antibody positivity rate is measured by detecting antibodies against an Anellovirus (e.g., as described herein) or an Anellovector 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 the method for determining anti-TTV IgG seropositivity described in Kakkola et al. (2008; Virology 382:182-189; incorporated herein by reference). Additionally, antibodies to anellovirus or anellovectors based thereon can also be detected by methods known in the art for detecting anti-viral antibodies, such as the methods for detecting anti-AAV antibodies described in Calcedo et al. (2013; Front. Immunol. 4(341):1-7; incorporated herein by reference).

[0317] In some embodiments, a replication-deficient, replication-impaired, or replication-deficient genetic element does not encode all of the machinery or components required for replication of the genetic element. In some embodiments, a replication-deficient genetic element does not encode a replication factor. In some embodiments, a replication-deficient genetic element does not encode one or more ORFs (e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, and / or ORF2t / 3, e.g., as described herein). In some embodiments, the machinery or components not encoded by the genetic element are provided in trans (e.g., encoded in a helper, e.g., a helper virus or helper plasmid, or in a nucleic acid contained in the host cell, e.g., integrated into the genome of the host cell), e.g., such that the genetic element can undergo replication in the presence of the machinery or components provided in trans.

[0318] In some embodiments, the packaging-deficient, packaging-defective, or packageable genetic element cannot be packaged into the proteinaceous exoskeleton (e.g., where the proteinaceous exoskeleton comprises a capsid or a portion thereof that comprises, e.g., a polypeptide encoded by an ORF1 nucleic acid described herein). In some embodiments, the packaging-deficient genetic element is packaged into the proteinaceous exoskeleton with less than 10% (e.g., less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, or 0.001%) efficiency compared to a wild-type Anellovirus (e.g., as described herein). In some embodiments, the packaging-defective genetic element cannot be packaged into a proteinaceous exosomal region 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 the genetic element of a wild-type Anellovirus (e.g., as described herein). In some embodiments, the packaging-defective genetic element is packaged into the proteinaceous exosporium with an efficiency of less than 10% (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 enable packaging of the genetic element of a wild-type Anellovirus (e.g., as described herein).

[0319] In some embodiments, the packagable genetic element may be packaged into a proteinaceous exterior (e.g., where the proteinaceous exterior comprises a capsid or a portion thereof that includes, e.g., a polypeptide encoded by an ORF1 nucleic acid described herein). In some embodiments, the packagable genetic element is packaged into a proteinaceous exterior with an efficiency of 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 packagable genetic elements may be packaged into a proteinaceous exterior in the presence of factors (e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, or ORF2t / 3) that allow packaging of the genetic elements of a wild-type Anellovirus (e.g., as described herein). In some embodiments, the packagable genetic element is packaged into the proteinaceous exosomal with an efficiency of 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 enable packaging of the genetic element of a wild-type Anellovirus (e.g., as described herein).

[0320] Anello VLP In some embodiments, the AnelloVLP 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 set forth in any one of Tables A1-A25), or a fragment or portion thereof, or another substantially non-pathogenic virus, e.g., a mutualistic virus, a commensal virus, a naturally occurring virus. In some embodiments, an Anellovirus-based AnelloVLP comprises at least one element exogenous to the Anellovirus, e.g., an exogenous effector or a nucleic acid sequence encoding an exogenous effector. In some embodiments, an AnelloVLP comprises a surface portion that comprises an exogenous effector. In some embodiments, the Anellovirus-based AnelloVLP comprises at least one element heterologous to another element from the Anellovirus, e.g., an effector-encoding nucleic acid sequence heterologous to another linked nucleic acid sequence, such as a promoter element. The AnelloVLP may be a delivery vehicle (e.g., a substantially non-pathogenic delivery vehicle) of a payload to a host, e.g., a human. In some embodiments, the AnelloVLP is incapable of replicating in a eukaryotic cell, e.g., a mammalian cell, e.g., a human cell. In some embodiments, the AnelloVLP is substantially non-pathogenic in mammalian (e.g., human) cells and / or substantially non-integrating. In some embodiments, the AnelloVLP is substantially non-immunogenic in a mammal, e.g., a human.

[0321] In some aspects, the invention includes an anaeroVLP comprising a proteinaceous outer portion and an effector (e.g., an exogenous effector); wherein the anaeroVLP is capable of delivering the exogenous effector to a eukaryotic cell. In some embodiments, the exogenous effector is encapsulated within the proteinaceous outer portion. In some embodiments, the exogenous effector is included in a surface portion that is on the surface of the anaeroVLP (e.g., as described herein). In some embodiments, the proteinaceous outer portion comprises one or more ORF1 molecules (e.g., an anaerovirus 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).

[0322] In some embodiments, anelloVLPs comprise sequences or expression products from (or with greater than 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% homology to) non-enveloped circular single-stranded DNA viruses. Animal circular single-stranded DNA viruses generally refer to a group of single-stranded DNA (ssDNA) viruses with circular genomes that infect eukaryotic hosts other than plants. Thus, animal circular ssDNA viruses are distinguishable from ssDNA viruses that infect prokaryotes (i.e., Microviridae and Inoviridae) and ssDNA viruses that infect plants (i.e., Geminiviridae and Nanoviridae). They are also distinguishable from linear ssDNA viruses that infect eukaryotes other than plants (i.e., Parvoviridiae).

[0323] In some embodiments, the anelloVLPs modulate a host cell function, e.g., transiently or long term. In certain embodiments, the cell function is stably altered, such as modulation lasting 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 more, or any time in between. In certain embodiments, the cellular function is altered transiently, e.g., by modulation that does not persist for more than about 30 minutes to about 7 days, or does not persist for 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 in between.

[0324] The anaeroVLPs, compositions comprising anaeroVLPs, methods of using such anaeroVLPs, etc. as described herein are in part based on examples illustrating how different effectors, such as miRNAs (e.g., anti-IFN or miR-625), shRNAs, etc., and protein binding sequences, such as DNA sequences that bind capsid proteins, such as Q99153, can be combined with proteinaceous outer parts, such as capsids disclosed in Arch Virol (2007) 152:1961-1975, to create anaeroVLPs that can then be used to deliver effectors to cells (e.g., animal cells, such as human cells or non-human animal cells, such as porcine or mouse cells). In embodiments, the effectors can silence expression of factors such as interferons. These examples further describe how anaeroVLPs can be created, for example, by insertion of effectors into sequences derived from the Anellovirus genus. It is on the basis of these examples that the following description contemplates various variations of the specific findings and combinations discussed in these examples. For example, the skilled artisan will understand from these examples that the specific miRNA is used only as an example of an effector, and that other effectors may be, for example, other regulatory nucleic acids or therapeutic peptides. Similarly, the specific capsids used in these examples may be replaced with the substantially non-pathogenic proteins described below. The specific Anellovirus sequences described in these examples may also be replaced with the Anellovirus sequences described below. These considerations apply similarly to protein binding sequences, regulatory sequences such as promoters, etc. Apart from that, the skilled artisan will consider in detail such embodiments closely related to these examples.

[0325] In some embodiments, the anelloVLP is introduced into a cell (e.g., a human cell). In some embodiments, an exogenous effector is delivered to a cell. In some embodiments, delivery of an exogenous effector to a cell modulates (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, delivery of an exogenous effector to a cell modulates (e.g., increases or decreases) a function of the cell. In some embodiments, delivery of an exogenous effector to a cell modulates (e.g., increases or decreases) the viability of the cell. In some embodiments, delivery of an exogenous effector to a cell decreases the viability of the cell (e.g., a cancer cell).

[0326] In some embodiments, the anelloVLPs described herein (e.g., synthetic anelloVLPs) induce an antibody prevalence of less than 70% (e.g., an antibody prevalence of less than about 60%, 50%, 40%, 30%, 20%, or 10%). In some embodiments, the antibody prevalence is determined according to methods known in the art. In some embodiments, the antibody prevalence is determined by detecting antibodies against an Anellovirus (e.g., as described herein) or an anelloVLP 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 the anti-TTV IgG seroprevalence determination method described in Kakkola et al. (2008; Virology 382:182-189; incorporated herein by reference). Antibodies against Anellovirus or AnelloVLPs based thereon can also be detected by methods in the art for detecting anti-viral antibodies, for example, methods for detecting anti-AAV antibodies as described, for example, in Calcedo et al. (2013; Front. Immunol. 4(341):1-7; incorporated herein by reference).

[0327] Anellovirus In some embodiments, an anaerovector or an anaeroVLP, for example as described herein, comprises a sequence or expression product derived from an Anellovirus. In some embodiments, an anaerovector or an anaeroVLP comprises one or more sequences or expression products exogenous to an Anellovirus. In some embodiments, an anaerovector or an anaeroVLP comprises one or more sequences or expression products endogenous to an Anellovirus. In some embodiments, an anaerovector or an anaeroVLP comprises one or more sequences or expression products that are heterologous to one or more other sequences or expression products in the anaerovector. Anelloviruses generally have single-stranded circular DNA genomes with negative polarity. Anelloviruses have not been associated with human disease. However, attempts to link Anellovirus infection to human disease have been hampered by the high incidence of asymptomatic Anellovirus viremia in control cohort populations, the remarkable genomic diversity of the Anellovirus virus family, the inability to previously grow the virus in vitro, and the lack of animal models of Anellovirus disease (Yzebe et al., Panminerva Med. (2002) 44:167-177; Biagini, P., Vet. Microbiol. (2004) 98:95-101).

[0328] Anelloviruses are generally thought to be transmitted by oronasal or fecal-oral, maternal-fetal and / or uterine infections (Gerner et al., Ped. Infect. Dis. J. (2000) 19:1074-1077). Infected individuals may have a prolonged (months to years) course of Anellovirus viremia. Humans may be co-infected with two or more genogroups or lineages (Saback, et al., Scad. J. Infect. Dis. (2001) 33:121-125). It has been suggested that these genogroups may recombine in infected humans (Rey et al., Infect. (2003) 31:226-233). Double-stranded isoform (replicative) intermediates have been found in several tissues, including 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-Ilnigo et al., Am. J. Pathol. (2000) 156:1227-1234).

[0329] In some embodiments, an anaerovector or anaeroVLP 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 an Anellovirus sequence, e.g., as described herein, or a fragment thereof. In embodiments, the polypeptide comprises an amino acid sequence encoded by a nucleic acid sequence selected from a sequence as set forth in any one of Tables N1-N25, 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 set forth in any one of Tables A1-A25, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0330] In some embodiments the genetic element comprises a nucleic acid sequence encoding an amino acid sequence or a functional fragment thereof, or a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the amino acid sequences described herein, for example an Anellovirus amino acid sequence.

[0331] In some embodiments, the anaerovectors described herein comprise one or more nucleic acid molecules (e.g., genetic elements described herein) that comprise a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to, for example, an Anellovirus sequence described herein, or a fragment thereof. In embodiments, the anaerovector comprises a nucleic acid sequence selected from a sequence as set forth in any one of Tables N1-N25, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In embodiments, the anaerovector comprises a polypeptide comprising a sequence as set forth in any one of Tables A1-A25, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0332] In some embodiments, the anaerovectors described herein comprise one or more nucleic acid molecules (e.g., genetic elements described herein) that comprise a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more TATA boxes, initiation elements, cap sites, transcription start sites, 5'UTR conserved domains, ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, 3 open reading frame regions, poly(A) signals, GC-rich regions, or any combination thereof, of any of the Anelloviruses described herein (e.g., an Anellovirus sequence annotated in or encoded by a sequence listed in any one of Tables N1-N25). 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, ORF2t / 3 sequence of any of the Anelloviruses described herein (e.g., an Anellovirus sequence annotated in or encoded by a sequence listed in any one of Tables N1-N25). 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 one of Tables N1-N25, or an ORF1 or ORF2 amino acid sequence encoded by a nucleic acid sequence set forth in any one of Tables N1-N25).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 protein (e.g., an ORF1 amino acid sequence set forth in any one of Tables N1-N25, or an ORF1 amino acid sequence encoded by a nucleic acid sequence set forth in any one of Tables N1-N25).

[0333] Nucleic acid sequence 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 through N25. 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 through N25. 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 through N25. 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 through N25. 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 through N25.

[0334] Amino acid sequences encoded by nucleic acid sequences 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 an Anellovirus ORF1 amino acid sequence of any one of Tables A1-A25. 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 an Anellovirus ORF2 amino acid sequence of any one of Tables A1-A25. 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 an Anellovirus ORF3 amino acid sequence of any one of Tables A1 through A25.

[0335] Proteins containing amino acid sequences In embodiments, the anaerovectors 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 an Anellovirus ORF1 amino acid sequence of any one of Tables A1-A25. In embodiments, the anaerovectors 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 an Anellovirus ORF2 amino acid sequence of any one of Tables A1-A25. In embodiments, an anaerovector described herein comprises 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 an Anellovirus ORF3 amino acid sequence of any one of Tables A1-A25. In some embodiments, an ORF1 molecule (e.g., contained in an anaerovector) comprises an anellovirus ORF1 protein of any one of Tables A1-A25, or a splice variant or post-translationally processed (e.g., proteolytically processed) variant thereof. In some embodiments, an ORF2 molecule (e.g., contained in an anaerovector) comprises an anellovirus ORF2 protein of any one of Tables A1-A25, or a splice variant or post-translationally processed (e.g., proteolytically processed) variant thereof. In some embodiments, the ORF3 molecule (e.g., contained in an anellovector) comprises an anellovirus ORF3 protein of any one of Tables A1-A25, or a splice variant or post-translationally processed (e.g., proteolytically processed) variant thereof.

[0336] Polypeptides containing amino acid sequences In some embodiments, a polypeptide described herein comprises 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, a polypeptide described herein comprises 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-A25.

[0337] 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 as listed in any one of Tables A1-A25.

[0338] In some embodiments, a polypeptide described herein comprises 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, a polypeptide described herein comprises 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-A25.

[0339] 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 as listed in any one of Tables A1-A25.

[0340] In some embodiments, a polypeptide described herein comprises 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, a polypeptide described herein comprises 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-A25.

[0341] 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 as listed in any one of Tables A1-A25.

[0342] In some embodiments, the polypeptide comprises an amino acid sequence as set forth in any one of Tables A1-A25 (e.g., an 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.

[0343] [Table 1]

[0344] [Table 2]

[0345] [Table 3]

[0346] [Table 4]

[0347] [Table 5]

[0348]

Table 6

[0349]

Table 7

[0350]

Table 8

[0351]

Table 9

[0352]

Table 10

[0353]

Table 11

[0354]

Table 12

[0355]

Table 13

[0356]

Table 14

[0357]

Table 15

[0358]

Table 16

[0359]

Table 17

[0360]

Table 18

[0361]

Table 19

[0362]

Table 20

[0363]

Table 21

[0364]

Table 22

[0365]

Table 23

[0366]

Table 24

[0367]

Table 25

[0368]

Table 26

[0369]

Table 27

[0370]

Table 28

[0371]

Table 29

[0372]

Table 30

[0373]

Table 31

[0374]

Table 32

[0375]

Table 33

[0376]

Table 34

[0377]

Table 35

[0378]

Table 36

[0379]

Table 37

[0380]

Table 38

[0381]

Table 39

[0382]

Table 40

[0383]

Table 41

[0384]

Table 42

[0385]

Table 43

[0386]

Table 44

[0387]

Table 45

[0388]

Table 46

[0389]

Table 47

[0390]

Table 48

[0391]

Table 49

[0392]

Table 50

[0393]

Table 51

[0394]

Table 52

[0395]

Table 53

[0396]

Table 54

[0397]

Table 55

[0398]

Table 56

[0399]

Table 57

[0400]

Table 58

[0401]

Table 59

[0402]

Table 60

[0403]

Table 61

[0404]

Table 62

[0405]

Table 63

[0406]

Table 64

[0407]

Table 65

[0408]

Table 66

[0409]

Table 67

[0410]

Table 68

[0411]

Table 69

[0412]

Table 70

[0413]

Table 71

[0414]

Table 72

[0415]

Table 73

[0416]

Table 74

[0417]

Table 75

[0418]

Table 76

[0419]

Table 77

[0420]

Table 78

[0421]

Table 79

[0422]

Table 80

[0423]

Table 81

[0424]

Table 82

[0425]

Table 83

[0426]

Table 84

[0427]

Table 85

[0428]

Table 86

[0429]

Table 87

[0430]

Table 88

[0431]

Table 89

[0432]

Table 90

[0433]

Table 91

[0434]

Table 92

[0435]

Table 93

[0436]

Table 94

[0437]

Table 95

[0438]

Table 96

[0439]

Table 97

[0440]

Table 98

[0441]

Table 99

[0442]

Table 100

[0443]

Table 101

[0444]

Table 102

[0445]

Table 103

[0446]

Table 104

[0447]

Table 105

[0448]

Table 106

[0449]

Table 107

[0450]

Table 108

[0451]

Table 109

[0452]

Table 110

[0453]

Table 111

[0454]

Table 112

[0455]

Table 113

[0456]

Table 114

[0457]

Table 115

[0458]

Table 116

[0459]

Table 117

[0460]

Table 118

[0461]

Table 119

[0462]

Table 120

[0463]

Table 121

[0464]

Table 122

[0465]

Table 123

[0466]

Table 124

[0467]

Table 125

[0468]

Table 126

[0469]

Table 127

[0470]

Table 128

[0471]

Table 129

[0472] In some embodiments, an anaerovector or anaeroVLP as described herein is a chimeric anaerovector or anaeroVLP. In some embodiments, a chimeric anaerovector or anaeroVLP further comprises one or more elements, polypeptides, or nucleic acids from a virus other than an Anellovirus.

[0473] In some embodiments, a chimeric anaerovector or anaeroVLP comprises multiple polypeptides (e.g., anaerovirus ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, and / or ORF2t / 3) that comprise sequences from multiple different anaeroviruses (e.g., as described herein).

[0474] In some embodiments, the anaellovector or anaelloVLP comprises, for example, a chimeric polypeptide comprising at least one portion from an anaellovirus (e.g., as described herein) and at least one portion from a different virus (e.g., as described herein) (e.g., anellovirus ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, and / or ORF2t / 3).

[0475] In some embodiments, the anaellovector or anaelloVLP comprises, for example, a chimeric polypeptide comprising at least one portion from one anaellovirus (e.g., as described herein) and at least one portion from a different anaellovirus (e.g., as described herein) (e.g., anaellovirus ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, and / or ORF2t / 3). In some embodiments, an anaerovector or anaeroVLP comprises an ORF1 molecule comprising at least a portion of an ORF1 molecule from one anaerovirus (e.g., as described herein), or an ORF1 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, and at least a portion of an ORF1 molecule from a different anaerovirus (e.g., as described herein), or an ORF1 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto. In some embodiments, a chimeric ORF1 molecule comprises an ORF1 jellyroll domain from one anellovirus, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and an ORF1 amino acid subsequence (e.g., as described herein) from a different anellovirus, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, a chimeric ORF1 molecule comprises an ORF1 arginine-rich region from one anellovirus, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and an ORF1 amino acid subsequence (e.g., as described herein) from a different anellovirus, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.In some embodiments, a chimeric ORF1 molecule comprises an ORF1 hypervariable domain from one anellovirus, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and an ORF1 amino acid subsequence (e.g., as described herein) from a different anellovirus, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, a chimeric ORF1 molecule comprises an ORF1 N22 domain from one anellovirus, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and an ORF1 amino acid subsequence (e.g., as described herein) from a different anellovirus, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, a chimeric ORF1 molecule comprises an ORF1 C-terminal domain from one anellovirus, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and an ORF1 amino acid subsequence (e.g., as described herein) from a different anellovirus, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0476] In some embodiments, the anaerovector or anaeroVLP comprises a chimeric ORF1 / 1 molecule comprising an ORF1 / 1 molecule from one anaerovirus (e.g., as described herein), or at least a portion of an ORF1 / 1 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, and an ORF1 / 1 molecule from a different anaerovirus (e.g., as described herein), or at least a portion of an ORF1 / 1 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto. In some embodiments, the anaerovector or anaeroVLP comprises an ORF1 / 2 molecule from one anaerovirus (e.g., as described herein), or at least a portion of an ORF1 / 2 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, and a chimeric ORF1 / 2 molecule from a different anaerovirus (e.g., as described herein), or at least a portion of an ORF1 / 2 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto. In some embodiments, an anaerovector or anaeroVLP comprises an ORF2 molecule from one anaerovirus (e.g., as described herein), or at least a portion of an ORF2 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, and a chimeric ORF2 molecule comprising an ORF2 molecule from a different anaerovirus (e.g., as described herein), or at least a portion of an ORF2 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.In some embodiments, an anaerovector or anaeroVLP comprises an ORF2 / 2 molecule comprising at least a portion of an ORF2 / 2 molecule from one anaerovirus (e.g., as described herein), or an ORF2 / 2 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, and at least a portion of an ORF2 / 2 molecule from a different anaerovirus (e.g., as described herein), or an ORF2 / 2 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto. In some embodiments, an anaerovector or anaeroVLP comprises a chimeric ORF2 / 3 molecule comprising an ORF2 / 3 molecule from one anaerovirus (e.g., as described herein), or at least a portion of an ORF2 / 3 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, and an ORF2 / 3 molecule from a different anaerovirus (e.g., as described herein), or at least a portion of an ORF2 / 3 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto. In some embodiments, the anaerovector or anaeroVLP comprises a chimeric ORF2T / 3 molecule comprising an ORF2T / 3 molecule from one anaerovirus (e.g., as described herein), or at least a portion of an ORF2T / 3 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, and an ORF2T / 3 molecule from a different anaerovirus (e.g., as described herein), or at least a portion of an ORF2T / 3 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto.

[0477] In some embodiments, the anaerovector comprises a nucleic acid comprising a sequence listed in PCT / US2018 / 037379, which is incorporated by reference in its entirety. In some embodiments, the anaerovector or anaeroVLP comprises a polypeptide comprising a sequence listed in PCT / US2018 / 037379, which is incorporated by reference in its entirety.

[0478] In some embodiments, the anaerovector comprises an Anellovirus genome, for example, as identified by the methods described in Example 9 of WO 2020 / 123816, which is incorporated by reference in its entirety. In some embodiments, the anaerovector or anaeroVLP comprises an Anellovirus sequence, or a portion thereof, as described in Example 30.

[0479] In some embodiments, an anaerovector comprises a genetic element comprising a consensus Anellovirus motif, e.g., as shown in Table 19. In some embodiments, an anaerovector comprises a genetic element comprising a consensus Anellovirus ORF1 motif, e.g., as shown in Table 19. In some embodiments, an anaerovector comprises a genetic element comprising a consensus Anellovirus ORF1 / 1 motif, e.g., as shown in Table 19. In some embodiments, an anaerovector comprises a genetic element comprising a consensus Anellovirus ORF1 / 2 motif, e.g., as shown in Table 19. In some embodiments, an anaerovector comprises a genetic element comprising a consensus Anellovirus ORF2 / 2 motif, e.g., as shown in Table 19. In some embodiments, an anaerovector comprises a genetic element comprising a consensus Anellovirus ORF2 / 3 motif, e.g., as shown in Table 19. In some embodiments, the anaerovector comprises a genetic element comprising a consensus Anellovirus ORF2t / 3 motif, e.g., as shown in Table 19. In some embodiments, X designates any amino acid as shown in Table 19. In some embodiments, Z designates glutamic acid or glutamine as shown in Table 19. In some embodiments, B designates aspartic acid or asparagine as shown in Table 19. In some embodiments, J designates leucine or isoleucine as shown in Table 19.

[0480] [Table 130]

[0481] [Table 131]

[0482] ORF1 molecule In some embodiments, an anaerovector or anaeroVLP comprises an ORF1 molecule and / or a nucleic acid encoding an ORF1 molecule. Generally, the ORF1 molecule comprises a polypeptide having structural features and / or activity of an anellovirus ORF1 protein (e.g., an anellovirus ORF1 protein as described herein, e.g., as listed in any one of Tables A1-A25), or a functional fragment thereof. In some embodiments, the ORF1 molecule comprises a truncation relative to an anellovirus ORF1 protein (e.g., an anellovirus ORF1 protein as described herein, e.g., as listed in any one of Tables A1-A25). In some embodiments, the ORF1 molecule is truncated by at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 amino acids from anellovirus ORF1 protein. In some embodiments, the ORF1 molecule comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to anellovirus ORF1 protein sequence as set forth in any one of Tables A1-A25. In some embodiments, the ORF1 molecule comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a BetaTorque virus ORF1 protein, e.g., as described herein. The ORF1 molecule is generally capable of binding to a nucleic acid molecule, such as DNA (e.g., a genetic element, e.g., as described herein). In some embodiments, the ORF1 molecule is localized to the nucleus of the cell. In certain embodiments, the ORF1 molecule is localized to the nucleolus of the cell.

[0483] In some embodiments, an ORF1 molecule as described herein comprises an amino acid sequence (e.g., an ORF1 sequence, or an arginine-rich region, a jelly-roll domain, an HVR, an N22, or a C-terminal domain sequence) as set forth in any of Table A2, Table A4, Table A6, Table A8, Table A10, Table A12, Tables C1-C5, Table 2, Table 4, Table 6, Table 8, Table 10, Table 12, Table 14, Table 16, Table 18, Table 20-37, or Tables D1-D10 of WO 2020 / 123816 (herein incorporated by reference in its entirety), or a sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% nucleotide sequence identity thereto.

[0484] Without wishing to be bound by theory, an ORF1 molecule may have the ability to bind to other ORF1 molecules to form, for example, a proteinaceous outer part (e.g., as described herein). Such an ORF1 molecule may be described as having the ability to form a capsid. In some embodiments, the proteinaceous outer part may encapsidate a nucleic acid molecule (e.g., a genetic element as described herein). In some embodiments, multiple ORF1 molecules may form a multimer, for example, to create a proteinaceous outer part. In some embodiments, the multimer may be a homomultimer. In other embodiments, the multimer may be a heteromultimer (e.g., comprising multiple different ORF1 molecules). It is also contemplated that an ORF1 molecule may have replicase activity.

[0485] An ORF1 molecule, in some embodiments, may include one or more of the following: a first region including an arginine-rich region, e.g., a region having at least 60% basic residues (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% basic residues; e.g., 60%-90%, 60%-80%, 70%-90%, or 70-80% basic residues), and a second region including a jelly-roll domain, e.g., at least six beta strands (e.g., 4, 5, 6, 7, 8, 9, 10, 11, or 12 beta strands).

[0486] Arginine-rich region The arginine-rich region has 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 or a sequence of at least about 40 amino acids that contains at least 60%, 70%, or 80% basic residues (e.g., arginine, lysine, or a combination thereof).

[0487] Jellyroll Domain Jellyroll domains or regions have the following characteristics: (i) at least 30% (e.g., at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or more) of the amino acids of the jelly-roll domain are part of one or more β-sheets; (ii) the secondary structure of the jellyroll domain comprises at least four (e.g., 4, 5, 6, 7, 8, 9, 10, 11, or 12) β-strands; and / or (iii) the tertiary structure of the jelly-roll domain comprises at least two (e.g., two, three, or four) β-sheets; and / or (iv) the jelly-roll domain comprises a ratio of β-sheets:α-helices of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. The term "antibody" refers to a polypeptide (e.g., a domain or region contained in a larger polypeptide) that comprises one or more (e.g., 1, 2, or 3) of:

[0488] In certain embodiments, the jellyroll domain comprises two beta sheets.

[0489] In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) beta sheets comprise about 8 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, or 12) beta strands. In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) beta sheets comprise 8 beta strands. In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) beta sheets comprise 7 beta strands. In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) beta sheets comprise 6 beta strands. In certain embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) beta sheets comprise 5 beta strands. In certain embodiments, one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) beta sheets comprise four beta strands.

[0490] In some embodiments, the jellyroll domain comprises a first β-sheet in an anti-parallel orientation relative to the second β-sheet. In certain embodiments, the first β-sheet comprises about four (e.g., 3, 4, 5, or 6) β-strands. In certain embodiments, the second β-sheet comprises about four (e.g., 3, 4, 5, or 6) β-strands. In some embodiments, the first and second β-sheets comprise, in total, about eight (e.g., 6, 7, 8, 9, 10, 11, or 12) β-strands.

[0491] In certain embodiments, the jelly-roll domain is a component of a capsid protein (e.g., an ORF1 molecule described herein). In certain embodiments, the jelly-roll domain has self-assembly activity. In some embodiments, a polypeptide comprising a jelly-roll domain binds to another copy of a polypeptide comprising a jelly-roll domain. In some embodiments, the jelly-roll domain of a first polypeptide binds to the jelly-roll domain of a second copy of the polypeptide.

[0492] The ORF1 molecule may also include a third region that includes the structure or activity of an Anellovirus N22 domain (e.g., as described herein, e.g., an N22 domain from an Anellovirus ORF1 protein described herein), and / or a fourth region that includes the structure or activity of an Anellovirus C-terminal domain (CTD) (e.g., as described herein, e.g., a CTD from an Anellovirus ORF1 protein described herein). In some embodiments, the ORF1 molecule includes, in order from the N-terminus to the C-terminus, the first region, the second region, the third region, and the fourth region.

[0493] The ORF1 molecule, in some embodiments, further comprises a hypervariable region (HVR), e.g., an HVR from, e.g., an Anellovirus ORF1 protein, e.g., as described herein. In some embodiments, the HVR is located between the second and third regions. In some embodiments, the HVR comprises at least about 55 (e.g., at least about 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 65) amino acids (e.g., about 45-160, 50-160, 55-160, 60-160, 45-150, 50-150, 55-150, 60-150, 45-140, 50-140, 55-140, or 60-140 amino acids).

[0494] In some embodiments, the first region is capable of binding to a nucleic acid molecule (e.g., DNA). In some embodiments, the basic residue is selected from arginine, histidine, or lysine, or a combination thereof. In some embodiments, the first region comprises at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% arginine residues (e.g., 60%-90%, 60%-80%, 70%-90%, or 70-80% arginine residues). In some embodiments, the first region comprises about 30-120 amino acids (e.g., about 40-120, 40-100, 40-90, 40-80, 40-70, 50-100, 50-90, 50-80, 50-70, 60-100, or 60-80 amino acids). In some embodiments, the first region comprises a structure or activity of a viral ORF1 arginine-rich region (e.g., an arginine-rich region from an anellovirus ORF1 protein, e.g., as described herein). In some embodiments, the first region comprises a nuclear localization signal.

[0495] In some embodiments, the second region comprises the structure or activity of a jelly-roll domain, e.g., a viral ORF1 jelly-roll domain (e.g., a jelly-roll domain from anellovirus ORF1 protein, e.g., as described herein). In some embodiments, the second region can combine with a second region of another ORF1 molecule to form a proteinaceous exterior (e.g., capsid) or portion thereof.

[0496] In some embodiments, the fourth region is exposed at the surface of a proteinaceous exterior (eg, a proteinaceous exterior that comprises a multimer of ORF1 molecules, as described herein).

[0497] In some embodiments, the first region, the second region, the third region, the fourth region, and / or the HVR each comprise three or fewer (eg, 0, 1, 2, or 3) beta sheets.

[0498] In some embodiments, one or more of the first region, second region, third region, fourth region, and / or HVR may be replaced with a heterologous amino acid sequence (e.g., the corresponding region from a heterologous ORF1 molecule). In some embodiments, the heterologous amino acid sequence comprises a desired functional fragment, e.g., as described herein.

[0499] In some embodiments, the ORF1 molecule comprises multiple conserved motifs (e.g., motifs comprising about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 amino acids or more) (e.g., as shown in Figure 34 of WO2020 / 123816). In some embodiments, the conserved motifs may exhibit 60, 70, 80, 85, 90, 95, or 100% sequence identity to the ORF1 protein of one or more wild-type anellovirus clades (e.g., Betatorquevirus). In some embodiments, the conserved motifs may each have a length of 1 to 1000 amino acids (e.g., 5 to 10, 5 to 15, 5 to 20, 10 to 15, 10 to 20, 15 to 20, 5 to 50, 5 to 100, 10 to 50, 10 to 100, 10 to 1000, 50 to 1000, or 100 to 1000 amino acids). In certain embodiments, the conserved motifs are comprised of about 2 to 4% (e.g., about 1 to 8%, 1 to 6%, 1 to 5%, 1 to 4%, 2 to 8%, 2 to 6%, 2 to 5%, or 2 to 4%) of the sequence of the ORF1 molecule, each of which exhibits 100% sequence identity to the corresponding motif in the ORF1 protein of a wild-type Anellovirus clade. In certain embodiments, the conserved motif comprises about 5-10% (e.g., about 1-20%, 1-10%, 5-20%, or 5-10%) of the sequence of the ORF1 molecule, each exhibiting 80% sequence identity to a corresponding motif in the ORF1 protein of a wild-type Anellovirus clade. In certain embodiments, the conserved motif comprises about 10-50% (e.g., about 10-20%, 10-30%, 10-40%, 10-50%, 20-40%, 20-50%, or 30-50%) of the sequence of the ORF1 molecule, each exhibiting 60% sequence identity to a corresponding motif in the ORF1 protein of a wild-type Anellovirus clade. In some embodiments, the conserved motif comprises one or more amino acid sequences listed in Table 19.

[0500] In some embodiments, the ORF1 molecule includes at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) compared to a wild-type ORF1 protein described herein (e.g., as shown in any one of Tables N1 to N25).

[0501] A conserved ORF1 motif within the N22 domain In some embodiments, a polypeptide described herein (e.g., an ORF1 molecule) has the amino acid sequence YNPX 2 DXGX 2 N (SEQ ID NO: 829) (X n is any consecutive sequence of n amino acids. For example, X n represents any two consecutive amino acid sequences. 2 DXGX 2 N (SEQ ID NO: 829) is, for example, contained within the N22 domain of the ORF1 molecule described herein. In some embodiments, the genetic element described herein has the amino acid sequence YNPX 2 DXGX 2 N (SEQ ID NO: 829) (X n is a contiguous sequence of any n amino acids (e.g., a nucleic acid sequence encoding, for example, an ORF1 molecule described herein).

[0502] In some embodiments, a polypeptide (e.g., an ORF1 molecule) can contain, e.g., a nucleotide sequence similar to, e.g., YNPX, e.g., in the N22 domain. 2 DXGX 2 In some embodiments, the conserved secondary structure comprises a first β strand and / or a second β strand. In some embodiments, the first β strand is about 5-6 (e.g., 3, 4, 5, 6, 7, or 8) amino acids in length. In some embodiments, the first β strand comprises a conserved secondary structure comprising a first β strand and / or a second β strand. In some embodiments, the first β strand is about 5-6 (e.g., 3, 4, 5, 6, 7, or 8) amino acids in length. In some embodiments, the first β strand comprises a conserved secondary structure comprising a first β strand and / or a second β strand. 2 DXGX 2 The N-terminus of the N (SEQ ID NO: 829) motif contains a tyrosine (Y) residue.2 DXGX 2 The N (SEQ ID NO: 829) motif comprises a random coil (e.g., about 8-9 amino acids of a random coil). In some embodiments, the second β strand is about 7-8 (e.g., 5, 6, 7, 8, 9, or 10) amino acids in length. In some embodiments, the second β strand is YNPX 2 DXGX 2 The N (SEQ ID NO: 829) motif contains an asparagine (N) residue at its C-terminus.

[0503] Example YNPX 2 DXGX 2 The N (SEQ ID NO: 829) motif adjacent secondary structure is described in Example 47 and Figure 48 of WO 2020 / 123816 (incorporated herein by reference in its entirety). In some embodiments, the ORF1 molecule comprises a region that includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the secondary structure elements (e.g., β-strands) shown in Figure 48 of WO 2020 / 123816. In some embodiments, the ORF1 molecule comprises a region that includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the secondary structure elements (e.g., β-strands) shown in Figure 48 of WO 2020 / 123816 (incorporated herein by reference in its entirety) adjacent to YNPX. 2 DXGX 2 N (SEQ ID NO: 829) motif (e.g., as described herein).

[0504] Conserved secondary structure within the ORF1 jelly-roll domain In some embodiments, a polypeptide described herein (e.g., an ORF1 molecule) comprises one or more secondary structure elements contained in an Anellovirus ORF1 protein (e.g., as described herein). In some embodiments, an ORF1 molecule comprises one or more secondary structure elements contained in a jellyroll domain of an Anellovirus ORF1 protein (e.g., as described herein). In general, the ORF1 jellyroll domain comprises a secondary structure comprising, in N-terminal to C-terminal order, a first β strand, a second β strand, a first α helix, a third β strand, a fourth β strand, a fifth β strand, a second α helix, a sixth β strand, a seventh β strand, an eighth β strand, and a ninth β strand. In some embodiments, the ORF1 molecule comprises a secondary structure comprising, in N-terminal to C-terminal order, a first beta strand, a second beta strand, a first alpha helix, a third beta strand, a fourth beta strand, a fifth beta strand, a second alpha helix, a sixth beta strand, a seventh beta strand, an eighth beta strand, and / or a ninth beta strand.

[0505] In some embodiments, a pair of conserved secondary structure elements (i.e., β-strands and / or α-helices) are separated by an intervening amino acid sequence, including, for example, a random coil sequence, a β-strand, or an α-helix, or a combination thereof. The intervening amino acid sequence between the conserved secondary structure elements may include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. In some embodiments, the ORF1 molecule may further include one or more additional β-strands and / or α-helices (e.g., within a jelly-roll domain). In some embodiments, consecutive β-strands or consecutive α-helices may be combined. In some embodiments, the first and second β-strands are included in a larger β-strand. In some embodiments, the third and fourth β-strands are included in a larger β-strand. In some embodiments, the 4th and 5th beta strands are comprised in a larger beta chain. In some embodiments, the 6th and 7th beta strands are comprised in a larger beta chain. In some embodiments, the 7th and 8th beta strands are comprised in a larger beta chain. In some embodiments, the 8th and 9th beta strands are comprised in a larger beta chain.

[0506] In some embodiments, the first β strand is about 5-7 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) amino acids in length. In some embodiments, the second β strand is about 15-16 (e.g., 13, 14, 15, 16, 17, 18, or 19) amino acids in length. In some embodiments, the first α helix is ​​about 15-17 (e.g., 13, 14, 15, 16, 17, 18, 19, or 20) amino acids in length. In some embodiments, the third β strand is about 3-4 (e.g., 1, 2, 3, 4, 5, or 6) amino acids in length. In some embodiments, the fourth β strand is about 10-11 (e.g., 8, 9, 10, 11, 12, or 13) amino acids in length. In some embodiments, the fifth β strand is about 6-7 (e.g., 4, 5, 6, 7, 8, 9, or 10) amino acids in length. In some embodiments, the second α-helix is ​​about 8-14 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) amino acids long. In some embodiments, the second α-helix may be truncated into two smaller α-helices (e.g., separated by a random coil sequence). In some embodiments, each of the two smaller α-helices is about 4-6 (e.g., 2, 3, 4, 5, 6, 7, or 8) amino acids long. In some embodiments, the sixth β-strand is about 4-5 (e.g., 2, 3, 4, 5, 6, or 7) amino acids long. In some embodiments, the seventh β-strand is about 5-6 (e.g., 3, 4, 5, 6, 7, 8, or 9) amino acids long. In some embodiments, the eighth β-strand is about 7-9 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acids long. In some embodiments, the ninth beta strand is about 5 to 7 (eg, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids in length.

[0507] Exemplary jelly-roll domain secondary structures are described in Example 47 and Figure 47 of WO 2020 / 123816 (incorporated herein by reference in its entirety). In some embodiments, the ORF1 molecule comprises a region that includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all) of the secondary structure elements (e.g., β-strands and / or α-helices) of any of the jelly-roll domain secondary structures shown in Figure 47 of WO 2020 / 123816 (incorporated herein by reference in its entirety).

[0508] Exemplary ORF1 Sequence In some embodiments, a polypeptide (e.g., an ORF1 molecule) described herein comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more Anellovirus ORF1 partial sequences (e.g., as described herein). In some embodiments, an anaerovector or anaeroVLP described herein comprises an ORF1 molecule that comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more Anellovirus ORF1 partial sequences (e.g., as described herein). In some embodiments, an anaerovector or anaeroVLP described herein comprises a nucleic acid molecule (e.g., a genetic element) encoding an ORF1 molecule comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more Anelloviridae ORF1 subsequences, e.g., as described herein.

[0509] In some embodiments, one or more Anellovir ORF1 subsequences include one or more of an Arg-rich domain, a jelly-roll domain, a hypervariable region (HVR), an N22 domain, or a C-terminal domain (CTD) (e.g., as listed herein), or a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, an ORF1 molecule includes multiple subsequences from different Anelloviruses. In some embodiments, an ORF1 molecule includes one or more of an Arg-rich domain, a jelly-roll domain, an N22 domain, and a CTD from one Anellovirus and an HVR from another. In some embodiments, the ORF1 molecule comprises one or more of a jelly-roll domain, HVR, N22 domain, and CTD from one Anellovirus and an Arg-rich domain from another. In some embodiments, the ORF1 molecule comprises one or more of an Arg-rich domain, HVR, N22 domain, and CTD from one Anellovirus and a jelly-roll domain from another. In some embodiments, the ORF1 molecule comprises one or more of an Arg-rich domain, jelly-roll domain, HVR, and CTD from one Anellovirus and an N22 domain from another. In some embodiments, the ORF1 molecule comprises one or more of an Arg-rich domain, jelly-roll domain, HVR, and CTD from one Anellovirus and a CTD from another.

[0510] In some embodiments, the one or more anellovirus ORF1 subsequences include one or more of an arginine (Arg)-rich domain, a jelly-roll domain, a hypervariable region (HVR), an N22 domain, or a C-terminal domain (CTD) as described in WO 2020 / 123816, which is incorporated by reference in its entirety.

[0511] In some embodiments, a polypeptide (e.g., an ORF1 molecule) described herein comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more Anelloviru ORF1 partial sequences (e.g., as set forth in any of Tables 20-37, or Tables D1-D11). In some embodiments, an anellosome described herein comprises an ORF1 molecule comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more Anelloviru ORF1 partial sequences (e.g., as set forth in any of Tables 20-37, or Tables D1-D11). In some embodiments, the anellosomes described herein comprise a nucleic acid molecule (e.g., a genetic element) encoding an ORF1 molecule comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more Anelloviruru ORF1 partial sequences, e.g., as set forth in any of Tables 20-37, or Tables D1-D11.

[0512] In some embodiments, one or more Anelloviru ORF1 subsequences comprise one or more of an arginine (Arg)-rich domain, a jelly-roll domain, a hypervariable region (HVR), an N22 domain, or a C-terminal domain (CTD) (e.g., as listed in any of Tables 20-37, or Tables D1-D11), or a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the ORF1 molecule comprises multiple subsequences from different Anellovirus genera (e.g., any combination of ORF1 subsequences selected from the Alphatorquevirus clades 1-7 subsequences listed in Tables 20-37, or Tables D1-D11). In an embodiment, the ORF1 molecule comprises one or more of an Arg-rich domain, a jelly-roll domain, an N22 domain, and a CTD from one Anellovirus and an HVR from another. In an embodiment, the ORF1 molecule comprises one or more of a jelly-roll domain, an HVR, an N22 domain, and a CTD from one Anellovirus and an Arg-rich domain from another. In an embodiment, the ORF1 molecule comprises one or more of an Arg-rich domain, an HVR, an N22 domain, and a CTD from one Anellovirus and a jelly-roll domain from another. In an embodiment, the ORF1 molecule comprises one or more of an Arg-rich domain, a jelly-roll domain, an HVR, and a CTD from one Anellovirus and an N22 domain from another. In an embodiment, the ORF1 molecule comprises one or more of the Arg-rich domain, the jelly-roll domain, the HVR, and the N22 domain from one Anellovirus and the CTD from another.

[0513] [Table 132]

[0514]

Table 133

[0515]

Table 134

[0516]

Table 135

[0517]

Table 136

[0518]

Table 137

[0519]

Table 138

[0520]

Table 139

[0521]

Table 140

[0522]

Table 141

[0523]

Table 142

[0524]

Table 143

[0525]

Table 144

[0526]

Table 145

[0527]

Table 146

[0528]

Table 147

[0529]

Table 148

[0530]

Table 149

[0531]

Table 150

[0532]

Table 151

[0533]

Table 152

[0534]

Table 153

[0535] [Table 154]

[0536] [Table 155]

[0537] [Table 156]

[0538] [Table 157]

[0539] [Table 158]

[0540] [Table 159]

[0541] [Table 160]

[0542] Consensus ORF1 domain sequence In some embodiments, for example, an ORF1 molecule described herein comprises one or more of a jelly-roll domain, an N22 domain, and / or a C-terminal domain (CTD). In some embodiments, the jelly-roll domain comprises an amino acid sequence having a jelly-roll domain consensus sequence described herein (e.g., listed in any of Tables 37A-37C). In some embodiments, the N22 domain comprises an amino acid sequence having an N22 domain consensus sequence described herein (e.g., listed in any of Tables 37A-37C). In some embodiments, the CTD domain comprises an amino acid sequence having a CTD domain consensus sequence described herein (e.g., listed in any of Tables 37A-37C). In some embodiments, the ORF1 molecule described herein comprises one or more of a jelly-roll domain, an N22 domain, and / or a C-terminal domain (CTD ... a-b Amino acids listed under "a" include a contiguous series of amino acids, where the series includes at least a and at most b amino acids. In certain embodiments, the amino acids in the series are all the same. In other embodiments, the series includes at least 2 (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21) different amino acids.

[0543] [Table 161]

[0544] [Table 162]

[0545] [Table 163]

[0546] In some embodiments, the jellyroll domain comprises a jellyroll domain amino acid sequence as listed in any of Tables 37A-C, or an amino acid sequence having at least 70%, 75%, 80%, 8%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the N22 domain comprises a N22 domain amino acid sequence as listed in any of Tables 37A-C, or an amino acid sequence having at least 70%, 75%, 80%, 8%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In some embodiments, the CTD domain comprises a CTD domain amino acid sequence as listed in any of Tables 37A-37C, or an amino acid sequence having at least 70%, 75%, 80%, 8%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0547] Exemplary ORF1 mutant constructs The following table provides exemplary anellovirus ORF1 mutant and variant sequences.

[0548] [Table 164]

[0549] Identification of ORF1 protein sequence In some embodiments, an Anellovirus ORF1 protein sequence, or a nucleic acid sequence encoding an ORF1 protein, can be identified from an Anellovirus genome (e.g., a putative Anellovirus genome identified by a nucleic acid sequencing technique, e.g., a deep sequencing technique). In some embodiments, the ORF1 protein sequence is identified by one or more (e.g., one, two, or all three) of the following selection criteria:

[0550] (i) Length Selection: Protein sequences (e.g., putative Anellovirus ORF1 sequences passing the criteria set forth in (ii) or (iii) below) can be size-selected for greater than about 600 amino acid residues to identify putative Anellovirus ORF1 proteins. In some embodiments, Anellovirus ORF1 protein sequences are at least about 600, 650, 700, 750, 800, 850, 900, 950, or 1000 amino acid residues in length. In some embodiments, Alphatorquevirus ORF1 protein sequences are at least about 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 900, or 1000 amino acid residues in length. In some embodiments, the Betatorquevirus ORF1 protein sequence is at least about 650, 660, 670, 680, 690, 700, 750, 800, 900, or 1000 amino acid residues in length. In some embodiments, the Gammatorquevirus ORF1 protein sequence is at least about 650, 660, 670, 680, 690, 700, 750, 800, 900, or 1000 amino acid residues in length. In some embodiments, the nucleic acid sequence encoding the Anellovirus ORF1 protein is at least about 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 nucleotides in length. In some embodiments, the nucleic acid sequence encoding the Alphatorquevirus ORF1 protein sequence is at least about 2100, 2150, 2200, 2250, 2300, 2400, or 2500 nucleotides in length. In some embodiments, the nucleic acid sequence encoding the Betatorquevirus ORF1 protein sequence is at least about 1900, 1950, 2000, 2500, 2100, 2150, 2200, 2250, 2300, 2400, or 2500 or 1000 nucleotides in length.In some embodiments, the nucleic acid sequence encoding the Gammatorquevirus ORF1 protein sequence is at least about 1900, 1950, 2000, 2500, 2100, 2150, 2200, 2250, 2300, 2400, or 2500 or 1000 nucleotides in length.

[0551] (ii) Presence of an ORF1 motif: Protein sequences (e.g., putative Anellovirus ORF1 sequences that pass the criteria set forth in (i) above or (iii) below) can be filtered to identify those that contain the conserved ORF1 motif in the N22 domain described above. In some embodiments, a putative Anellovirus ORF1 sequence comprises the sequence YNPXXDXGXXN. In some embodiments, a putative Anellovirus ORF1 sequence comprises the sequence Y[NCS]PXXDX[GASKR]XX[NTSVAK].

[0552] (iii) Presence of Arginine-Rich Regions: Protein sequences (e.g., putative Anellovirus ORF1 sequences that pass the criteria set forth in (i) and / or (ii) above) can be filtered for those that contain arginine-rich regions (e.g., as described herein). In some embodiments, the putative Anellovirus ORF1 sequence comprises a contiguous sequence of at least about 30, 35, 40, 45, 50, 55, 60, 65, or 70 amino acids that contain at least 30% (e.g., at least about 20%, 25%, 30%, 35%, 40%, 45%, or 50%) arginine residues. In some embodiments, the putative Anellovirus ORF1 sequence comprises a contiguous sequence of about 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, or 65-70 amino acids that contain at least 30% (e.g., at least about 20%, 25%, 30%, 35%, 40%, 45%, or 50%) arginine residues. In some embodiments, the arginine-rich region is located at least about 30, 40, 50, 60, 70, or 80 amino acids downstream of the start codon of the putative Anellovirus ORF1 protein. In some embodiments, the arginine-rich region is located at least about 50 amino acids downstream of the start codon of the putative Anellovirus ORF1 protein.

[0553] In some embodiments, the ORF1 protein is identified in an anellovirus genome sequence as described in Example 36 of WO 2020 / 123816 (herein incorporated by reference in its entirety).

[0554] ORF2 molecule In some embodiments, the anaerovector or anaeroVLP comprises an ORF2 molecule and / or a nucleic acid encoding an ORF2 molecule. Generally, the ORF2 molecule comprises a polypeptide having structural features and / or activity of an Anellovirus ORF2 protein (e.g., an Anellovirus ORF2 protein listed in any one of Tables A1-A25 described herein), or a functional fragment thereof. In some embodiments, the ORF2 molecule comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus ORF2 protein sequence set forth in any one of Tables A1-A25.

[0555] In some embodiments, the ORF2 molecule comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an Alphatorquevirus, Betatorquevirus, or Gammatorquevirus ORF2 protein, e.g., as described herein. In some embodiments, the ORF2 molecule (e.g., an ORF2 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an Alphatorquevirus ORF2 protein) has a length of 250 amino acids or less (e.g., about 150-200 amino acids). In some embodiments, the ORF2 molecule (e.g., an ORF2 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Betatorquevirus ORF2 protein) has a length of about 50-150 amino acids. In some embodiments, the ORF2 molecule (e.g., an ORF2 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Gammatorquevirus ORF2 protein) has a length of about 100-200 amino acids (e.g., about 100-150 amino acids). In some embodiments, the ORF2 molecule includes a helix-turn-helix motif (e.g., a helix-turn-helix motif including two alpha helices flanking a turn region). In some embodiments, the ORF2 molecule comprises the amino acid sequence of the ORF2 protein of TTV isolate TA278 or TTV isolate SANBAN. In some embodiments, the ORF2 molecule has protein phosphatase activity. In some embodiments, the ORF2 molecule comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) compared to a wild-type ORF2 protein described herein (e.g., as set forth in any one of Tables A1-A25).

[0556] Conserved ORF2 motif In some embodiments, the polypeptides described herein (e.g., ORF2 motifs) have the amino acid sequence [W / F]X 7 HX 3 CX 1 CX 5 H (SEQ ID NO: 949), where X n is a contiguous sequence of any n amino acids. 7 represents any consecutive sequence of 7 amino acids. 3 represents any consecutive sequence of 3 amino acids. 1 represents any single amino acid. 5 represents any sequence of 5 consecutive amino acids. In some embodiments, [W / F] can be either tryptophan or phenylalanine. In some embodiments, [W / F]X 7 HX 3 CX 1 CX 5 H (SEQ ID NO: 949), for example, is contained within the N22 domain of ORF2 described herein. In some embodiments, the genetic elements described herein have the amino acid sequence [W / F]X 7 HX 3 CX 1 CX 5 H(SEQ ID NO:949)(X n is a contiguous sequence of any n amino acids (eg, a nucleic acid sequence encoding an ORF2 molecule, as described herein).

[0557] Genetic elements In some embodiments, the anelovectin comprises a genetic element. In some embodiments, the genetic element has one or more of the following characteristics: does not substantially interact with the host cell genome, is an episomal nucleic acid, is single stranded DNA, is circular, is about 1-10 kb, is present in the nucleus of the cell, can be bound by endogenous proteins, produces an effector such as a polypeptide or nucleic acid (e.g., RNA, iRNA, microRNA) that targets a gene, activity, or function of the host or target cell. In one embodiment, the genetic element is substantially non-interacting DNA. In some embodiments, the genetic element comprises a packaging signal, e.g., a sequence that binds to a capsid protein. In some embodiments, outside the packaging or capsid binding sequences, the genetic elements have less than 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% sequence identity to a wild-type Anellovirus nucleic acid sequence, for example, less than 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% sequence identity to an Anellovirus nucleic acid sequence described herein. In some embodiments, outside the packaging or capsid binding sequences, the genetic elements have less than 500, 450, 400, 350, 300, 250, 200, 150, or 100 contiguous nucleotides that are at least 70%, 75%, 80%, 8%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an Anellovirus nucleic acid sequence. In a particular embodiment, the genetic element is a circular single-stranded DNA comprising a promoter sequence, a sequence encoding a therapeutic effector, and a capsid binding protein.

[0558] In some embodiments, the genetic element has at least about 70%, 75%, 80%, 8%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus nucleic acid sequence, e.g., as described herein (e.g., as described in any one of Tables N1-N25), or a fragment thereof, or encodes an amino acid sequence having at least about 70%, 75%, 80%, 8%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus amino acid sequence (e.g., as described in any one of Tables A1-A25), or a fragment thereof. In some embodiments, the genetic element comprises a sequence encoding an effector (e.g. an endogenous effector or an exogenous effector, e.g. a payload), such as a polypeptide effector (e.g. a protein) or a nucleic acid effector (e.g. a non-coding RNA, e.g. miRNA, siRNA, mRNA, lncRNA, RNA, DNA, antisense RNA, gRNA).

[0559] In some embodiments, the genetic elements have a length of less than 20 kb (e.g., less than about 19 kb, 18 kb, 17 kb, 16 kb, 15 kb, 14 kb, 13 kb, 12 kb, 11 kb, 10 kb, 9 kb, 8 kb, 7 kb, 6 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb). In some embodiments, the genetic elements are independently or in addition more than 1000 b (e.g., at least about 1.1 kb, 1.2 kb, 1.3 kb, 1.4 kb, 1.5 kb, 1.6 kb, 1.7 kb, 1.8 kb, 1.9 kb, 2 kb, 2.1 kb, 2.2 kb, 2.3 kb, 2.4 kb, 2.5 kb, 2.6 kb, 2.7 kb, 2.8 kb, 2.9 kb, 3.1 kb, 3.2 kb, 3.3 kb, 3.4 kb, 3.5 kb, 3.6 kb, 3.7 kb, 3.8 kb, 3 ...1 kb, 3.2 kb, 3 In some embodiments the genetic element has a length of about 2.5-4.6, 2.8-4.0, 3.0-3.8, or 3.2-3.7 kb. In some embodiments, the genetic element has a length of about 1.5-2.0, 1.5-2.5, 1.5-3.0, 1.5-3.5, 1.5-3.8, 1.5-3.9, 1.5-4.0, 1.5-4.5, or 1.5-5.0 kb. In some embodiments, the genetic element has a length of about 2.0-2.5, 2.0-3.0, 2.0-3.5, 2.0-3.8, 2.0-3.9, 2.0-4.0, 2.0-4.5, or 2.0-5.0 kb. In some embodiments, the genetic element has a length of about 2.5-3.0, 2.5-3.5, 2.5-3.8, 2.5-3.9, 2.5-4.0, 2.5-4.5, or 2.5-5.0 kb. In some embodiments, the genetic element has a length of about 3.0-5.0, 3.5-5.0, 4.0-5.0, or 4.5-5.0 kb. In some embodiments, the genetic element has a length of about 1.5-2.0, 2.0-2.5, 2.5-3.0, 3.0-3.5, 3.1-3.6, 3.2-3.7, 3.3-3.8, 3.4-3.9, 3.5-4.0, 4.0-4.5 kb, or 4.5-5.0 kb.

[0560] In some embodiments, the genetic element comprises one or more of the features described herein, e.g., a sequence encoding a substantially non-pathogenic protein, a protein binding sequence, one or more sequences encoding regulatory nucleic acids, one or more regulatory sequences, one or more sequences encoding replication proteins, and other sequences. In some embodiments, the substantially non-pathogenic protein comprises an amino acid sequence or a functional fragment thereof or a sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90% 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of the amino acid sequences described herein, e.g., an Anellovirus amino acid sequence as listed in any one of Tables A1-A25.

[0561] In some embodiments, the genetic element is produced from double stranded circular DNA (e.g., produced by in vitro circularization). In some embodiments, the genetic element is produced by rolling circle replication from double stranded circular DNA. In some embodiments, the rolling circle replication occurs in a cell (e.g., a host cell, e.g., a mammalian cell, e.g., a human cell, e.g., a HEK293T, A549 cell, or Jurkat cell). In some embodiments, the genetic element may be exponentially amplified by rolling circle replication in the cell. In some embodiments, the genetic element may be linearly amplified by rolling circle replication in the cell. In some embodiments, the double stranded circular DNA or genetic element may be produced at least 2, 4, 8, 16, 32, 64, 128, 256, 518, or 1024 times or more of the initial amount by rolling circle replication in the cell. In some embodiments, the double stranded circular DNA has been introduced into the cell, e.g., as described herein.

[0562] In some embodiments, the double stranded circular DNA and / or genetic elements do not include one or more bacterial plasmid elements (e.g., a bacterial origin of replication or a selection marker, e.g., a bacterial resistance gene). In some embodiments, the double stranded circular DNA and / or genetic elements do not include a bacterial plasmid backbone.

[0563] In some embodiments, the invention includes a genetic element comprising a nucleic acid sequence (e.g., a DNA sequence) encoding (i) a substantially non-pathogenic foreign protein, (ii) an foreign protein binding sequence that binds the genetic element to the substantially non-pathogenic foreign protein, and (iii) a regulatory nucleic acid. In such embodiments, the genetic element may comprise one or more sequences having at least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% nucleotide sequence identity to any one of the nucleotide sequences compared to a native viral sequence (e.g., a native Anellovirus sequence, e.g., as described herein).

[0564] In some embodiments, a genetic element as described herein comprises a sequence (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region sequence) as listed in any of Table A1, Table A3, Table A5, Table A7, Table A9, Table A11, Tables B1-B5, Table 1, Table 3, Table 5, Table 7, Table 9, Table 11, Table 13, Table 15, or Table 17 of WO 2020 / 123816 (herein incorporated by reference in its entirety), or a sequence having at least 70%, 80%, 85%, 90% 95%, 96%, 97%, 98% and 99% nucleotide sequence identity thereto.

[0565] In some embodiments, the genetic element comprises a sequence encoding an effector (e.g., an exogenous effector). In some embodiments, the effector coding sequence is inserted into an anellovirus genomic sequence (e.g., as described herein). In some embodiments, the effector coding sequence replaces contiguous sequence (e.g., at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 nucleotides, or more) from an anellovirus genomic sequence. In some embodiments, the effector coding sequence is a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region sequence, or a portion thereof (e.g., a portion consisting of at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 nucleotides, or more), as set forth in any of Table A1, Table A3, Table A5, Table A7, Table A9, Table A11, Tables B1-B5, Table 1, Table 3, Table 5, Table 7, Table 9, Table 11, Table 13, Table 15, or Table 17 of WO 2020 / 123816, which is incorporated by reference in its entirety, or at least 70% thereof. Replace sequences with 80%, 85%, 90% 95%, 96%, 97%, 98% and 99% nucleotide sequence identity.

[0566] In some embodiments, the sequence of a first nucleic acid element (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region) comprised in a genetic element overlaps with the sequence of a second nucleic acid element (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region) by, for example, at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, or 500 nucleotides. In some embodiments, the sequence of a first nucleic acid element (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region) comprised in a genetic element does not overlap with the sequence of a second nucleic acid element (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region).

[0567] Protein Binding Sequences The strategy used by many viruses is that the viral capsid protein recognizes a specific protein binding sequence in its genome. For example, in the case of viruses with a non-segmented genome, such as the yeast LA virus, there is a secondary structure (stem-loop) and a specific sequence at the 5' end of the genome, both of which are used to bind with the viral capsid protein. However, viruses with segmented genomes, such as Reoviridae, Orthomyxoviridae (influenza), Bunyaviruse and Arenaviruses, need to package each of the genome segments. Some viruses use the complementary regions of the segments to facilitate the virus containing one of each genome molecule. Other viruses have specific binding sites for each of the various segments. See, e.g., Curr Opin Struct Biol. 2010 Feb;20(1):114-120; and Journal of Virology (2003), 77(24), 13036-13041.

[0568] In some embodiments, the genetic element encodes a protein binding sequence that binds a substantially non-pathogenic protein. In some embodiments, the protein binding sequence facilitates packaging of the genetic element into a proteinaceous exterior. In some embodiments, the protein binding sequence specifically binds to an arginine-rich region of a substantially non-pathogenic protein. In some embodiments, the genetic element comprises a protein binding sequence as described in Example 8 of WO 2020 / 123816 (hereby incorporated by reference in its entirety). In some embodiments, the genetic element comprises a protein binding sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a 5'UTR conserved domain or a GC-rich domain of an Anellovirus sequence (e.g., as set forth in any one of Tables N1-N25).

[0569] In embodiments, the protein binding sequence has 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 through N25.

[0570] 5'UTR region In some embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a nucleic acid sequence shown in Table 38 and / or Figure 20 of WO 2020 / 123816. In some embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence of a consensus 5'UTR sequence shown in Table 38 (wherein 1 , X 2 , X 3 , X 4 , and X 5 are each independently any nucleotide, for example, 1 = G or T, X 2 = C or A, X 3 = G or A, X 4 = T or C and X 5=A, C, or T). In some embodiments, the genetic element (e.g., the protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the consensus 5'UTR sequences shown in Table 38. In some embodiments, the genetic element (e.g., the protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the exemplary TTV 5'UTR sequences shown in Table 38. In some embodiments, the genetic element (e.g., the protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the TTV-CT30F 5'UTR sequence shown in Table 38. In some embodiments, the genetic element (e.g., the protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the TTV-HD23a 5'UTR sequence shown in Table 38. In some embodiments, the genetic element (e.g., the protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the TTV-JA20 5'UTR sequence shown in Table 38. In some embodiments, the genetic element (e.g., the protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the TTV-TJN02 5'UTR sequence shown in Table 38.In some embodiments, the genetic element (e.g. the protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g. at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the TTV-tth8 5'UTR sequence shown in Table 38.

[0571] In some embodiments, the genetic element (e.g., the protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to an Alphatorque virus consensus 5'UTR sequence shown in Table 38. In some embodiments, the genetic element (e.g., the protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to an Alphatorque virus clade 1 5'UTR sequence shown in Table 38. In some embodiments, the genetic element (e.g., the protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to an Alphatorque virus clade 2 5'UTR sequence shown in Table 38. In some embodiments, the genetic element (e.g., the protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to an Alphatorque virus clade 3 5'UTR sequence shown in Table 38. In some embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to an Alphatorquevirus Clade 4 5'UTR sequence shown in Table 38. In some embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to an Alphatorquevirus Clade 5 5'UTR sequence shown in Table 38.In some embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to an Alphatorque virus clade 6 5'UTR sequence shown in Table 38. In some embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to an Alphatorque virus clade 7 5'UTR sequence shown in Table 38.

[0572] In some embodiments, the genetic element 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 to N25.

[0573] In some embodiments, the genetic element 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 to N25.

[0574] [Table 165]

[0575] [Table 166]

[0576] [Table 167]

[0577] Identification of 5'UTR sequences In some embodiments, an Anellovirus 5'UTR sequence may be identified within an Anellovirus genome (e.g., a putative Anellovirus genome identified by a nucleic acid sequencing technique, e.g., a deep sequencing technique). In some embodiments, an Anellovirus 5'UTR sequence is identified by one or both of the following steps:

[0578] (i) Identification of circularization junction: In some embodiments, the 5'UTR will be located near the circularization junction of the full-length circularized Anellovirus genome. The circularization junction can be identified, for example, by identifying overlapping regions of the sequence. In some embodiments, the overlapping regions of the sequence can be trimmed from the sequence to create a circularized full-length Anellovirus genome sequence. In some embodiments, the genome sequence is circularized in this manner using software. Without wishing to be bound by theory, computationally circularizing the genome can provide a starting position for the non-biologically oriented sequence. Landmarks in the sequence can be used to re-orient the sequence in the appropriate direction. For example, the landmark sequence can include a sequence having substantial homology to one or more elements within an Anellovirus genome described herein (e.g., one or more of the Anellovirus TATA box, cap site, initiator element, transcription start site, 5'UTR conserved domain, ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, 3 open reading frame regions, poly(A) signal, or GC-rich region, as described herein).

[0579] (ii) Identification of 5'UTR Sequences: Once a putative Anellovirus genomic sequence is obtained, the sequence (or a portion thereof, e.g., having a length of about 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 nucleotides) can be compared to one or more Anellovirus 5'UTR sequences (e.g., as described herein) to identify sequences having substantial homology thereto. In some embodiments, the putative Anellovirus 5'UTR region has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the Anellovirus 5'UTR sequences described herein.

[0580] GC-rich regions In some embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the nucleic acid sequences shown in Table 39 and / or Figures 20 and 32 of PCT Publication WO 2020 / 123816. In some embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the GC-rich sequences shown in Table 39.

[0581] In some embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a 36 nucleotide GC rich sequence set forth in Table 39 (e.g., a 36 nucleotide consensus GC rich region sequence 1, a 36 nucleotide consensus GC rich region sequence 2, a TTV clade 1 36 nucleotide region, a TTV clade 3 36 nucleotide region, a TTV clade 3 isolate GH1 36 nucleotide region, a TTV clade 3 sle1932 36 nucleotide region, a TTV clade 4 ctdc002 36 nucleotide region, a TTV clade 5 36 nucleotide region, a TTV clade 6 36 nucleotide region, or a TTV clade 7 36 nucleotide region). In some embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence that comprises at least 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, or 36 contiguous nucleotides of a 36 nucleotide GC rich sequence set forth in Table 39 (e.g., a 36 nucleotide consensus GC rich region sequence 1, a 36 nucleotide consensus GC rich region sequence 2, a TTV clade 1 36 nucleotide region, a TTV clade 3 36 nucleotide region, a TTV clade 3 isolate GH1 36 nucleotide region, a TTV clade 3 sle1932 36 nucleotide region, a TTV clade 4 ctdc002 36 nucleotide region, a TTV clade 5 36 nucleotide region, a TTV clade 6 36 nucleotide region, or a TTV clade 7 36 nucleotide region).

[0582] In some embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to an Alphatorquevirus GC rich region sequence selected from, e.g., TTV-CT30F, TTV-P13-1, TTV-tth8, TTV-HD20a, TTV-16, TTV-TJN02, or TTV-HD16d, e.g., as shown in Table 39. In some embodiments, the genetic element (e.g. the protein binding sequence of the genetic element) comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 104, 105, 108, 110, 111, 115, 120, 122, 130, 140, 145, 150, 155, or 156 contiguous nucleotides of an Alphatorquevirus GC rich region sequence selected from, e.g., TTV-CT30F, TTV-P13-1, TTV-tth8, TTV-HD20a, TTV-16, TTV-TJN02, or TTV-HD16d, e.g., as shown in Table 39.

[0583] In some embodiments, the 36 nucleotide GC rich region has the following structure: (i) CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC (SEQ ID NO: 160); (ii) GCGCTX 1 CGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 164) (X 1 is selected from T, G, or A. (iii) GCGCTCGCGCGCCGCCCACTAGGGGGCGTTGCGCCG (SEQ ID NO: 165); (iv) GCGCTGCGCCGCCGCCCAGTAGGGGGCGCAATGCG (SEQ ID NO: 166); (v) GCGCTGCGCCGCGGCCCCCGGGGGAGGCATTGCCT (SEQ ID NO: 167); (vi)GCGCTGCGCGCGCGCGCCGGGGGGGCGCCAGCGCCC (SEQ ID NO: 168); (vii) GCGCTTCGCGCGCGCGCCGGGGGGCTCCGCCCCCCC (SEQ ID NO: 169); (viii) GCGCTTCGCGCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 170); (ix) GCGCTACGCGCCGCGCGCCGGGGGGCTGCGCCCCCCC (SEQ ID NO: 171); or (x) GCGCTACGCGCCGCGCGCCGGGGGGCTCTGCCCCCCC (SEQ ID NO: 172); is selected from.

[0584] In some embodiments, the genetic element (eg the protein binding sequence of the genetic element) comprises the nucleic acid sequence CGCGCTGCGCGCGCCGCCCAGTAGGGGGAGCCATGC (SEQ ID NO: 160).

[0585] In some embodiments, the genetic element (e.g. the protein binding sequence of the genetic element) comprises a nucleic acid sequence of a consensus GC-rich sequence as shown in Table 39, wherein X 4 , X 5 , X 6 , X 7 , X 12 , X 13 , X 14 , X 15 , X 20 , X 21 , X 22 , X 26 , X 29、 X 30 , and X 33 are each independently any nucleotide, and X 2 , X 3 , X 8 , X 9 , X 10 , X 11 , X 16 , X 17 , X 18 , X 19 , X 23, X 24 , X 25 , X 27 , X 28 , X 31 , X 32 , and X 34 are each independently absent or any nucleotide. 1 ~X 34wherein one or more (e.g. all) are each independently a nucleotide (or absent) set forth in Table 39. In some embodiments, the genetic element (e.g. the protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g. at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the exemplary TTV GC rich sequences set forth in Table 39 (e.g. the entire sequence, fragment 1, fragment 2, fragment 3, or any combination thereof, e.g. fragments 1-3, in order). In some embodiments, the genetic element (e.g., the protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a TTV-CT30F GC-rich sequence shown in Table 39 (e.g., the entire sequence, fragment 1, fragment 2, fragment 3, fragment 4, fragment 5, fragment 6, fragment 7, fragment 8, or any combination thereof, e.g., fragments 1-7, in order). In some embodiments, the genetic element (e.g., the protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a TTV-HD23a GC-rich sequence shown in Table 39 (e.g., the entire sequence, fragment 1, fragment 2, fragment 3, fragment 4, fragment 5, fragment 6, or any combination thereof, e.g., fragments 1-6, in order). In some embodiments, the genetic element (e.g., the protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the TTV-JA20 GC-rich sequence shown in Table 39 (e.g., the entire sequence, fragment 1, fragment 2, or any combination thereof, e.g., fragment 1 and 2, in order).In some embodiments, the genetic element (e.g., the protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a TTV-TJN02 GC-rich sequence shown in Table 39 (e.g., the entire sequence, fragment 1, fragment 2, fragment 3, fragment 4, fragment 5, fragment 6, fragment 7, fragment 8, or any combination thereof, e.g., fragments 1-8, in order). In some embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a TTV-tth8 GC rich sequence (e.g., the entire sequence, fragment 1, fragment 2, fragment 3, fragment 4, fragment 5, fragment 6, fragment 7, fragment 8, fragment 9, or any combination thereof, e.g., fragments 1-6, in order) shown in Table 39. In some embodiments, the genetic element (e.g., a protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to fragment 7 shown in Table 39. In some embodiments, the genetic element (e.g., the protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to fragment 8 shown in Table 39. In some embodiments, the genetic element (e.g., the protein binding sequence of the genetic element) comprises a nucleic acid sequence having at least about 75% (e.g., at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to fragment 9 shown in Table 39.

[0586] [Table 168]

[0587] [Table 169]

[0588] [Table 170]

[0589] [Table 171]

[0590] [Table 172]

[0591] [Table 173]

[0592] [Table 174]

[0593] In some embodiments, the genetic element 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 nucleotide sequence of any one of Tables N1 to N25.

[0594] effector In some embodiments, the genetic element may include one or more sequences encoding a functional effector, e.g., an endogenous effector or an exogenous effector, e.g., a therapeutic polypeptide or nucleic acid, e.g., a cytotoxic or cytolytic RNA or protein. In some embodiments, the functional nucleic acid is a non-coding RNA. In some embodiments, the functional nucleic acid is a coding RNA. The effector may modulate a biological activity, e.g., increasing or decreasing an enzyme activity, gene expression, cell signaling, and cell or organ function. Effector activity may also include binding to a regulatory protein to modulate the activity of a regulator, such as transcription or translation. Effector activity may also include activator or inhibitor function. For example, an effector may induce an enzyme activity by triggering increased substrate affinity in an enzyme, e.g., fructose 2,6-bisphosphate activates phosphofructokinase 1 to increase the rate of glycolysis in response to insulin. In another example, an effector can inhibit the substrate from binding to the receptor, blocking its activation, for example, naltrexone and naloxone bind to opioid receptors without activating them, blocking the receptor's ability to bind opioids. Effector activity can also include modulation of protein stability / degradation and / or transcript stability / degradation. For example, proteins can be targeted for degradation by a polypeptide cofactor, ubiquitin, on the protein, marking them for degradation. In another example, an effector inhibits enzyme activity by blocking the active site of the enzyme, for example, methotrexate is a structural analog of tetrahydrofolate, i.e., a coenzyme of the enzyme dihydrofolate reductase, which binds to dihydrofolate reductase more than 1000-fold stronger than the natural substrate, inhibiting nucleotide base synthesis.

[0595] In some embodiments, the sequence encoding the effector is part of a genetic element, e.g., it can be inserted into an insertion site as described in Examples 10, 12, or 22 of WO 2020 / 123816 (hereby incorporated by reference in its entirety) and Example 28 herein. In some embodiments, the sequence encoding the effector is inserted into a non-coding region within the genetic element, e.g., a non-coding region located 3' to the open reading frame and 5' to a GC-rich region of the genetic element, a 5' non-coding region upstream of the TATA box, a 5' UTR, a 3' non-coding region downstream of a poly-A signal, or a 3' non-coding region upstream of a GC-rich region. In some embodiments, the sequence encoding the effector is inserted into a genetic element, e.g., near nucleotide 3588 of the TTV-tth8 plasmid, e.g., as described herein, or near nucleotide 2843 of the TTMV-LY2 plasmid, e.g., as described herein. In some embodiments, the effector-encoding sequence is inserted within a genetic element, e.g., at or within nucleotides 336-3015 of a TTV-tth8 plasmid, as described herein, or at or within nucleotides 242-2812 of a TTV-LY2 plasmid, as described herein. In some embodiments, the effector-encoding sequence replaces part or all of an open reading frame (e.g., an ORF described herein, e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, and / or ORF2t / 3 as shown in any one of Tables A1-A25 or N1-N25).

[0596] In some embodiments, the sequence encoding the effector comprises 100-2000, 100-1000, 100-500, 100-200, 200-2000, 200-1000, 200-500, 500-1000, 500-2000, or 1000-2000 nucleotides. In some embodiments, the effector is a nucleic acid or protein payload, e.g., as described in Example 28.

[0597] regulatory nucleic acids In some embodiments, the effector is a regulatory nucleic acid. The regulatory nucleic acid modifies the expression of endogenous and / or exogenous genes. In one embodiment, the regulatory nucleic acid targets a host gene. Regulatory nucleic acids include, but are not limited to, nucleic acids that hybridize with exogenous genes (e.g., miRNA, siRNA, mRNA, lncRNA, RNA, DNA, antisense RNA, gRNA, as described elsewhere herein), nucleic acids that hybridize with exogenous nucleic acids such as viral DNA or RNA, nucleic acids that hybridize with RNA, nucleic acids that disrupt gene transcription, nucleic acids that disrupt RNA translation, nucleic acids that stabilize or destabilize RNA, such as by targeting degradation, and nucleic acids that regulate DNA or RNA binding factors. In some embodiments, the regulatory nucleic acid encodes a miRNA.

[0598] In some embodiments, the regulatory nucleic acid comprises an RNA or RNA-like structure that typically contains 5-500 base pairs (depending on the particular RNA structure, e.g., miRNA 5-30 bp, lncRNA 200-500 bp) and may have a nucleobase sequence that is identical (or complementary) or nearly identical (or substantially complementary) to a coding sequence in an expressed target gene in a cell, or a sequence that encodes an expressed target gene in a cell.

[0599] In some embodiments, the regulatory nucleic acid comprises a nucleic acid sequence, e.g., a guide RNA (gRNA). In some embodiments, the DNA targeting moiety comprises a guide RNA or a nucleic acid encoding a guide RNA. The short synthetic RNA of the gRNA may consist of a "scaffold" sequence required to bind to the incomplete effector moiety and a user-defined targeting sequence of about 20 nucleotides to the genomic target. In practice, guide RNA sequences are generally designed to have a length of 17-24 nucleotides (e.g., 19, 20, or 21 nucleotides) and are complementary to the targeting nucleic acid sequence. Custom gRNA generation machines and algorithms are commercially available for use in designing effective guide RNAs. Gene editing has also been achieved using chimeric "single guide RNAs" ("sgRNAs"), which are engineered (synthetic) single RNA molecules that mimic the naturally occurring crRNA-tracrRNA complex and contain both tracrRNA (which binds to nucleases) and at least one crRNA (which guides the nuclease to the sequence targeted for editing). Chemically modified sgRNAs have also been demonstrated to be effective for genome editing; see, for example, Hendel et al. (2015) Nature Biotechnol., 985-991.

[0600] Regulatory nucleic acids include gRNAs that recognize specific DNA sequences (e.g., sequences adjacent to or within a gene's promoter, enhancer, silencer, or repressor).

[0601] Some regulatory nucleic acids can inhibit gene expression by the biological process of RNA interference (RNAi). RNAi molecules typically contain 15-50 base pairs (e.g., about 18-25 base pairs) and comprise RNA or RNA-like structures with identical (complementary) or nearly identical (substantially complementary) nucleobase sequences to coding sequences in expressed target genes in cells. RNAi molecules include, but are not limited to, small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), small hairpin RNA (shRNA), meroduplex, and dicer substrates (U.S. Pat. Nos. 8,084,599, 8,349,809, and 8,513,207).

[0602] Long non-coding RNAs (lncRNAs) are defined as non-protein-coding transcripts longer than 100 nucleotides. This somewhat arbitrary restriction distinguishes lncRNAs from small regulatory RNAs such as microRNAs (miRNAs), small interfering RNAs (siRNAs), and other small RNAs. In general, the majority of lncRNAs (about 78%) are characterized as tissue-specific. Branched lncRNAs (which account for a significant proportion of all lncRNAs in mammalian genomes, about 20%) that are transcribed in the opposite direction to adjacent protein-coding genes are likely to regulate the transcription of adjacent genes.

[0603] A genetic element encodes a regulatory nucleic acid having a sequence that is substantially complementary or completely complementary to all or a fragment of an endogenous gene or gene product (e.g., mRNA). The regulatory nucleic acid can complement sequences at boundaries between introns and exons to prevent the nascent nuclear RNA transcript of a particular gene from maturing into an mRNA for transcription. A regulatory nucleic acid complementary to a particular gene hybridizes with the mRNA of that gene to prevent its translation. An antisense regulatory nucleic acid can be DNA, RNA, or derivatives or hybrids thereof.

[0604] The length of the regulatory nucleic acid that hybridizes to the transcript of interest can be 5-30 nucleotides, about 10-30 nucleotides, or about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides. The percent identity of the regulatory nucleic acid to the target transcript should be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0605] The genetic element may encode a regulatory nucleic acid, e.g., a microRNA (miRNA) molecule identical to about 5 to about 25 contiguous nucleotides of the target gene. In some embodiments, the miRNA sequence targets an mRNA, starts with the dinucleotide AA, contains about 30-70% (about 30-60%, about 40-60%, or about 45%-55%) GC content, and does not have a high percent identity to any non-target nucleotide sequences in the genome of the mammal into which it is to be introduced, as determined, e.g., by a standard BLAST search.

[0606] In some embodiments, the regulatory nucleic acid is at least one miRNA, e.g., 2, 3, 4, 5, 6 or more miRNAs. In some embodiments, the genetic element comprises a sequence encoding a miRNA with at least about 75%, 80%, 85%, 90% 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of those nucleotide sequences or a sequence complementary to a sequence described herein.

[0607] siRNA and shRNA are similar to intermediates in the processing pathway of endogenous microRNA (miRNA) genes (Bartel, Cell 116:281-297, 2004). In some embodiments, siRNA can function as miRNA and vice versa (Zeng et al., Mol Cell 9:1327-1333, 2002; Doench et al., Genes Dev 17:438-442, 2003). MicroRNAs, like siRNAs, use RISC to downregulate target genes, but unlike siRNAs, most animal miRNAs do not cleave mRNAs. Instead, miRNAs reduce protein production by translational repression or polyA removal and mRNA degradation (Wu et al., Proc Natl Acad Sci USA 103:4034-4039, 2006). Known miRNA binding sites are within the mRNA 3'UTR; miRNAs appear to target sites with near perfect complementarity to nucleotides 2-8 from the 5' end of the miRNA (Rajewsky, Nat Genet 38 Suppl:S8-13, 2006; Lim et al., Nature 433:769-773, 2005). This region is known as the seed region. Because siRNAs and miRNAs are replaceable, exogenous siRNAs downregulate mRNAs with seed complementarity to the siRNA (Birmingham et al., Nat Methods 3:199-204, 2006. Multiple target sites within the 3'UTR confer stronger downregulation (Doench et al., Genes Dev 17:438-442, 2003).

[0608] Lists of known miRNA sequences can be found in databases maintained by research institutions such as the Wellcome Trust Sanger Institute, the Penn Center for Bioinformatics, the Memorial Sloan Kettering Cancer Center, and the European Molecule Biology Laboratory, among others. Known effective siRNA sequences and cognate binding sites are also well described in the relevant literature. RNAi molecules are easily designed and produced by techniques known in the art. In addition, there are computational tools that increase the chances of finding effective and specific sequence motifs (Lagana et al., Methods Mol. Bio., 2015, 1269:393-412).

[0609] Regulatory nucleic acids can regulate the expression of RNA encoded by genes. In some embodiments, because multiple genes share a degree of sequence homology with each other, regulatory nucleic acids can be designed to target classes of genes with sufficient sequence homology. In some embodiments, regulatory nucleic acids can include sequences that are shared among various gene targets or have complementarity to sequences that are unique for a particular gene target. In some embodiments, regulatory nucleic acids can be designed to target conserved regions of RNA sequences that have homology among several genes, thereby targeting several genes within a gene family (e.g., different gene isoforms, splice variants, mutant genes, etc.). In some embodiments, regulatory nucleic acids can be designed to target sequences that are unique to a particular RNA sequence of a single gene.

[0610] In some embodiments, the genetic elements may comprise one or more sequences encoding regulatory nucleic acids that regulate expression of one or more genes.

[0611] In some embodiments, gRNA described elsewhere herein is used as part of a CRISPR system for gene editing.For gene editing purposes, annelovectors or anneloVLPs can be designed to contain one or more guide RNA sequences corresponding to desired target DNA sequences; see, for example, Cong et al. (2013) Science, 339:819-823; Ran et al. (2013) Nature Protocols, 8:2281-2308.Generally, at least about 16 or 17 nucleotides of gRNA allow Cas9-mediated DNA cleavage; in the case of Cpf1, at least about 16 nucleotides of gRNA sequence are required to achieve detectable DNA cleavage.

[0612] Therapeutic effectors (e.g., peptides or polypeptides) In some embodiments, the genetic element comprises a therapeutic expression sequence, e.g., a sequence encoding a therapeutic peptide or polypeptide, e.g., an intracellular peptide or intracellular polypeptide, a secreted polypeptide, or a protein replacement therapeutic, e.g., a wild-type protein or a functional fragment or variant thereof. In some embodiments, the genetic element comprises a sequence encoding a protein, e.g., a therapeutic protein. Some examples of therapeutic proteins include, but are not limited to, hormones, cytokines, enzymes, antibodies (e.g., one or more polypeptides encoding at least a heavy or light chain), transcription factors, receptors (e.g., membrane receptors), ligands, membrane transporters, secreted proteins, peptides, carrier proteins, structural proteins, nucleases, or components thereof.

[0613] In some embodiments, the genetic element comprises a sequence encoding a peptide, for example a therapeutic peptide. The peptide may be linear or branched. The peptide has a length of about 5 to about 500 amino acids, about 15 to about 400 amino acids, about 20 to about 325 amino acids, about 25 to about 250 amino acids, about 50 to about 200 amino acids, or any range therebetween.

[0614] In some embodiments, the polypeptide encoded by the therapeutic expression sequence may be any of the above functional variants or fragments thereof, e.g., a protein having at least 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to a protein sequence disclosed in the tables herein by reference to its UniProt ID.

[0615] In some embodiments, the therapeutic expression sequence may encode an antibody or antibody fragment that binds to any of the above, e.g., an antibody to a protein having at least 80%, 85%, 90%, 95%, 967%, 98%, 99% identity to a protein sequence disclosed in the tables herein by reference to its UniProt ID. The term "antibody" is used broadly herein to encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. An "antibody fragment" refers to a molecule that contains at least one heavy or light chain and binds to an antigen. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab') 2 linear antibodies; single-chain antibody molecules (e.g., scFV); and multispecific antibodies formed from antibody fragments.

[0616] In some embodiments, an effector as described herein comprises a cytoplasmic polypeptide or peptide, such as a wild-type protein or a functional fragment or variant thereof.

[0617] In some embodiments, the effector comprises a regulatory intracellular polypeptide, such as a wild-type protein or a functional fragment or variant thereof. In some embodiments, the regulatory intracellular polypeptide binds to one or more molecules (e.g., proteins or nucleic acids) endogenous to the target cell. In some embodiments, the regulatory intracellular polypeptide increases the level or activity of one or more molecules (e.g., proteins or nucleic acids) endogenous to the target cell. In some embodiments, the regulatory intracellular polypeptide decreases the level or activity of one or more molecules (e.g., proteins or nucleic acids) endogenous to the target cell.

[0618] In some embodiments, an effector as described herein comprises a secreted polypeptide effector, e.g., a wild-type protein or a functional fragment or variant thereof. Exemplary secreted therapeutic agents include cytokines and cytokine receptors.

[0619] Exemplary cytokines and cytokine receptors are described, for example, in Akdis et al., "Interleukins (from IL-1 to IL-38), interferons, transforming growth factor β, and TNF-α: Receptors, functions, and roles in diseases," October 2016 Volume 138, Issue 4, Pages 984-1010.

[0620] Additional exemplary secreted therapeutic agents include polypeptide hormones and receptors, eg, wild-type proteins or functional fragments or mutants thereof.

[0621] Additional exemplary secreted therapeutic agents include growth factors, e.g., wild-type proteins or functional fragments or variants thereof. Exemplary growth factors and growth factor receptors are described, for example, in Bafico et al., "Classification of Growth Factors and Their Receptors" Holland-Frei Cancer Medicine. 6th edition.

[0622] Additional exemplary secreted therapeutic agents include coagulation-related factors, such as wild-type proteins or functional fragments or variants thereof.

[0623] In some embodiments, an effector described herein comprises a protein replacement therapeutic, e.g., a wild-type protein or a functional fragment or variant thereof. Exemplary protein replacement therapeutics are described herein.

[0624] In some embodiments, the effectors described herein include enzymatic effectors, such as wild-type proteins or functional fragments or variants thereof.

[0625] In some embodiments, the effectors described herein include non-enzymatic effectors, such as wild-type proteins or functional fragments or variants thereof.

[0626] In some embodiments, the effectors described herein include proteins that, when mutated, cause a lysosomal storage disorder, such as a wild-type protein or a functional fragment or mutant thereof. In some embodiments, the effectors described herein include transport proteins, such as a wild-type protein or a functional fragment or mutant thereof.

[0627] In some embodiments, a functional variant of a wild-type protein includes a protein having one or more activities of the wild-type protein, e.g., the functional variant catalyzes the same reaction as the corresponding wild-type protein, e.g., at a rate at least 10%, 20%, 30%, 40%, or 50% lower than the wild-type protein. In some embodiments, the functional variant binds to the same binding partner as that bound by the wild-type protein, e.g., with a Kd that is at most 10%, 20%, 30%, 40%, or 50% higher than the Kd of the corresponding wild-type protein for the same binding partner under the same conditions. In some embodiments, the functional variant has a polypeptide sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of the wild-type polypeptide. In some embodiments, the functional variant includes a homolog (e.g., an ortholog or paralog) of the corresponding wild-type protein. In some embodiments, the functional variant is a fusion protein. In some embodiments, the fusion comprises a first region having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a corresponding wild-type protein and a second, heterologous region. In some embodiments, the functional variant comprises or consists of a fragment of the corresponding wild-type protein.

[0628] In some embodiments, the effector as described herein comprises a transforming factor, e.g., a wild-type protein or fragment or variant thereof. In embodiments, the transforming factor is a protein factor that transforms fibroblasts into differentiated cells. In some embodiments, the effector as described herein comprises a protein that stimulates cell regeneration, e.g., a wild-type protein or fragment or variant thereof.

[0629] In some embodiments, the effectors described herein modulate STING / cGAS signaling, e.g., wild-type protein or fragments or mutants thereof. In some embodiments, the STING modulator is a polypeptide, e.g., a viral polypeptide or a functional mutant thereof. For example, the effector may include a STING modulator (e.g., an inhibitor) described in Maringer et al. "Message in a bottle: lessons learned from antagonism of STING signalling during RNA viral infection" Cytokine & Growth Factor Reviews Volume 25, Issue 6, December 2014, Pages 669-679. Additional STING modulators (e.g., activators) are described, for example, in Wang et al. "STING activator c-di-GMP enhances the anti-tumor effects of peptide vaccines in melanoma-bearing mice." Cancer Immunol Immunother. 2015 Aug; 64(8): 1057-66. doi: 10.1007 / s00262-015-1713-5. Epub 2015 May 19; Bose "cGAS / STING Pathway in Cancer: Jekyll and Hyde Story of Cancer Immune Response" Int J Mol Sci. 2017 Nov; 18(11): 2456; and Fu et al. "STING agonist formulated cancer vaccines can cure established tumors resistant to PD-1 blockade" Sci Transl Med. 2015 Apr 15; 7(283): 283ra52.Some examples of peptides include, but are not limited to, fluorescent tags or markers, antigens, peptide therapeutics, synthetic or analog peptides derived from natural bioactive peptides, agonist or antagonist peptides, antimicrobial peptides, targeting or cytotoxic peptides, degradative or self-destructive peptides, and degradative or self-destructive peptides. Peptides useful in the invention described herein also include antigen-binding peptides, such as antigen-binding antibodies or antibody-like fragments, such as single-chain antibodies, nanobodies (see, e.g., Steeland et al. 2016. Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today: 21(7): 1076-113). Such antigen-binding peptides may bind to cytosolic, nuclear, or intraorganellar antigens.

[0630] In some embodiments, the genetic elements include sequences encoding small peptides, peptidomimetics (e.g., peptoids), amino acids, and amino acid analogs. Such therapeutic agents generally have a molecular weight of less than about 5,000 grams per mole, less than about 2,000 grams per mole, less than about 1,000 grams per mole, less than about 500 grams per mole, as well as salts, esters, and other pharma- ceutically acceptable forms of such compounds. Such therapeutic agents include, but are not limited to, neurotransmitters, hormones, drugs, toxins, viral or microbial particles, synthetic particles, and agonists or antagonists thereof.

[0631] In some embodiments, the compositions, anelovectors, or anelovLPs described herein comprise a polypeptide linked to a ligand capable of targeting to a particular location, tissue, or cell.

[0632] Gene editing constructs The genetic elements of the anaerovector may include one or more genes encoding a component of a gene editing system. Alternatively, the anaerovector or anaeroVLP as described herein may include components of a gene editing system. Exemplary gene editing systems include clustered regulatory interspaced short palindromic repeat (CRISPR) systems, zinc finger nucleases (ZFNs), and transcription activator-like effector-based nucleases (TALENs). ZFN-, TALEN-, and CRISPR-based methods are described, for example, in Gaj et al. Trends Biotechnol. 31.7(2013):397-405; CRISPR methods of gene editing are described, for example, in Guan et al., Application of CRISPR-Cas system in gene therapy: Pre-clinical progress in animal model. DNA Repair 2016 Oct;46:1-8. doi:10.1016 / j.dnarep.2016.07.004; Zheng et al., Precise gene deletion and replacement using the CRISPR / Cas9 system in human cells. BioTechniques, Vol. 57, No. 3, September 2014, pp. 115-124.

[0633] The CRISPR system is an adaptive defense system originally discovered in bacteria and archaea. CRISPR systems cleave foreign DNA using RNA-guided nucleases called CRISPR-associated or "Cas" endonucleases (e.g., Cas9 or Cpf1). In a typical CRISPR / Cas system, the endonuclease is directed to a target nucleotide sequence (e.g., a site in the genome where sequence editing is to be performed) by a sequence-specific non-coding "guide RNA" that targets a single-stranded or double-stranded DNA sequence. Three classes (I-III) of CRISPR systems have been identified. Class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). Some class II CRISPR systems include a type II Cas endonuclease, s...

Claims

1. A mixture comprising degraded particles and a modifier, said mixture comprising: (i) a plurality of anellovirus ORF1 molecules formed by degradation of the particle; and (ii) Nucleic acid molecule A mixture comprising:

2. The mixture described in claim 1, wherein the nucleic acid molecule is endogenous to the host cell and is substantially dissociated from the proteinaceous outer portion.

3. The mixture described in claim 1, wherein the nucleic acid molecule is exogenous to the host cell.

4. The mixture described in claim 2, wherein the nucleic acid molecule is an anellovirus genome. (i) the anellovirus ORF1 molecule is produced in a mammalian cell; (ii) the mixture comprises a population of the degraded particles; and / or (iii) the host cell is a human cell; The mixture of claim 1.

6. A method of making the mixture of claim 2, said method comprising: (a) providing a mixture comprising particles and a modifier, the particles comprising: (i) a proteinaceous outer region comprising multiple anellovirus ORF1 molecules; and (ii) Nucleic acid molecule containing; and (b) the mixture; Degradation of the proteinaceous outer portion; and Dissociation of the nucleic acid molecule from the proteinaceous exterior Incubating under conditions suitable for A method comprising:

7. A plurality of anellovirus ORF1 molecules, and Chaotropic Agents A mixture comprising: at least 75% of the plurality of anellovirus ORF1 molecules in the mixture are contained in capsomers; A mixture, wherein host cell DNA in said mixture is substantially dissociated from said plurality of anellovirus ORF1 molecules.

8. The mixture comprising: (i) a plurality of pentamers comprising said anellovirus ORF1 molecule; and / or (ii) a plurality of decamers comprising the anellovirus ORF1 molecule; The mixture of claim 1 comprising (i) does not contain a proteinaceous outer region comprising about 40 to 80 copies of anellovirus ORF1 molecules; and / or (ii) It is a cell-free system; The mixture of claim 1.

10. The anellovirus ORF1 molecule, (i) comprises an arginine-rich region; (ii) does not contain a C-terminal domain, and / or (iii) each comprising a tagging moiety, optionally wherein the tagging moiety comprises a chitin-binding protein (CBP), a maltose-binding protein (MBP), a glutathione-S-transferase (GST), or a poly(His) tag; The mixture of claim 1.

11. The anellovirus ORF1 molecule, (i) an Alphatorque virus, Betatorque virus, or Gammatorque virus ORF1 molecule; (ii) comprises the amino acid sequence of SEQ ID NO: 1024 or 1028, or an amino acid sequence having at least 70% sequence identity thereto; or (iii) comprising the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence having at least 70% sequence identity thereto; A mixture according to any one of claims 1 to 5 or 7 to 10.

12. 1. A method for generating an anellovector, comprising: (a) providing a mixture comprising a plurality of anellovirus ORF1 molecules, wherein at least 75% of the plurality of ORF1 molecules are not contained in particles comprising about 40-80 (e.g., about 60) copies of ORF1 molecules; (b) subjecting the mixture to conditions suitable for the in vitro assembly of the anellovirus ORF1 molecules; and (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 anellovectors, each encapsulating one or more of the genetic elements. A method comprising: (i) the mixture provided in (a) is under denaturing conditions, e.g., the mixture contains a denaturant at a level sufficient to degrade complexes (e.g., proteinaceous outer portions) comprising at least about 20 copies, or 20-60 copies, of the anellovirus ORF1 molecule; and / or 13. The method of claim 12, wherein (ii) the conditions suitable for in vitro assembly comprise reducing the concentration of a denaturing agent or removing the mixture from denaturing conditions.

14. 1. A method for generating an anellovector, 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% of the plurality of ORF1 molecules are not contained in particles comprising about 40-80 (e.g., about 60) copies of ORF1 molecules; (b) subjecting the mixture to non-denaturing conditions suitable for in vitro assembly of the anellovirus ORF1 molecules (e.g., by dialysis) (e.g., reducing the concentration of the denaturant to a certain level); and (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 anellovectors, each encapsulating one or more of the genetic elements. A method comprising:

15. The method of claim 14, wherein (b) and (c) are performed simultaneously.

16. The method of claim 14, wherein (b) is performed before (c).

17. (i) the genetic element is introduced into a mixture containing the anellovirus ORF1 molecule prior to, simultaneously with, or after (b). (ii) at least 75% of the plurality of ORF1 molecules in the mixture of (a) are comprised in capsomers (e.g., decamers having a diameter of 25-40 nm, e.g., a diameter of 25-40 nm or about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nm); (iii) the ratio of ORF1 molecules in said mixture of (a) that are contained in capsomers (e.g., decamers) compared to ORF1 molecules in said mixture of (a) that are contained in particles is at least 2:1; and / or (iv) The method of claim 14, wherein after the incubation of (c), at least 75% of the plurality of ORF1 molecules are contained in an anellovector (e.g., a 60-mer or a particle at least 30 nm in diameter).

18. The genetic element comprising: (i) encoding an exogenous effector; (ii) is an oligonucleotide, and / or (iii) does not encode a polypeptide or functional nucleic acid. (i) the concentration of the denaturant after step (b) does not exceed about 2 M. (ii) after the incubation of (c), at least 75% of the particles in the mixture contain at least 50 copies of anellovirus ORF1 molecules; (iii) at least 90% of the particles in the mixture have a diameter of at least 30 nm; (iv) at least 90% of the particles in the mixture have a symmetrical morphology; and / or (v) The method of claim 14, wherein the denaturing agent is selected from a chaotropic agent (e.g., urea), heat (e.g., a temperature above about 85°C), or pH (e.g., an acidic or basic pH).

20. (i) the genetic element comprises one or more of the following characteristics: single-stranded, circular, negative-stranded, and / or DNA; (ii) the anellovirus ORF1 molecule is an alphatorque virus, a betatorque virus, or a gammatorque virus ORF1 molecule; (iii) the anellovirus ORF1 molecule comprises an arginine-rich region; and / or (iv) The method of any one of claims 12 to 19, wherein the anellovirus ORF1 molecule does not include a C-terminal domain.

21. The anellovirus ORF1 molecule, (i) the amino acid sequence of SEQ ID NO: 1024 or 1028, or an amino acid sequence having at least 70% sequence identity thereto; or (ii) the amino acid sequence of SEQ ID NO: 58, or an amino acid sequence having at least 70% sequence identity thereto; The method according to any one of claims 12 to 19, comprising: