Novel Anelloviridae Vector Compositions and Methods

Abstract

The present invention generally relates to Anelloviridae vectors (e.g., anellovectors), and compositions and uses thereof. Anelloviridae vectors can be used, for example, to deliver exogenous effectors to the retinal pigment epithelium (RPE) of a subject. In some embodiments, the vectors can be used to treat age-related macular degeneration (AMD).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 320,515, filed March 16, 2022, and International Application No. PCT / US2022 / 077923, filed October 11, 2022. The contents of the aforementioned applications are incorporated herein by reference in their entireties. [Background technology]

[0002] There is a continuing need to develop suitable vectors for delivering therapeutic genetic material to patients. Summary of the Invention [Means for solving the problem]

[0003] The present disclosure provides Anelloviridae vectors (e.g., anellovectors), e.g., synthetic Anelloviridae vectors (e.g., anellovectors), that can be used, e.g., for delivery of genetic material, for delivery of an effector, e.g., a payload, or as a delivery vehicle for delivery of a therapeutic agent or therapeutic effector to a eukaryotic cell (e.g., a human cell or tissue). In some embodiments, an Anelloviridae vector (e.g., an Anellovector) (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 exoskeleton (e.g., a proteinaceous exoskeleton comprising an Anelloviridae virus capsid protein (e.g., an Anellovirus capsid protein, e.g., an Anellovirus ORF1 protein or a polypeptide encoded by an Anellovirus ORF1 nucleic acid; or, e.g., a Chicken Anemia Virus (CAV) VP1 protein or a polypeptide encoded by a CAV VP1 nucleic acid, as described herein), which is capable of introducing the genetic element into a cell (e.g., a mammalian cell, e.g., a human cell). In some embodiments, an Anelloviridae vector (e.g., an anellovector) is a particle that comprises a proteinaceous exterior that includes a polypeptide encoded by an Anellovirus ORF1 nucleic acid (e.g., as described herein, e.g., an ORF1 nucleic acid of an Alphatorquevirus, Betatorquevirus, or Gammatorquevirus) or a polypeptide encoded by a CAV VP1 nucleic acid (e.g., as described herein).The genetic elements of the Anelloviridae vectors (e.g., Anellovectors) 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 binds to the proteinaceous exterior that confines it, or a polypeptide bound thereto, which may facilitate the entrapment of the genetic element within the proteinaceous exterior and / or the enrichment of the genetic element relative to other nucleic acids within the proteinaceous exterior. In some cases, the genetic element is circular or linear. In some cases, 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) that can be expressed, for example, in a cell. In some embodiments, the effector is a therapeutic agent or therapeutic effector, e.g., as described herein. In some cases, the effector is an endogenous or exogenous effector, e.g., to a wild-type Anellovirus or to a target cell. In some embodiments, the effector is exogenous to the wild-type Anellovirus or to the target cell. In some embodiments, an Anelloviridae vector (e.g., 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 by, for example, an Anelloviridae vector (e.g., anellovector)). In other cases, the effector is an exogenous effector. The effector can, in some cases, modulate a function of the cell or modulate the activity or level of a target molecule in the cell. For example, the effector can reduce the level of a target protein in the cell (e.g., as described in Examples 3 and 4).In another example, anelloviridae vectors (e.g., anellovectors) can deliver and express effectors, e.g., exogenous proteins, in vivo (e.g., as described in Examples 19 and 28). Anelloviridae vectors (e.g., 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 to treat diseases and disorders, for example, by delivering effectors that can act as therapeutic agents to a desired cell, tissue, or subject.

[0004] The present invention further provides synthetic Anelloviridae vectors (e.g., anellovectors). Synthetic Anelloviridae vectors (e.g., anellovectors) have at least one structural difference compared to a wild-type virus (e.g., a wild-type Anellovirus such as those described herein), such as a deletion, insertion, substitution, modification (e.g., enzymatic modification) relative to the wild-type virus. Generally, synthetic Anelloviridae vectors (e.g., anellovectors) comprise an exogenous genetic element enclosed within a proteinaceous exterior, which can be used to deliver the genetic element or an effector (e.g., an exogenous effector or an endogenous effector) encoded therein (e.g., a polypeptide or nucleic acid effector) into a eukaryotic (e.g., human) cell. In some embodiments, the Anelloviridae vectors (e.g., anellovectors) do not elicit a detectable and / or unwanted immune or inflammatory response, e.g., do not elicit greater than a 1%, 5%, 10%, 15% increase in molecular markers of inflammation, e.g., TNF-α, IL-6, IL-12, IFN, and B cell responses, e.g., reactive or neutralizing antibodies, e.g., the Anelloviridae vectors (e.g., anellovectors) can be substantially non-immunogenic to a target cell, tissue, or subject.

[0005] In one aspect, the invention features an Anelloviridae vector (e.g., anellovector) that includes: (i) a promoter element and a sequence encoding an effector (e.g., an endogenous or exogenous effector), and a protein binding sequence (e.g., an exogenous protein binding sequence, e.g., a packaging signal); and (ii) a proteinaceous exoskeleton; where the genetic element is encapsulated within the proteinaceous exoskeleton (e.g., a capsid); and where the Anelloviridae vector (e.g., anellovector) is capable of delivering the genetic element to a eukaryotic (e.g., mammalian, e.g., human) cell. 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 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, the integration frequency is determined as described, for example, 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, Anelloviridae vectors (e.g., anellovectors) are capable of delivering genetic elements to 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, or a wild-type CAV, e.g., a wild-type Anellovirus sequence listed in Tables N1-N4). 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 Anelloviridae virus sequence (e.g. a wild-type Anellovirus sequence or a CAV sequence listed in Tables N1-N4). 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 mammalian (e.g., human) cells.

[0006] In one aspect, the invention features an infectious particle (e.g., for a human cell) that includes an Anelloviridae virus capsid, e.g., an Anellovirus capsid (e.g., a capsid including an Anellovirus ORF, e.g., an ORF1 polypeptide) or a CAV capsid (e.g., a capsid including a CAV VP1 polypeptide) that encapsulates a genetic element that includes a protein binding sequence that binds to the capsid and a heterologous (relative to the Anellovirus) sequence that encodes a therapeutic effector. In some embodiments, the particle is capable of delivering the genetic element to a mammalian, e.g., human cell. In some embodiments, the genetic element has less than about 6% identity to a wild-type Anellovirus or CAV (e.g., less than 6%, less than 5.5%, less than 5%, less than 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%). In some embodiments, the genetic element has no more than 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, or 6% identity to a wild-type Anellovirus or CAV. 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 or CAV. In some embodiments, the genetic element has more than about 2000, 3000, 4000, 4500, or 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 4000, 4500 (e.g. more than 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, which is incorporated herein by reference in its entirety).

[0007] Also described herein are viral vectors and viral particles based on Anelloviridae viruses (e.g., Anelloviruses or CAVs), which can be used to deliver an agent (e.g., an exogenous effector or an endogenous effector, e.g., a therapeutic effector) to a cell (e.g., a cell of a subject to be therapeutically treated). In some embodiments, Anelloviridae viruses (e.g., Anelloviruses or CAVs) can be used as effective delivery vehicles to introduce an agent, such as an effector described herein, into a target cell, e.g., a target cell of a subject to be therapeutically or prophylactically treated.

[0008] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: (i) a first region that includes an amino acid sequence that has at least 70% (e.g., at least about 70, 80, 90, 95, 96, 97, 98, 99, or 100%) sequence identity to an arginine-rich region, e.g., an arginine-rich region described herein, or a sequence of at least about 40 amino acids, at least 60%, 70%, or 80% of which are basic residues (e.g., arginine, lysine, or a combination thereof); (ii) 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 a jellyroll domain, e.g., a jellyroll domain sequence described herein, or a sequence comprising at least six beta 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 or CAV VP1 C-terminal domain (CTD) sequence described herein. The present invention also features a polypeptide (e.g., a synthetic polypeptide, e.g., an ORF1 molecule) that comprises (e.g., consecutively): (v) Optionally, the polypeptide comprises an amino acid sequence having less than 100%, 99%, 98%, 95%, 90%, 85%, 80% sequence identity to a wild-type anellovirus ORF1 or CAV VP1 protein described herein.

[0009] In some embodiments, the present invention provides a method for producing a composition comprising the steps of: (i) an arginine-rich region, e.g., a first region comprising 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 that includes a jelly-roll domain, e.g., a sequence having at least six β-strands, e.g., six, seven, or eight β-strands, arranged in two antiparallel β-sheets that are packed together across a hydrophobic interface. The present invention also features a polypeptide (e.g., a synthetic polypeptide, e.g., a VP1 molecule) that comprises (e.g., consecutively): (iii) Optionally, the polypeptide comprises an amino acid sequence having, e.g., less than 100%, 99%, 98%, 95%, 90%, 85%, 80% sequence identity to a wild-type CAV VP1 protein described herein.

[0010] 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 or CAV VP1 molecule described herein (e.g., as listed in any of Tables A1-A3). In some embodiments, the polypeptide comprises at least about 70, 80, 90, 95, 96, 97, 98, 99, or 100% sequence identity to a subsequence of an anellovirus ORF1 molecule or CAV VP1 molecule described herein (e.g., as listed in any of Tables A1-A3) (e.g., an arginine (Arg)-rich domain, a jellyroll domain, a hypervariable region (HVR), an N22 domain, or a C-terminal domain (CTD)). In one embodiment, the amino acid sequences of regions (i), (ii), (iii), and (iv) have at least 90% sequence identity to their respective reference sequences, wherein the polypeptide has an amino acid sequence that has less than 100%, 99%, 98%, 95%, 90%, 85%, 80% sequence identity to a wild-type anellovirus ORF1 or CAV VP1 protein described herein.

[0011] In one aspect, the invention features a complex that includes a polypeptide described herein (e.g., an anellovirus ORF1 molecule or a CAV VP1 molecule described herein) and a genetic element that includes a promoter element, 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.

[0012] The disclosure further provides nucleic acid molecules (e.g., nucleic acid molecules comprising the genetic elements described herein, or nucleic acid molecules comprising a sequence encoding a proteinaceous external protein described herein). A nucleic acid molecule of the invention may comprise one or both of: (a) the genetic elements described herein, and (b) a nucleic acid sequence encoding a proteinaceous external protein described herein.

[0013] 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 exoprotein binding sequence. In some embodiments, the exoprotein 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 or CAV 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 an embodiment, the effector is not derived from TTV and is not SV40-miR-S1. In an embodiment, the nucleic acid molecule does not include the polynucleotide sequence of TTMV-LY2. In an embodiment, the promoter element can direct the expression of the effector in eukaryotic (e.g., mammalian, e.g., human) cells.

[0014] 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).

[0015] 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 a VP1 molecule (e.g., a CAV VP1 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 or CAV 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); or at least 72% (e.g., at least at least 100 (e.g., at least 300, 500, 1000, 1500) contiguous nucleic acids having at least 72, 73, 74, 75, 76, 77, 78, 79, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) sequence identity with the nucleic acid construct; and (iii) a protein binding sequence, e.g., an exogenous protein binding sequence, wherein the nucleic acid construct comprises: 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 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 genetic element encoding the 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 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). In some embodiments, the genetic element comprises a sequence encoding a VP1 molecule (e.g., a CAV VP1 protein, e.g., as described herein).

[0016] In one aspect, 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, ORF3, VP1, VP2, or VP3 molecule (e.g., a sequence encoding an anellovirus ORF1 polypeptide or a CAV VP1 polypeptide described herein), where the nucleic acid is a plasmid, a viral nucleic acid, or integrated into a helper cell chromosome; and (b) a genetic element that includes (i) a promoter operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector), and (ii) a protein binding sequence that binds the polypeptide of (a), optionally where the genetic element does not encode an ORF1 or VP1 polypeptide (e.g., an ORF1 protein or a VP1 protein). For example, the host or helper cell includes (a) and (b) in either cis (both are part of the same nucleic acid molecule) or in trans (each is part 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 cell or helper cell is adherent or in suspension, or both. In some embodiments, the host cell or helper cell is grown in a microcarrier. In some embodiments, the host cell or helper cell is compatible with cGMP manufacturing. In some embodiments, the host cell or helper cell is grown in a medium suitable for promoting cell growth. In certain embodiments, once the host cell or helper cell has 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 cell or helper cell.

[0017] In one aspect, the invention features a pharmaceutical composition that includes an Anelloviridae vector (e.g., anellovector) (e.g., a synthetic Anelloviridae vector (e.g., anellovector)) described herein. 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 100 mg / kg of the subject. 5 ~10 14 The pharmaceutical composition may include a unit dose that contains a genome equivalent of an Anelloviridae vector (e.g., anellovector). In some embodiments, the pharmaceutical composition including the preparation is stable for an acceptable period of time and temperature, and / or is compatible with a desired route of administration and / or any device required by this route of administration, e.g., a needle or syringe. In some embodiments, the pharmaceutical composition is formulated for administration as a single dose or multiple doses. In some embodiments, the pharmaceutical composition is formulated at the site of administration, e.g., by a medical professional. In some embodiments, the pharmaceutical composition includes a desired concentration of an Anelloviridae vector (e.g., anellovector) genome or genome equivalent (e.g., defined by the number of genomes per volume).

[0018] 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 Anelloviridae vector (e.g., anellovector), e.g., a synthetic Anelloviridae vector (e.g., anellovector), e.g., a synthetic Anelloviridae vector (e.g., anellovector), described herein. In one aspect, the invention features a method of treating a disease or disorder in a subject, the method includes administering to an eye of the subject an Anelloviridae vector (e.g., anellovector), e.g., a synthetic Anelloviridae vector (e.g., anellovector), described herein.

[0019] In one aspect, the invention features a method of delivering an effector or payload (e.g., an endogenous or exogenous effector) to a cell, tissue, or subject, the method includes administering to a subject an Anelloviridae vector (e.g., an anellovector), e.g., a synthetic Anelloviridae vector (e.g., anellovector), e.g., as described herein, to a subject, where the anellovector includes a nucleic acid sequence encoding an effector. In embodiments, the payload is a nucleic acid. In embodiments, the payload is a polypeptide. In some embodiments, the cell is an ocular cell. In certain embodiments, the ocular cell is a photoreceptor cell, a retinal cell, a cell of the posterior ocular cup (PEC), a cell of the optic nerve, a cell of the optic nerve head, a retinal ganglion cell, or a retinal pigmented epithelium (RPE) cell. In some embodiments, the tissue is an ocular tissue. In certain embodiments, the ocular tissue is the retina, posterior cup, retinal ganglion, retinal pigment epithelium, optic nerve, optic nerve head, subretinal space, or intravitreal space.

[0020] In one aspect, the invention provides a method of delivering an Anelloviridae vector (e.g., anellovector) to a cell, comprising contacting an Anelloviridae vector (e.g., anellovector), e.g., a synthetic Anelloviridae vector (e.g., anellovector) described herein, with a cell, e.g., a eukaryotic cell, e.g., a mammalian cell, e.g., in vivo or ex vivo. In some embodiments, the cell is an ocular cell. In certain embodiments, the ocular cell is a photoreceptor cell, a retinal cell, a cell of the posterior ocular cup (PEC), a cell of the optic nerve, a cell of the optic nerve head, a retinal ganglion cell, or a retinal pigmented epithelial (RPE) cell.

[0021] In one aspect, the invention features a method of making an Anelloviridae vector (e.g., anellovector), e.g., a synthetic anellovector. The method includes: a)Below: (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; (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, ORF1 / 2, VP1, VP2, or VP3, 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, e.g., as set forth in Tables A1-A3; providing a host cell comprising the b) incubating the host cells under conditions suitable for production of an Anelloviridae vector (e.g., anellovector). Includes.

[0022] 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 genome of a helper plasmid or a helper virus).

[0023] In another aspect, the invention features a method of producing an Anelloviridae vector (e.g., anellovector) composition, comprising: The method includes providing a host cell that contains, e.g., expresses, one or more components (e.g., all components) of, e.g., an Anelloviridae vector (e.g., an anellovector), e.g., a synthetic Anelloviridae vector (e.g., an anellovector) described herein. For example, the host cell includes: (a) a nucleic acid that includes a sequence encoding an Anellovirus ORF1 or CAV VP1 polypeptide described herein, where the nucleic acid is a plasmid, a viral nucleic acid, or is integrated into a helper cell chromosome; and (b) a genetic element, where the genetic element 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 (i) a protein binding sequence (e.g., a packaging sequence) that binds the polypeptide of (a), where the host cell or helper cell includes (a) and (b) in either cis or trans. In one embodiment, the genetic element of (b) is circular, single-stranded DNA. In some embodiments, the host cell is a manufacturing cell line; b) culturing the host cell under conditions suitable for producing a preparation of Anelloviridae vectors (e.g., anellovectors) from the host cell, wherein the Anelloviridae vectors (e.g., anellovectors) of the preparation comprise a proteinaceous exterior (e.g., comprising an ORF1 molecule) that encapsulates genetic elements (e.g., as described herein), thereby producing a preparation of Anelloviridae vectors (e.g., anellovectors); and Optionally, c) formulating the Anelloviridae vector (e.g., anellovirus) preparation, e.g., as a pharmaceutical composition suitable for administration to a subject.

[0024] In some embodiments, components of an Anelloviridae vector (e.g., anellovector) are introduced into a host cell (e.g., by transient transfection) during production. In some embodiments, a host cell stably expresses components of an Anelloviridae vector (e.g., anellovector) (e.g., where one or more nucleic acids encoding components of an Anelloviridae vector (e.g., anellovector) are introduced into the host cell, or a progenitor cell thereof, e.g., by stable transfection).

[0025] 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 is stable over an acceptable period and temperature, and / or is compatible with the desired route of administration and / or any device required by this route of administration, e.g., an injection needle or syringe.

[0026] In one aspect, the invention features a method of producing an Anelloviridae vector (e.g., anellovirus) composition, including: a) providing a plurality of Anelloviridae vectors (e.g., anellovectors) described herein, or a preparation of an Anelloviridae vector (e.g., anellovector) described herein; and formulating the Anelloviridae vector (e.g., anellovector) or preparation thereof, e.g., as a pharmaceutical composition suitable for administration to a subject.

[0027] In one aspect, the invention features a method of generating a host cell, e.g., a first host cell or producer cell (e.g., as shown in FIG. 12), e.g., a population of first host cells, that includes an Anelloviridae vector (e.g., an anellovector), the method includes, e.g., introducing a genetic element described herein into the host cell, and culturing the host cell under conditions suitable for production of the Anelloviridae vector (e.g., an anellovector). 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 Anelloviridae vector (e.g., an anellovector).

[0028] In one aspect, the invention features a method of producing an Anelloviridae vector (e.g., anellovector), including providing a host cell, e.g., a first host cell or producer cell (e.g., as shown in FIG. 12 ), that includes an Anelloviridae vector (e.g., anellovector), e.g., as described herein, and purifying the Anelloviridae vector (e.g., anellovector) from the host cell. In some embodiments, the method further includes, prior to the providing step, contacting the host cell with an Anelloviridae vector (e.g., anellovector), e.g., as described herein, and incubating the host cell under conditions suitable for production of the Anelloviridae vector (e.g., anellovector). 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 an Anelloviridae vector (e.g., anellovector) from a host cell comprises lysing the host cell.

[0029] In some embodiments, the method further comprises a second step of contacting the Anelloviridae vector (e.g., anellovector) 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. 12), 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 Anelloviridae vector (e.g., anellovector). In some embodiments, the method further comprises purifying the Anelloviridae vector (e.g., anellovector) from the second host cell, e.g., thereby producing an Anelloviridae vector (e.g., anellovector) seed population. In some embodiments, at least about 2-100 times more Anelloviridae vector (e.g., anellovector) is produced from the population of second host cells compared to that from the first population of host cells. In some embodiments, purifying the Anelloviridae vector (e.g., anellovector) from the second host cell comprises lysing the second host cell. In some embodiments, the method further comprises a second step of contacting the Anelloviridae vector (e.g., anellovector) produced by the second host cell with a third host cell, e.g., a permissive cell (e.g., as shown in FIG. 12), e.g., a population of third host cells. In some embodiments, the method further comprises incubating the third host cell under conditions suitable for production of the Anelloviridae vector (e.g., anellovector). In some embodiments, the method further comprises purifying the Anelloviridae vector (e.g., anellovector) from the third host cell, e.g., thereby producing a Anelloviridae vector (e.g., anellovector) stock population.In some embodiments, purifying the Anelloviridae vector (e.g., anellovector) from the third host cells includes lysing the third host cells. In some embodiments, at least about 2-100 fold more Anelloviridae vector (e.g., anellovector) is produced from the population of third host cells compared to that from the population of second host cells.

[0030] In some embodiments, the host cells are grown in a medium suitable for promoting cell growth. In certain embodiments, once the host cells have grown sufficiently (e.g., to a suitable cell density), the medium may be replaced with a medium suitable for the production of the Anelloviridae vector (e.g., anellovector) by the host cells. In some embodiments, the Anelloviridae vector (e.g., anellovector) produced by the host cells is separated from the host cells (e.g., by lysing the host cells) before contacting with a second host cell. In some embodiments, the Anelloviridae vector (e.g., anellovector) produced by the host cells is contacted with a second host cell without a purification step.

[0031] In one aspect, the invention features a method for making a pharmaceutical Anelloviridae vector (e.g., anellovector) preparation. The method includes the steps of (a) producing an Anelloviridae vector (e.g., anellovector) preparation described herein; (b) measuring one or more pharmaceutical quality control parameters, e.g., identity, purity, titer, potency (e.g., genome equivalents per Anelloviridae vector (e.g., anellovector) particle), and / or nucleic acid sequence, e.g., nucleic acid sequence of an Anelloviridae vector (e.g., an Anelloviridae vector), and / or ... and (c) formulating the preparation for pharmaceutical use for which it is evaluated to meet predetermined criteria, e.g., pharmaceutical specifications. In some embodiments, assessing identity comprises evaluating (e.g., confirming) a sequence of a genetic element of the Anelloviridae vector (e.g., anellovector), e.g., a sequence encoding an effector.In some embodiments, assessing purity includes assessing the amount of impurities, e.g., 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), replication-competent agents (RCA), e.g., replication-competent viruses or unwanted Anelloviridae vectors (e.g., anellovectors) (e.g., an Anelloviridae vector (e.g., anellovector) other than a desired Anelloviridae vector (e.g., anellovector), e.g., a synthetic Anelloviridae vector (e.g., anellovector) described herein), free viral capsid proteins, adventitious material, and aggregates. In some embodiments, assessing titer includes assessing the percentage of functional and non-functional (e.g., infectious and non-infectious) Anelloviridae vectors (e.g., anellovectors) in the preparation (e.g., by HPLC, etc.). In some embodiments, assessing efficacy includes assessing the level of Anelloviridae vector (e.g., anellovector) function detectable in the preparation (e.g., expression and / or function of an effector encoded therein or genome equivalent).

[0032] 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 Anelloviridae vectors (e.g., anellovectors) can be produced in a single batch. In some embodiments, the level of Anelloviridae vectors (e.g., anellovectors) produced in a batch can be assessed (e.g., individually or together).

[0033] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising the steps of: (i) a first nucleic acid molecule comprising a nucleic acid sequence of a genetic element of an Anelloviridae vector (e.g., anellovector) 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, ORF1 / 2, VP1, VP2, or VP3, as listed in Tables A1-A3, or 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 thereto. The present invention features a host cell comprising:

[0034] In one aspect, the invention features a reaction mixture that includes an Anelloviridae vector (e.g., an anellovector) described herein and a helper virus, where the helper virus includes a polynucleotide, e.g., a polynucleotide encoding an outer protein (e.g., an outer protein capable of binding to an outer protein binding sequence, and optionally, a lipid envelope), a polynucleotide encoding a replication protein (e.g., a polymerase), or any combination thereof.

[0035] In some embodiments, the Anelloviridae vector (e.g., anellovector) (e.g., a synthetic Anelloviridae vector (e.g., anellovector)) is isolated, e.g., isolated from a host cell and / or isolated from other components in a solution (e.g., a supernatant). In some embodiments, the Anelloviridae vector (e.g., anellovector) (e.g., a synthetic Anelloviridae vector (e.g., anellovector)) is purified, e.g., from a solution (e.g., a supernatant). In some embodiments, the Anelloviridae vector (e.g., anellovector) is concentrated in the solution relative to other components in the solution.

[0036] In some embodiments of any of the Anelloviridae vectors (e.g., anellovectors), compositions, or methods described above, providing the Anelloviridae vector (e.g., anellovector) comprises isolating (e.g., harvesting) the Anelloviridae vector (e.g., anellovector) from a composition comprising an Anelloviridae vector (e.g., anellovector) producing cell, such as those described herein. In other embodiments, providing the Anelloviridae vector (e.g., anellovector) comprises obtaining the Anelloviridae vector (e.g., anellovector) or a preparation thereof, e.g., from a third party.

[0037] In some embodiments of any of the Anelloviridae vectors (e.g., anellovectors), compositions, or methods described above, the genetic element comprises an Anelloviridae vector (e.g., anellovector) genome, e.g., as identified according to the methods described in Example 9. In several embodiments, the Anelloviridae vector (e.g., anellovector) genome is an Anelloviridae vector (e.g., anellovector) genome capable of self-replication and / or self-amplification. In some embodiments, the Anelloviridae vector (e.g., anellovector) genome is incapable of self-replication and / or self-amplification. In some embodiments, the Anelloviridae vector (e.g., anellovector) genome is capable of replicating and / or amplifying in trans, e.g., in the presence of a helper, e.g., a helper virus.

[0038] It is understood that the applicable embodiments described herein with respect to anellovectors may also be applied to Anelloviridae vectors (e.g., vectors based on or derived from Chicken Anemia Virus (CAV) as described herein).

[0039] Additional features of any of the Anelloviridae vectors (e.g., anellovectors), compositions, or methods described above include one or more of the embodiments listed below.

[0040] 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.

[0041] Enumerated Embodiments 1. The following: (i) a proteinaceous exterior comprising an anellovirus ORF1 protein listed in Table A1 or A2, or a CAV VP1 protein listed in Table A3, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and (ii) a genetic element enclosed by a proteinaceous exterior, said genetic element comprising a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an exogenous effector; Anelloviridae vectors (e.g., anellovectors), including:

[0042] 2. The following: (i) a proteinaceous exterior comprising an anellovirus ORF1 protein listed in Table A1 or A2, or a CAV VP1 protein listed in Table A3, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and (ii) a genetic element enclosed by a proteinaceous envelope, said genetic element comprising 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); An Anelloviridae vector (e.g., anellovector) comprising: the proteinaceous external and / or genetic element comprises at least one difference (e.g., a mutation, a chemical modification, or an epigenetic alteration), e.g., an insertion, a substitution, a chemical or enzymatic modification, and / or a deletion, e.g., a deletion of a domain (e.g., one or more of the arginine-rich region, the jelly-roll domain, the HVR, the N22, or the CTD, as described herein) or a genomic region (e.g., one or more of the TATA box, the cap site, the transcription start site, the 5'UTR, the open reading frame (ORF), the poly(A) signal, or the GC-rich region, as described herein), compared to a wild-type anellovirus ORF1 protein and / or a wild-type anellovirus genome, respectively, or compared to a wild-type CAV VP1 protein and / or a wild-type CAV genome, respectively (e.g., as described herein); Anelloviridae vectors (e.g., anellovectors).

[0043] 3. The following: (i) a proteinaceous exosomal portion comprising a polypeptide encoded by an anellovirus ORF1 nucleic acid sequence listed in any of Tables N1-N2, or a CAV VP1 nucleic acid sequence of Tables N3 or N4, or a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the anellovirus ORF1 nucleic acid sequence or the CAV VP1 nucleic acid sequence; (ii) a genetic element enclosed by a proteinaceous exterior, said genetic element comprising a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an exogenous effector; Anelloviridae vectors (e.g., anellovectors), including:

[0044] 4. The following: (i) a proteinaceous exosomal region comprising a polypeptide encoded by an anellovirus ORF1 nucleic acid sequence listed in any of Tables N1-N2, or a CAV VP1 nucleic acid sequence of Tables N3 or N4, or a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an anellovirus ORF1 nucleic acid sequence or a CAV nucleic acid sequence; (ii) a genetic element enclosed by a proteinaceous exterior, said genetic element comprising 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); An Anelloviridae vector (e.g., anellovector) comprising: the proteinaceous external and / or genetic element comprises at least one difference (e.g., a mutation, a chemical modification, or an epigenetic alteration), e.g., an insertion, a substitution, a chemical or enzymatic modification, and / or a deletion, e.g., a deletion of a domain (e.g., one or more of the arginine-rich region, the jelly-roll domain, the HVR, the N22, or the CTD, as described herein) or a genomic region (e.g., one or more of the TATA box, the cap site, the transcription start site, the 5'UTR, the open reading frame (ORF), the poly(A) signal, or the GC-rich region, as described herein), compared to a wild-type anellovirus ORF1 protein and / or a wild-type anellovirus genome, respectively, or compared to a wild-type CAV VP1 protein and / or a wild-type CAV genome, respectively (e.g., as described herein); Anelloviridae vectors (e.g., anellovectors).

[0045] 5. The following: (i) a proteinaceous exterior (e.g., an anellovirus ORF1 molecule or VP1 molecule described herein, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto); and (ii) a genetic element enclosed by a proteinaceous exterior, said genetic element comprising: (a) a 5'UTR conserved domain listed in any of Tables N1 to N4, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto, or a complement thereof; and (b) a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an exogenous effector; Anelloviridae vectors (e.g., anellovectors), including:

[0046] 6. Below: (i) a proteinaceous exterior (e.g., an anellovirus ORF1 molecule or VP1 molecule described herein, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto); and (ii) a genetic element enclosed by a proteinaceous exterior, said genetic element comprising: (a) a 5'UTR conserved domain listed in any of Tables N1 to N4, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto, or a complement thereof; and (b) a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector). An Anelloviridae vector (e.g., anellovector) comprising: the proteinaceous external and / or genetic element comprises at least one difference (e.g., a mutation, a chemical modification, or an epigenetic alteration), e.g., an insertion, a substitution, a chemical or enzymatic modification, and / or a deletion, e.g., a deletion of a domain (e.g., one or more of the arginine-rich region, the jelly-roll domain, the HVR, the N22, or the CTD, e.g., as described herein) or a genomic region (e.g., one or more of the TATA box, the cap site, the transcription start site, the 5'UTR, the open reading frame (ORF), the poly(A) signal, or the GC-rich region, e.g., as described herein), compared to a wild-type anellovirus ORF1 protein and / or a wild-type anellovirus genome, respectively, or compared to a wild-type CAV VP1 protein and / or a wild-type CAV genome, respectively (e.g., as described herein); Anelloviridae vectors (e.g., anellovectors).

[0047] 7. Below: (i) a proteinaceous exterior (e.g., comprising an Anelloviridae capsid protein, e.g., an Anellovirus ORF1 molecule or a CAV VP1 protein described herein, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto); and (ii) a genetic element enclosed by the proteinaceous exterior, the genetic element comprising a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an exogenous effector, the genetic element having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anelloviridae virus genomic sequence listed in any of Tables N1-N4, or a complement thereof; Anelloviridae vectors (e.g., anellovectors), including:

[0048] 8. Below: (i) a proteinaceous exterior (e.g., comprising an Anelloviridae capsid protein, e.g., an Anellovirus ORF1 molecule or a CAV VP1 protein described herein, or a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto); and (ii) a genetic element enclosed by the proteinaceous exterior, the genetic element comprising 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), the genetic element having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anelloviridae virus (e.g., anellovirus or CAV) genomic sequence listed in any of Tables N1-N4, or a complement thereof; An Anelloviridae vector (e.g., anellovector) comprising: the proteinaceous external and / or genetic element comprises at least one difference (e.g., a mutation, a chemical modification, or an epigenetic alteration), e.g., an insertion, a substitution, a chemical or enzymatic modification, and / or a deletion, e.g., a deletion of a domain (e.g., one or more of the arginine-rich region, the jelly-roll domain, the HVR, the N22, or the CTD, e.g., as described herein) or a genomic region (e.g., one or more of the TATA box, the cap site, the transcription start site, the 5'UTR, the open reading frame (ORF), the poly(A) signal, or the GC-rich region, e.g., as described herein), respectively, compared to a wild-type Anelloviridae virus (e.g., anellovirus or CAV) ORF1 protein and / or a wild-type Anelloviridae (e.g., anellovirus or CAV) genome (e.g., as described herein); Anelloviridae vectors (e.g., anellovectors).

[0049] 9. The Anelloviridae vector (e.g., anellovector) of any of the previous embodiments, wherein at least one difference relative to a wild-type Anelloviridae virus (e.g., anellovirus or CAV) ORF1 protein and / or a wild-type Anelloviridae virus (e.g., anellovirus or CAV) genome encodes an exogenous effector.

[0050] 10. The proteinaceous exterior has the amino acid sequence YNPX 2 DXGX 2 N (SEQ ID NO: 829), where X n

[0023] The Anelloviridae vector (e.g., anellovector) of any of the preceding embodiments, wherein:

[0051] 11. An isolated ORF1 molecule comprising an amino acid sequence of ORF1 listed in Table A1 or A2, or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto; An isolated ORF1 molecule, wherein the ORF1 molecule comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) 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., as described herein, such as one or more of the arginine-rich region, jelly-roll domain, HVR, N22, or CTD).

[0052] 12. An isolated ORF1 molecule comprising an amino acid sequence of the Jellyroll Domain of ORF1 listed in Table A1 or A2, or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto; An isolated ORF1 molecule, wherein the ORF1 molecule comprises at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) 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., as described herein, such as one or more of the arginine-rich region, jelly-roll domain, HVR, N22, or CTD).

[0053] 13. An isolated VP1 molecule comprising an amino acid sequence of VP1 listed in Table A3, or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto; An isolated VP1 molecule, wherein the VP1 molecule comprises at least one difference (e.g., a mutation, a chemical modification, or an epigenetic alteration), e.g., an insertion, substitution, chemical or enzymatic modification, and / or a deletion, e.g., 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), compared to a wild-type VP1 protein (e.g., as described herein).

[0054] 14. An isolated VP1 molecule comprising an amino acid sequence of the VP1 jellyroll domain listed in Table A3, or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto; An isolated VP1 molecule, wherein the VP1 molecule comprises at least one difference (e.g., a mutation, a chemical modification, or an epigenetic alteration), e.g., an insertion, substitution, chemical or enzymatic modification, and / or a deletion, e.g., 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), compared to a wild-type VP1 protein (e.g., as described herein).

[0055] 15. The ORF1 molecule or the VP1 molecule has the amino acid sequence YNPX 2 DXGX 2 N (SEQ ID NO: 829), n is a consecutive sequence of any n amino acids.

[0056] 16. Amino acid sequence YNPX 2 DXGX 2 The ORF1 or VP1 molecule of embodiment 15, wherein N (sequence number 829) is included in the N22 domain of the ORF1 or VP1 molecule.

[0057] 17. The ORF1 or VP1 molecule of any one of embodiments 13-16, wherein the ORF1 or VP1 molecule comprises one or more (e.g., one, two, three, four, or all five) of the following anellovirus ORF1 or CAV VP1 subdomains: arginine-rich region, jelly-roll region, hypervariable region, N22 domain, C-terminal domain (CTD) (e.g., as described herein), e.g., the C-terminal domain (CTD) of an anellovirus ORF1 protein listed in Table A1 or A2 or a CAV VP1 protein listed in Table A3 (or a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto).

[0058] 18. An isolated ORF2 molecule comprising an amino acid sequence of ORF2 listed in Table A1 or A2, or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto; An isolated ORF2 molecule, wherein the ORF2 molecule contains at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration), such as an insertion, substitution, chemical or enzymatic modification, and / or a deletion, e.g., the deletion of a domain, compared to a wild-type ORF2 protein (e.g., as described herein).

[0059] 19. The above ORF2 molecule has the amino acid sequence [W / F]X 7 HX 3 CX 1 CX 5 H (SEQ ID NO: 949), n is a contiguous sequence of any n amino acids.

[0060] 20. An isolated nucleic acid molecule (e.g., a genetic element construct or a genetic element) comprising a nucleic acid sequence of a 5'UTR conserved domain listed in any of Tables N1-N4, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a complement thereof.

[0061] 21. An isolated nucleic acid molecule (genetic element construct, or, for example, a construct for providing an ORF1 molecule in trans, as described herein) comprising a nucleic acid sequence of an ORF1 gene or a VP1 gene listed in any of Tables N1-N4, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a complement thereof.

[0062] 22. An isolated nucleic acid molecule (genetic element construct, or, e.g., a construct for providing an ORF2 molecule in trans, as described herein) comprising a nucleic acid sequence of the ORF2 gene listed in any of Tables N1-N2, or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a complement thereof.

[0063] 23. An isolated nucleic acid molecule (e.g., a genetic element construct, a genetic element, or a construct for providing an ORF1, ORF2, VP1, or VP2 molecule in trans, e.g., as described herein) comprising an anellovirus genomic sequence listed in any of Tables N1-N4, or a nucleic acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a complement thereof.

[0064] 24. The isolated nucleic acid molecule of any of embodiments 20 to 23, wherein the isolated nucleic acid molecule comprises at least one difference (e.g., a mutation, a chemical modification, or an epigenetic alteration) compared to a wild-type anellovirus genomic sequence (e.g., as described herein).

[0065] 25. The isolated nucleic acid molecule of embodiment 24, wherein at least one difference comprises a deletion (e.g., lacking one or more of the following: a 5'UTR conserved domain, an ORF1 gene, an ORF2 gene, a VP1 gene, a VP2 gene, a GC-rich region, an ORF3 gene, a VP3 gene, or a functional fragment thereof).

[0066] 26. The isolated nucleic acid molecule of any of embodiments 20-25, wherein the isolated nucleic acid molecule is substantially incapable of being encapsulated in an anellovirus or CAV capsid (e.g., the proteinaceous exterior of an Anelloviridae vector (e.g., anellovector) described herein).

[0067] 27. The isolated nucleic acid molecule according to any of embodiments 20 to 26, wherein the isolated nucleic acid molecule encodes an effector (e.g. an exogenous effector or an endogenous effector).

[0068] 28. The following: (a) a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto, or a complement thereof, to a 5'UTR conserved domain listed in any of Tables N1-N4; (b) a promoter element operably linked to a nucleic acid sequence (e.g., a DNA sequence) encoding an exogenous effector; A genetic element comprising:

[0069] 29. Below: (i) nucleotides 1-71 of SEQ ID NO:1, or a nucleic acid sequence having at least 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (ii) a 5′ portion of the ORF2 nucleic acid sequence; (iii) a promoter element; (iv) a nucleic acid sequence encoding an exogenous effector (e.g., a therapeutic exogenous effector); and (v) the 3' portion of the ORF1 nucleic acid sequence; or the complement of (i) to (v) (e.g., in 5' to 3' order): the genetic element does not encode a full-length ORF1 polypeptide or a full-length ORF2 polypeptide; Genetic elements.

[0070] 30. The genetic element of embodiment 29, wherein the 3' portion of the ORF1 nucleic acid sequence comprises nucleotides 4367 to 5358 of SEQ ID NO:7, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0071] 31. The genetic element according to embodiment 29 or 30, wherein the 3' portion of the ORF1 nucleic acid sequence comprises 0-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 consecutive nucleotides of the sequence of nucleotides 283 to 2250 of SEQ ID NO:1, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0072] 32. The genetic element according to any of embodiments 29 to 31, wherein the genetic element does not comprise 1 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, or 900 to 1000 contiguous nucleotides from the 5' end of nucleotides 283 to 2250 of SEQ ID NO:1, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0073] 33. The genetic element of embodiment 29, wherein the 3' portion of the ORF1 nucleic acid sequence comprises nucleotides 4890 to 5284 of SEQ ID NO: 11, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0074] 34. The genetic element according to embodiment 29 or 30, wherein the 3' portion of the ORF1 nucleic acid sequence comprises 0-100, 100-200, 200-300, or 300-350, 350-360, 360-370, 370-380, 380-390, or 390-395 consecutive nucleotides of the sequence of nucleotides 4890-5284 of SEQ ID NO:11, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0075] 35. A genetic element according to any of embodiments 29 to 34, wherein the ORF2 nucleic acid sequence comprises nucleotides 101 to 391 of SEQ ID NO:1, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0076] 36. A genetic element according to any of embodiments 29 to 35, wherein the ORF2 nucleic acid sequence encodes an ORF2 molecule comprising SEQ ID NO:3, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0077] 37. A genetic element according to any of embodiments 29 to 36, wherein the 5' portion of the ORF2 nucleic acid sequence comprises nucleotides 3218 to 3385 of SEQ ID NO:7, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0078] 38. A genetic element according to any of embodiments 29 to 37, wherein the 5' portion of the ORF2 nucleic acid sequence comprises 0 to 50, 50 to 100, 100 to 150, 150 to 160, 160 to 165, or 165 to 168 consecutive nucleotides of the sequence of nucleotides 3218 to 3385 of SEQ ID NO: 7, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0079] 39. The genetic element according to any of embodiments 29-38, wherein the genetic element does not comprise 0-50, 50-100, 100-150, 150-160, 160-166, 166-170, 170-180, 180-190, 190-200, 200-225, 225-250, 250-275, 275-300, 300-310, 310-320, 320-330, 330-333 contiguous nucleotides from the 3' end of nucleotides 59-391 of SEQ ID NO:1, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0080] 40. The genetic element according to any of embodiments 29 to 39, further comprising at least one nucleotide (e.g., 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 75, 75 to 100, 100 to 110, 110 to 120, 120 to 130, 130 to 132, 132 to 135, 135 to 139, 139 to 140, 140 to 150, 150 to 160, 160 to 170, 170 to 180, 180 to 190, or 190 to 200 nucleotides) between the 5' portion of the ORF2 nucleic acid and the promoter.

[0081] 41. At least one nucleotide (e.g., 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 75, 75 to 100, 100 to 110, 110 to 120, 120 to 130, 130 to 135, 135 to 139, 139 to 140, 140 41. The genetic element of claim 29, further comprising:

[0082] 42. A genetic element according to any of embodiments 29 to 41, further comprising a Poly-A tail, e.g. a Poly-A tail located between the nucleic acid sequence encoding an exogenous effector and the 3' portion of the ORF1 nucleic acid sequence.

[0083] 43. At least one nucleotide (e.g., 1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-110, 110-120, 120-130, 130-135, 135-139, 139-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-280, 280-290, 290-300, 300-310, 310-320, 320-330, 330-340, 340-350, 340-360, 350-370, 350-380, 350-390, 360-400, 360-410, 360-420, 360-430, 360-440, 360-450, 360-460, 360-470, 360-480, 360-490, 360-500, 360-510, 360-520, 360-530, 360-540, 360-550, 360-560, 360-570, 360-580, 43. The genetic element of embodiment 42, further comprising between 50, 150 and 160, 160 and 170, 170 and 180, 180 and 190, 190 and 200, 200 and 250, 250 and 300, 300 and 310, 310 and 320, 320 and 323, 323 and 330, 330 and 340, 340 and 350, or 350 and 400 nucleotides.

[0084] 44. Below: (i) nucleotides 1-71 of SEQ ID NO:1, or a nucleic acid sequence having at least 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (ii) the 5′ portion of the ORF1 nucleic acid sequence; (iii) a promoter element; (iv) a nucleic acid sequence encoding an exogenous effector (e.g., a therapeutic exogenous effector); and (v) the 3' portion of the ORF1 nucleic acid sequence; or the complement of (i) to (v) (e.g., in 5' to 3' order): The genetic element does not encode the full-length ORF1 polypeptide, Genetic elements.

[0085] 45. The genetic element of embodiment 44, wherein the 5' portion of the ORF1 nucleic acid sequence comprises nucleotides 3400 to 3684 of SEQ ID NO:8, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0086] 46. ​​A genetic element according to any of embodiments 44 to 45, wherein the 5' portion of the ORF1 nucleic acid sequence comprises 0-100, 100-200, 200-300, 250-260, 260-270, 270-280, 280-284, 284-290, or 290-300 contiguous nucleotides of the sequence of nucleotides 283 to 2250 of SEQ ID NO:1, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0087] 47. A genetic element according to any of embodiments 44 to 46, wherein the 3' portion of the ORF1 nucleic acid sequence comprises nucleotides 4663 to 5358 of SEQ ID NO:8, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0088] 48. A genetic element according to any of embodiments 44 to 47, wherein the 3' portion of the ORF1 nucleic acid sequence comprises 0 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, or 600 to 700 consecutive nucleotides of the sequence from nucleotides 283 to 2250 of SEQ ID NO:1, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0089] 49. A genetic element comprising 1-100, 100-200, 200-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000, 2000-2100, 2100-2250, 2250-3000, 2300-2400, 2400-2500, 2500-3000, 2600-3000, 2700-3000, 2800-3000, 2900-3000, 3000-3500, 3100-3250, 3250-3300, 3300-3400, 3400-3500, 3500-3600, 3600-3700, 3700-3800, 3800-3900, 3900-4000, 4000-4500, 4000-4500, 4000-45 49. The genetic element according to any of embodiments 44 to 48, which does not comprise 0, 850-900, 900-950, 950-960, 960-970, 970-980, 980-987, 987-990, or 990-1000 contiguous nucleotides, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0090] 50. A genetic element according to any of embodiments 44 to 49, wherein the nucleic acid sequences of (iii) and (iv) are comprised in the part of nucleotides 283 to 2250 of SEQ ID NO: 1 which corresponds to the part of SEQ ID NO: 8 which is replaced by the nLuc expression cassette.

[0091] 51. The genetic element of embodiment 44, wherein the 5' portion of the ORF1 nucleic acid sequence comprises nucleotides 3400 to 3984 of SEQ ID NO:9, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0092] 52. The genetic element according to embodiment 44 or 51, wherein the 5' portion of the ORF1 nucleic acid sequence comprises 0-100, 100-200, 200-300, 300-400, 400-500, 500-600, 550-560, 560-570, 570-580, 580-584, 584-590, or 590-600 consecutive nucleotides of the sequence of nucleotides 283-2250 of SEQ ID NO:1, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0093] 53. A genetic element according to any of embodiments 44 or 51 to 52, wherein the 3' portion of the ORF1 nucleic acid sequence comprises nucleotides 4964 to 5358 of SEQ ID NO:9, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0094] 54. A genetic element according to any of embodiments 44 or 51 to 53, wherein the 3' portion of the ORF1 nucleic acid sequence comprises 0 to 100, 100 to 200, 200 to 300, 300 to 400, 350 to 360, 360 to 370, 370 to 380, 380 to 390, 390 to 394, or 394 to 400 consecutive nucleotides of the sequence of nucleotides 283 to 2250 of SEQ ID NO:1, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0095] 55. The genetic element according to any of embodiments 44 or 51 to 54, wherein the genetic element does not comprise 1 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, or 900 to 1000 consecutive nucleotides from the portion of nucleotides 283 to 2250 of SEQ ID NO: 1 corresponding to the portion of SEQ ID NO: 9 replaced by the nLuc expression cassette, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0096] 56. A genetic element according to any of embodiments 44 or 51 to 55, wherein the nucleic acid sequences of (iii) and (iv) are comprised in the part of nucleotides 283 to 2250 of SEQ ID NO: 1 which corresponds to the part of SEQ ID NO: 9 which is replaced by the nLuc expression cassette.

[0097] 57. The genetic element according to any of embodiments 44 to 56, further comprising at least one nucleotide (e.g., 1 to 5, 5 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 75, 75 to 100, 100 to 110, 110 to 120, 120 to 130, 130 to 135, 135 to 139, 139 to 140, 140 to 150, 150 to 160, 160 to 170, 170 to 180, 180 to 190, or 190 to 200 nucleotides) between the 5' portion of the ORF1 nucleic acid and the promoter.

[0098] 58. At least one nucleotide (e.g., 1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-110, 110-120, 120-130, 130-135, 135-139, 139-140, 58. The genetic element of embodiment 57, further comprising between 140 and 150, 150 and 160, 160 and 170, 170 and 180, 180 and 190, 190 and 200, 200 and 250, 250 and 300, 300 and 310, 310 and 320, 320 and 323, 323 and 330, 330 and 340, 340 and 350, or 350 and 400 nucleotides.

[0099] 59. A genetic element according to any of embodiments 44 to 58, further comprising a Poly-A tail, for example located between the nucleic acid sequence encoding an exogenous effector and the 3' portion of the ORF1 nucleic acid sequence.

[0100] 60. At least one nucleotide (e.g., 1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-110, 110-120, 120-130, 130-135, 135-139, 139-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-280, 280-290, 290-300, 300-310, 310-320, 320-330, 330-340, 340-350, 340-360, 350-370, 350-380, 350-390, 360-400, 360-410, 360-420, 360-430, 360-440, 360-450, 360-460, 360-470, 360-480, 360-490, 360-500, 360-510, 360-520, 360-530, 360-540, 360-550, 360-560, 360-570, 360-580, 60. The genetic element of embodiment 59, further comprising between 50, 150 and 160, 160 and 170, 170 and 180, 180 and 190, 190 and 200, 200 and 250, 250 and 300, 300 and 310, 310 and 320, 320 and 323, 323 and 330, 330 and 340, 340 and 350, or 350 and 400 nucleotides.

[0101] 61. A genetic element according to any of embodiments 44 to 60, further comprising an ORF2 nucleic acid sequence.

[0102] 62. The genetic element of embodiment 61, wherein the ORF2 nucleic acid sequence comprises nucleotides 101 to 391 of SEQ ID NO:1, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0103] 63. The genetic element of embodiment 61, wherein the ORF2 molecule comprises an amino acid sequence of SEQ ID NO:3 or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0104] 64. The genetic element of any of the preceding embodiments, wherein the ORF1 nucleic acid sequence comprises nucleotides 283 to 2250 of SEQ ID NO:1, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0105] 65. The genetic element of embodiment 64, wherein the 5' codon of the ORF1 nucleic acid sequence is ATG.

[0106] 66. The genetic element of embodiment 64, wherein the 5' codon of the ORF1 nucleic acid sequence is not ATG (e.g., the 5' codon of the ORF1 nucleic acid sequence is AAA).

[0107] 67. The genetic element of any of the previous embodiments, wherein the encoded ORF1 molecule comprises SEQ ID NO:2, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0108] 68. The genetic element of any of the preceding embodiments, further comprising nucleotides 2277-2462 of SEQ ID NO:1, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0109] 69. The genetic element of any of the previous embodiments, further comprising a sequence encoding SEQ ID NO:4, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0110] 70. The genetic element of any of the preceding embodiments, further comprising nucleotides 2515 to 2615 of SEQ ID NO:1, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0111] 71. A genetic element according to any of the preceding embodiments, further comprising a promoter.

[0112] 72. The genetic element of embodiment 71, wherein the promoter consists of a CMV promoter, e.g., a nucleic acid sequence of nucleotides 3525 to 3728 of SEQ ID NO: 8, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0113] 73. The genetic element of embodiment 71, wherein the promoter comprises a hEF1a promoter (e.g. a minimal hEF1a promoter), a UbC promoter, an MSCV promoter, an SFFV promoter, an hPGK promoter, a CMV promoter (e.g. a minimal CMV promoter), an INS84 promoter, or a U1a promoter.

[0114] 74. The genetic element of embodiment 71, wherein the promoter comprises an SV40 promoter, for example comprising the nucleic acid sequence of nucleotides 3417 to 3613 of SEQ ID NO: 11, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0115] 75. The genetic element of any of the preceding embodiments, further comprising a PolyA sequence (e.g., an SV40 Poly-A sequence comprising the nucleic acid sequence of nucleotides 4301-4349 of SEQ ID NO:7, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto).

[0116] 76. A genetic element according to any of the preceding embodiments, wherein the 5' codon of the ORF2 nucleic acid sequence is ATG.

[0117] 77. The genetic element of any of the preceding embodiments, wherein the 5' codon of the ORF2 nucleic acid sequence is not ATG (e.g. the 5' codon of the ORF2 nucleic acid sequence is AAA).

[0118] 78. A genetic element according to any of the preceding embodiments, wherein the 5' codon of the ORF1 nucleic acid sequence is ATG.

[0119] 79. The genetic element of any of the preceding embodiments, wherein the 5' codon of the ORF1 nucleic acid sequence is not ATG (e.g. the 5' codon of the ORF1 nucleic acid sequence is AAA).

[0120] 80. Below: (a) an anellovirus genomic sequence (e.g., the nucleic acid sequence of SEQ ID NO:1, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (b) a nucleic acid sequence of a genetic element according to any of the preceding embodiments; and (e.g., in 5' to 3' order).

[0121] 81. The nucleic acid molecule of embodiment 80, which is a plasmid.

[0122] 82. Below: (i) a proteinaceous exterior (e.g., a polypeptide comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, e.g., as listed in Table A1); (ii) a genetic element according to any of the preceding embodiments; and An anaerovector comprising: An anaerovector, in which the genetic elements are enclosed by a proteinaceous exterior.

[0123] 83. A method for producing an anellovector, comprising: (a) providing a cell, e.g., a host cell described herein; (b) introducing into the cell a nucleic acid molecule encoding an ORF1 polypeptide (e.g., comprising an amino acid sequence of an ORF1 protein set forth in Table A1, or a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto); (c) (e.g., before, after, or simultaneously with (b)), introducing into a cell a nucleic acid molecule of embodiment 80 or 81; (d) incubating the cells under conditions that allow the cells to produce the anaerovector; thereby producing the anaerovector. A method comprising:

[0124] 84. A method for producing an anellovector, comprising: (a) providing a cell (e.g., a host cell described herein) comprising a nucleic acid molecule encoding an ORF1 polypeptide (e.g., comprising an amino acid sequence of an ORF1 protein listed in Table A1, or a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto); (b) introducing into a cell the nucleic acid molecule of embodiment 80 or 81; (c) incubating the cells under conditions that allow the cells to produce the anaerovector; thereby producing the anaerovector. A method comprising:

[0125] 85. The method of embodiment 83 or 84, further comprising formulating the anellovector, for example, as a pharmaceutical composition suitable for administration to a subject.

[0126] 86. A pharmaceutical composition comprising an Anelloviridae vector (e.g., an anellovector), an ORF1 molecule, an ORF2 molecule, a VP1 molecule, a VP2 molecule, a genetic element, or a nucleic acid molecule according to any of the preceding embodiments, and a pharma- ceutical acceptable carrier and / or excipient.

[0127] 87. A pharmaceutical composition having the following characteristics: a) the pharmaceutical composition meets pharmaceutical or Good Manufacturing Practice (GMP) standards; b) the pharmaceutical composition is made in accordance with Good Manufacturing Practice (GMP); c) the pharmaceutical composition has a pathogen level below a predetermined threshold, e.g., is substantially free of pathogens; d) the pharmaceutical composition has a contaminant level below a predetermined threshold, e.g., is substantially free of contaminants; e) the pharmaceutical composition 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); or f) the pharmaceutical composition has low immunogenicity or is substantially non-immunogenic, e.g., as described herein; The pharmaceutical composition of embodiment 86, comprising one or more of:

[0128] 88. The pharmaceutical composition according to embodiment 86 or 87, wherein the pharmaceutical composition has a contaminant level below a predetermined reference value, e.g. is substantially free of contaminants.

[0129] 89. The pharmaceutical composition of embodiment 88, wherein the contaminant is selected from the group consisting of: mycoplasma, endotoxin, host cell nucleic acid (e.g., host cell DNA and / or host cell RNA), animal-derived impurities (e.g., serum albumin or trypsin), replication-competent agents (RCA), e.g., replication-competent viruses or unwanted Anelloviridae vectors (e.g., anellovectors) (e.g., an Anelloviridae vector (e.g., anellovector) other than a desired Anelloviridae vector (e.g., anellovector), e.g., a synthetic Anelloviridae vector (e.g., anellovector) described herein), free viral capsid proteins, adventitious material, and aggregates.

[0130] 90. The pharmaceutical composition of embodiment 88, wherein the contaminant is host cell DNA and the threshold amount is about 10 ng of host cell DNA per dose of the pharmaceutical composition.

[0131] 91. The pharmaceutical composition of any one of embodiments 86-90, wherein the pharmaceutical composition contains less than 10% by weight (e.g., less than about 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%) of contaminants.

[0132] 92. An intraocular delivery system comprising an Anelloviridae vector (e.g., an anellovector, such as those described herein).

[0133] 93. Below: (a) a nucleic acid molecule encoding an ORF1 and / or ORF2 polypeptide, or a VP1 and / or VP2 polypeptide according to any of the preceding embodiments, wherein the nucleic acid is a plasmid, a viral nucleic acid, or is integrated into a cellular chromosome; (b) a genetic element construct comprising a promoter element and a nucleic acid sequence (e.g., a DNA sequence) encoding an effector (e.g., an exogenous effector or an endogenous effector), and a protein binding sequence; In an isolated cell, e.g., a host cell, comprising Optionally, the isolated cell, e.g., a host cell, wherein the genetic element does not encode an ORF1 polypeptide (e.g., an ORF1 protein) or a VP1 polypeptide.

[0134] 94. Below: (i) a first nucleic acid molecule comprising a nucleic acid sequence of a genetic element of an Anelloviridae vector (e.g., anellovector) according to any of the preceding embodiments (optionally, the genetic element does not encode an ORF1 molecule or a VP1 molecule); (ii) a second nucleic acid molecule encoding an amino acid sequence of ORF1 or ORF2 listed in Table A1 or A2, or an amino acid sequence of VP1 or VP2 listed in Table A3, or an amino acid sequence having at least 70% (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity thereto; and An isolated cell, e.g., a host cell, comprising:

[0135] 95. A method for producing an Anelloviridae vector (e.g., anellovector) composition, the method comprising: (a) providing a cell, e.g., a host cell described herein; (b) introducing into the cell a genetic element construct encoding a genetic element of an Anelloviridae vector (e.g., anellovector) according to any of the preceding embodiments; (c) incubating the cells under conditions that allow the cells to produce an Anelloviridae vector (e.g., anellovector); and (d) formulating the anellovector, e.g., as a pharmaceutical composition suitable for administration to a subject; Including, This results in the production of an Anelloviridae vector (e.g., anellovector) composition.

[0136] 96. A method for producing an Anelloviridae vector (e.g., anellovector) composition, the method comprising: (a) providing a cell, e.g., a host cell described herein; (b) introducing into the cell a nucleic acid molecule encoding an ORF1 or ORF2 polypeptide listed in Table A1 or A2, or a VP1 polypeptide listed in Table A3 (or an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto); (c) introducing a genetic element construct into the cell (e.g. before, after or simultaneously with (b)); (d) incubating the cells under conditions that allow the cells to produce an Anelloviridae vector (e.g., anellovector); and (e) formulating the Anelloviridae vector (e.g., anellovector), e.g., as a pharmaceutical composition suitable for administration to a subject; This results in the production of an Anelloviridae vector (e.g., anellovector) composition.

[0137] 97. A method for producing an Anelloviridae vector (e.g., anellovirus vector) composition, the method comprising: (a) providing a cell, e.g., a host cell described herein; (b) introducing an ORF1, ORF2, VP1 or VP2 polypeptide into the cell; (c) introducing into the cell (e.g., before, after, or simultaneously with (b)) a genetic element construct listed in any of Tables N1-N4 (or a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto); and (d) incubating the cells under conditions that allow the cells to produce an Anelloviridae vector (e.g., anellovector); and (e) formulating the Anelloviridae vector (e.g., anellovector), e.g., as a pharmaceutical composition suitable for administration to a subject; This results in the production of an Anelloviridae vector (e.g., anellovector) composition.

[0138] 98. A method of making an Anelloviridae vector (e.g., anellovector), e.g., a synthetic Anelloviridae vector (e.g., anellovector), comprising: (a) The following: (i) a nucleic acid molecule, e.g., a first nucleic acid molecule, comprising a nucleic acid sequence of an anellovirus genome listed in any of Tables N1-N4 (or a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto); (ii) a nucleic acid molecule, e.g., a second nucleic acid molecule, encoding one or more of the amino acid sequences selected from, e.g., ORF1, ORF2, ORF2 / 2, ORF2 / 3, ORF1 / 1, ORF1 / 2, VP1, or VP2, listed in Tables A1-A3, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and providing a host cell comprising the (b) culturing the host cell under conditions suitable for production of an Anelloviridae vector (e.g., anellovector); A method comprising:

[0139] 99. The method of embodiment 98, further comprising, prior to step (a), introducing the first nucleic acid molecule and / or the second nucleic acid molecule into a host cell.

[0140] 100. The method of embodiment 98 or 99, wherein the second nucleic acid molecule is introduced into the host cell before, simultaneously with, or after the first nucleic acid molecule.

[0141] 101. The method of any of embodiments 95 to 100, further comprising isolating the Anelloviridae vector (e.g., the anellovector) from the cell.

[0142] 102. A method for producing an ORF1 molecule or a VP1 molecule, the method comprising: (a) providing a host cell (e.g., a host cell described herein) comprising a nucleic acid encoding an ORF1 polypeptide or a VP1 polypeptide of any of the preceding embodiments; and (b) maintaining the host cell under conditions that permit the cell to produce the polypeptide; Includes; This results in a method of producing an ORF1 or VP1 molecule.

[0143] 103. The method of any of embodiments 95 to 102, wherein the method comprises purifying the Anelloviridae vector using a CsCl gradient (e.g., as described in Example 20).

[0144] 104. The method of any of embodiments 95 to 103, wherein the method comprises purifying the Anelloviridae vector using a linear iodixanol gradient (e.g., as described in Example 20).

[0145] 105. A method of delivering an effector (e.g., an exogenous effector or an endogenous effector, e.g., overexpressing an endogenous effector) to a subject (e.g., to the subject's eye, e.g., to the subject's photoreceptors, retina, posterior cup (PEC), retinal ganglion, optic nerve, optic nerve head, retinal pigment epithelium (RPE), intravitreal space, or subretinal space), comprising administering to the subject (e.g., the subject's eye, e.g., to the subject's photoreceptors, retina, posterior cup (PEC), retinal ganglion, optic nerve head, subretinal space, intravitreal space, or retinal pigment epithelium (RPE)) an Anelloviridae vector (e.g., an anellovector) or pharmaceutical composition described in any of the preceding embodiments.

[0146] 106. A method of delivering an effector (e.g., an exogenous effector or an endogenous effector, e.g., overexpressing an endogenous effector) to a target cell (e.g., an ocular cell, e.g., a photoreceptor cell, a retinal cell, a cell of the posterior eye cup (PEC), a retinal ganglion cell, a cell of the optic nerve, a cell of the optic nerve head, or a retinal pigment epithelial (RPE) cell), the method comprising contacting the target cell with an Anelloviridae vector (e.g., an anellovector) described in any of the preceding embodiments.

[0147] 107. A method of ex vivo delivery of an effector (e.g., an exogenous effector or an endogenous effector, e.g., an overexpressing endogenous effector) to a target cell (e.g., a target cell isolated from a subject, e.g., a patient), the method comprising contacting the target cell with an Anelloviridae vector (e.g., an anellovector) described in any of the preceding embodiments.

[0148] 108. A method of modulating, e.g., enhancing or inhibiting, a biological function (e.g., as described herein) of a subject (e.g., an eye of the subject, e.g., the photoreceptors, retina, posterior cup (PEC), retinal ganglion, optic nerve, optic nerve head, subretinal space, intravitreal space, or retinal pigment epithelium (RPE) of the subject), the method comprising administering to the subject (e.g., the eye of the subject, e.g., the photoreceptors, retina, posterior cup (PEC), retinal ganglion, optic nerve, optic nerve head, subretinal space, intravitreal space, or retinal pigment epithelium (RPE) of the subject) an Anelloviridae vector (e.g., an anellovector) or pharmaceutical composition described in any of the preceding embodiments.

[0149] 109. A method of treating a disease or disorder (e.g., an ocular disease or disorder) in a subject in need thereof, the method comprising administering to the subject (e.g., an eye of the subject, e.g., to the subject's photoreceptors, retina, posterior cup (PEC), retinal ganglion, optic nerve, optic nerve head, subretinal space, intravitreal space, or retinal pigment epithelium (RPE)) an Anelloviridae vector (e.g., an anellovector) or pharmaceutical composition described in any of the preceding embodiments.

[0150] 110. Use of an Anelloviridae vector (e.g., anellovector) or pharmaceutical composition according to any of the preceding embodiments for treating a disease or disorder (e.g., as described herein) in a subject, optionally wherein the disease or disorder is an eye disease or disorder.

[0151] 111. An Anelloviridae vector (e.g., anellovector) or pharmaceutical composition according to any of the preceding embodiments for use in treating a disease or disorder (e.g., as described herein) in a subject, optionally wherein the disease or disorder is an eye disease or disorder.

[0152] 112. Use of an Anelloviridae vector (e.g., anellovector) or pharmaceutical composition of any of the preceding embodiments in the manufacture of a medicament for treating a disease or disorder (e.g., as described herein) in a subject, optionally wherein the disease or disorder is an eye disease or disorder.

[0153] 113. A method of delivering an effector (e.g., an exogenous effector or an endogenous effector, e.g., overexpressing an endogenous effector) to the eye of a subject (e.g., the photoreceptors, retina, posterior cup (PEC), retinal ganglion, optic nerve, optic nerve head, subretinal space, intravitreal space, or retinal pigment epithelium (RPE) of the subject), comprising administering an Anelloviridae vector (e.g., an anellovector) to the eye of the subject (e.g., the photoreceptors, retina, posterior cup (PEC), retinal ganglion, optic nerve, optic nerve head, subretinal space, intravitreal space, or retinal pigment epithelium (RPE) of the subject).

[0154] 114. A method of delivering an effector (e.g., an exogenous effector or an endogenous effector, e.g., overexpressing an endogenous effector) to an ocular cell (e.g., a photoreceptor cell, a retinal cell, a cell of the posterior ocular cup (PEC), a retinal ganglion cell, a cell of the optic nerve, a cell of the optic nerve head, or a retinal pigment epithelial (RPE) cell), the method comprising contacting an ocular cell with an Anelloviridae vector (e.g., an anellovector) described in any of the preceding embodiments.

[0155] 115. A method of ex vivo delivery of an effector (e.g., an exogenous effector or an endogenous effector, e.g., an overexpressing endogenous effector) to a target ocular cell (e.g., a target ocular cell isolated from a subject, e.g., a patient), the method comprising contacting the target ocular cell with an Anelloviridae vector (e.g., an anellovector) described in any of the preceding embodiments.

[0156] 116. A method of regulating, e.g., enhancing or inhibiting, a biological function (e.g., as described herein) of a subject's eye (e.g., the subject's photoreceptors, retina, posterior cup (PEC), retinal ganglion, optic nerve, optic nerve head, subretinal space, intravitreal space, or retinal pigment epithelium (RPE)), the method comprising administering to the subject's eye (e.g., the subject's photoreceptors, retina, posterior cup (PEC), retinal ganglion, optic nerve, optic nerve head, subretinal space, intravitreal space, or retinal pigment epithelium (RPE)) an Anelloviridae vector (e.g., anellovector) or pharmaceutical composition described in any of the preceding embodiments.

[0157] 117. The method of embodiment 116, wherein the biological function comprises one or more of best corrected visual acuity (BCVA), retinal sensitivity to light (e.g., as measured by visual field measurement or microperimetry, e.g., dark-adapted and light-adapted, full-field, multifocal, focal or pattern electroretinogram ERG), contrast sensitivity, reading speed, and / or color vision.

[0158] 118. The method of embodiment 116 or 117, wherein the biological function is measured using clinical biomicroscopy, fundus photography, optical coherence tomography (OCT), fundus autofluorescence (FAF), infrared and / or polychromatic imaging, fluorescein or ICG angiography, and / or adaptive optics.

[0159] 119. A method of treating a disease or disorder (e.g., an ocular disease or disorder) in a subject in need thereof, the method comprising administering to the subject's eye (e.g., to the subject's photoreceptors, retina, posterior cup (PEC), retinal ganglion, optic nerve, optic nerve head, subretinal space, intravitreal space, or retinal pigment epithelium (RPE)) an Anelloviridae vector (e.g., an anellovector) or pharmaceutical composition described in any of the preceding embodiments.

[0160] 120. Use of an Anelloviridae vector (e.g., anellovector) or pharmaceutical composition according to any of the preceding embodiments for treating a disease or disorder (e.g., as described herein) in a subject, wherein the disease or disorder is an eye disease or disorder.

[0161] 121. The Anelloviridae vector (e.g., anellovector) or pharmaceutical composition of any of the preceding embodiments for use in treating a disease or disorder (e.g., as described herein) in a subject, wherein the disease or disorder is an ocular disease or disorder.

[0162] 122. Use of an Anelloviridae vector (e.g., anellovector) or pharmaceutical composition of any of the preceding embodiments in the manufacture of a medicament for treating a disease or disorder (e.g., as described herein) in a subject, wherein the disease or disorder is an eye disease or disorder.

[0163] 123. A method, use, or an Anelloviridae vector (e.g. an Anellovirus vector) or pharmaceutical composition or use according to any one of claims 109 to 122, wherein the disease or disorder is a monofactorial disease.

[0164] 124. A method, use, or Anelloviridae vector (e.g., anellovector) or pharmaceutical composition or use according to any one of claims 109 to 123, wherein the disease or disorder is a multifactorial disease (e.g., glaucoma).

[0165] 125. A method, use, or Anelloviridae vector (e.g., anellovector) or pharmaceutical composition or use according to any one of claims 109 to 124, wherein the disease or disorder is macular degeneration (e.g., age-related macular degeneration (AMD), Stargardt's disease, or myopic macular degeneration).

[0166] 126. The method, use, or Anelloviridae vector (e.g., anellovector) or pharmaceutical composition or use according to claim 125, wherein the macular degeneration is wet AMD.

[0167] 127. The method, use, or Anelloviridae vector (e.g., anellovector) or pharmaceutical composition or use of claim 125, wherein the macular degeneration is dry AMD (e.g., AMD with geographic atrophy).

[0168] 128. A method, use, or Anelloviridae vector (e.g. anellovector) or pharmaceutical composition or use according to any one of claims 109 to 127, wherein the disease or disorder is a retinal disease.

[0169] 129. The method, use, or Anelloviridae vector (e.g., anellovector) or pharmaceutical composition or use of claim 128, wherein the retinal disease is an inherited retinal disease (IRD), such as a disease as described in Stone et al. (2017, Ophthalmology; incorporated herein by reference with respect to the diseases and disorders described therein).

[0170] 130. The method, use, or Anelloviridae vector (e.g., anellovector) or pharmaceutical composition or use of claim 128, wherein the retinal disease is retinitis pigmentosa (e.g., X-linked retinitis pigmentosa (XLRP)).

[0171] 131. The method, use, or Anelloviridae vector (e.g., anellovector) or pharmaceutical composition or use of any of claims 109 to 130, wherein the disease or disorder is a VEGF-related disorder (e.g., a cancer described herein; macular edema; or proliferative retinopathy).

[0172] 132. The method, use, or pharmaceutical composition or use of any of claims 109 to 131, wherein the disease or disorder is selected from the group consisting of: retinal leakage, Leber's congenital amaurosis (LCA) (e.g., wherein the genetic element comprises a human RPE65 sequence, e.g., a sequence encoding a human RPE65 protein, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto), congenital amaurosis, cone-rod dystrophy, choroideremia, vitelliform macular dystrophy, hyperferritinemia-cataract syndrome, optic atrophy, XLR retinoschisis, cytomegalovirus retinitis, color vision deficiency, Leber's hereditary optic neuropathy, keratitis, uveitis, Graeve's ophthalmopathy, diabetic retinopathy, or diabetic macular edema.

[0173] 133. A method, use, or Anelloviridae vector (e.g., an Anelloviridae vector) or pharmaceutical composition or use according to any of claims 109 to 132, wherein the Anelloviridae vector is administered to a subject in the subretinal or subretinal space, in the intravitreal or intravitreal space, or in the suprachoroidal or suprachoroidal space.

[0174] 134. A method, use, or Anelloviridae vector (e.g., an Anelloviridae vector) or pharmaceutical composition or use according to any one of claims 109 to 133, wherein the Anelloviridae vector is administered to the subject subretinaly or into the subretinal space.

[0175] 135. A method, use, or pharmaceutical composition or use according to any one of claims 109 to 134, wherein the Anelloviridae vector is administered to the subject intravitreally or into the intravitreal cavity.

[0176] 136. A method, use, or Anelloviridae vector (e.g., anellovector) or pharmaceutical composition or use according to any one of claims 109 to 135, wherein the Anelloviridae vector is administered to the subject above the choroid or into the suprachoroidal space.

[0177] 137. A method, use, or Anelloviridae vector (e.g., anellovector) or pharmaceutical composition or use according to any of claims 109 to 136, wherein the Anelloviridae vector is administered to the subject via an SCS microinjector, via a cannula, and / or via a needle.

[0178] 138. The genetic element, Anelloviridae vector (e.g. anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element is single stranded.

[0179] 139. The genetic element, Anelloviridae vector (e.g. anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element is circular.

[0180] 140. The genetic element, Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element comprises DNA.

[0181] 141. The genetic element, Anelloviridae vector (e.g. anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element is double-stranded.

[0182] 142. The genetic element, Anelloviridae vector (e.g. anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element is linear.

[0183] 143. The genetic element, Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element comprises RNA.

[0184] 144. The genetic element, Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element comprises a nucleic acid sequence encoding an Anelloviridae capsid protein, e.g., an Anellovirus ORF1 molecule or a CAV VP1 molecule (e.g., an ORF1 protein or VP1 protein listed in Tables A1-A3, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto).

[0185] 145. The genetic element, Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element does not comprise a nucleic acid sequence encoding an Anelloviridae capsid protein, such as an Anellovirus ORF1 molecule or a CAV VP1 molecule (e.g., an ORF1 protein or VP1 protein listed in Tables A1-A3, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto).

[0186] 146. The genetic element, Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element comprises a nucleic acid sequence encoding an anellovirus ORF2 molecule or VP2 molecule (e.g., an ORF2 protein listed in Table A1 or A2 or a VP2 protein listed in Table A3, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto).

[0187] 147. The genetic element, Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element does not comprise a nucleic acid sequence encoding an Anellovirus ORF2 molecule or a CAV VP2 molecule (e.g., an ORF2 protein or VP1 protein listed in Tables A1-A3, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto).

[0188] 148. The genetic element, Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element comprises at least 20, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides having a GC content of at least 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0189] 149. The proteinaceous exterior has the amino acid sequence YNPX. 2 DXGX 2 N (SEQ ID NO: 829), where X n is a contiguous sequence of any n amino acids.

[0190] 150. Amino acid sequence YNPX 2 DXGX 2 The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of embodiment 149, wherein N (SEQ ID NO: 829) is included in the N22 domain.

[0191] 151. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the ORF1 or VP1 molecule comprises an arginine-rich region (e.g., having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an arginine-rich region sequence of an ORF1 protein or VP1 protein listed in Tables A1-A3).

[0192] 152. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the proteinaceous exterior comprises an amino acid sequence of at least 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, or 50 contiguous nucleotides, at least 40% (e.g., at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 80%, 85%, 90%, or 95%) of which are arginine residues.

[0193] 153. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of embodiment 151 or 152, wherein the arginine-rich region is located at the N-terminus or C-terminus of the ORF1 molecule or VP1 molecule.

[0194] 154. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the ORF1 molecule or VP1 molecule comprises a jelly-roll domain having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a jelly-roll domain sequence of an ORF1 or VP1 protein listed in Tables A1-A3.

[0195] 155. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the ORF1 molecule or VP1 molecule comprises an N22 domain having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an N22 domain sequence of an ORF1 or VP1 protein listed in Tables A1-A3.

[0196] 156. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the ORF1 molecule or VP1 molecule comprises a C-terminal domain (CTD) having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a CTD domain sequence of an ORF1 or VP1 protein listed in Tables A1-A3.

[0197] 157. The genetic element, Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element comprises one or more of the following: a TATA box, an initiation element, a cap site, a transcription start site, an ORF1 / 1 coding sequence, an ORF1 / 2 coding sequence, an ORF2 / 2 coding sequence, an ORF2 / 3 coding sequence, an ORF2 / 3t coding sequence, three open reading frame regions, a poly(A) signal, and / or a GC-rich region from an Anellovirus or CAV described herein (e.g., as listed in any of Tables N1-N4), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0198] 158. The genetic element, Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element has at least 75% (e.g., at least 75, 76, 77, 78, 79, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) sequence identity to a 5'UTR conserved domain sequence listed in any of Tables N1-N4.

[0199] 159. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the proteinaceous exterior comprises one or more of the following: one or more glycosylated proteins, a hydrophilic DNA-binding region, an arginine-rich region, a threonine-rich region, a glutamine-rich region, an N-terminal polyarginine sequence, a variable region, a C-terminal polyglutamine / glutamic acid sequence, and one or more disulfide bridges.

[0200] 160. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the proteinaceous exterior comprises one or more of the following characteristics: icosahedral symmetry, recognition and / or binding to molecules that interact with one or more host cells and mediate entry into the host cells, lack of lipid molecules, lack of carbohydrates, inclusion of one or more desired carbohydrates (e.g., glycosylation), pH and temperature stability, detergent resistance, and being substantially non-immunogenic or substantially non-pathogenic in host cells.

[0201] 161. The genetic element, Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the promoter comprises 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).

[0202] 162. The genetic element, Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the effector encodes a therapeutic agent, e.g., a therapeutic peptide or polypeptide, or a therapeutic nucleic acid.

[0203] 163. The genetic element, Anelloviridae vector (e.g. anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the effector is an exogenous effector.

[0204] 164. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the effector is an endogenous effector (e.g., where the anellovector overexpresses an endogenous effector in the target cell).

[0205] 165. The effector may be any of the following: regulatory nucleic acids, e.g., miRNA, siRNA, mRNA, lncRNA, RNA, DNA, antisense RNA, gRNA; fluorescent tags or markers, antigens, peptides, synthetic or analogous peptides from natural bioactive peptides, agonist or antagonist peptides, antimicrobial peptides, pore-forming peptides, bicyclic peptides, targeting or cytotoxic peptides, degradative or self-destructing peptides, small molecules, immune effectors (e.g., affecting sensitivity to immune responses / signals), cell death proteins (e.g., inducers of apoptosis or necrosis), non-lytic inhibitors of tumors (e.g., oncoproteins), or a nucleic acid molecule or method according to any of the preceding embodiments, comprising an inhibitor of a protein, an epigenetic modifier, an epigenetic enzyme, a transcription factor, a DNA or protein modifying enzyme, a DNA intercalating agent, an efflux pump inhibitor, a nuclear receptor activator or inhibitor, a proteasome inhibitor, a competitive inhibitor for an enzyme, a protein synthesis effector or inhibitor, a nuclease, a protein fragment or domain, a ligand, an antibody, a receptor, or a CRISPR system or component.

[0206] 166. The genetic element, Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the effector comprises a miRNA, e.g., the miRNA reduces expression of a target gene.

[0207] 167. The genetic element, Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the effector modulates gene or protein expression or activity, e.g., increases or decreases gene or protein expression or activity.

[0208] 168. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the Anelloviridae vector is capable of autonomous replication.

[0209] 169. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the Anelloviridae vector is replication-deficient (e.g., unable to replicate autonomously).

[0210] 170. The genetic element, Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the genetic element is integrated 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.

[0211] 171. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the Anelloviridae vector is substantially non-pathogenic, e.g., does not induce detectable adverse symptoms in a subject (e.g., increased cell death or toxicity compared to a subject not exposed to the anellovector).

[0212] 172. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the Anelloviridae vector is substantially non-immunogenic, e.g., does not induce a detectable and / or unwanted immune response.

[0213] 173. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein a population of at least 1000 Anelloviridae vectors is capable of delivering at least about 100 copies (e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 copies) of a genetic element to one or more eukaryotic cells (e.g., mammalian cells, e.g., human cells).

[0214] 174. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein a population of Anelloviridae vectors (e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 genome equivalents of genetic elements per cell) is capable of delivering genetic elements to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more of a population of eukaryotic cells (e.g., mammalian cells, e.g., human cells).

[0215] 175. A population of Anelloviridae vectors (e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 genome equivalents of genetic elements per cell) that transmits at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 8,000, 1 x 10 4 , 1×10 5 , 1×10 6 , 1×10 7

[0023] The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, capable of delivering one or more of the vectors to a population of eukaryotic cells (e.g., mammalian cells, e.g., human cells).

[0216] 176. A population of Anelloviridae vectors (e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 genome equivalents of genetic elements per cell) is capable of transmitting 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 5-10, 10-20, 20-50, 50-100, 100-1000, 1000-1000 copies of the genetic element per cell. 4 , 1×10 4 ~1×10 5 , 1×10 4 ~1×10 6 , 1×10 4 ~1×10 7 , 1×10 5 ~1×10 6 , 1×10 5 ~1×10 7 , or 1 × 10 6 ~1×10 7The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, capable of being delivered to a population of eukaryotic cells (e.g., mammalian cells, e.g., human cells).

[0217] 177. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein target cells to which the genetic element is delivered each receive at least 10, 50, 100, 500, 1000, 10,000, 50,000, 100,000, or more copies of the genetic element.

[0218] 178. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the Anelloviridae vector is resistant to degradation by detergents (e.g., mild detergents, e.g., bile salts, e.g., sodium deoxycholate) compared to viral particles comprising an outer lipid bilayer, e.g., retroviruses.

[0219] 179. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the previous embodiments, wherein the genetic elements enclosed by the proteinaceous exterior are resistant to degradation by a nuclease enzyme (e.g., DNase).

[0220] 180. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the Anelloviridae vector is capable of infecting a mammalian cell, e.g., a human cell, e.g., in vitro, in vivo, or ex vivo.

[0221] 181. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, wherein the Anelloviridae vector selectively delivers the effector to, or is present at higher levels (e.g., selectively accumulates) in, a desired cell type, tissue, or organ (e.g., bone marrow, blood, heart, gastrointestinal tract, skin, retinal photoreceptors, epithelial lining, or pancreas).

[0222] 182. The genetic element or genetic element construct is replicable (e.g. by rolling circle replication) and can replicate at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 10, 25, 30, 35 ... 2 , 2×10 2 , 5×10 2 , 10 3 , 2×10 3 , 5×10 3 , or 10 4 The genetic element, Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the preceding embodiments, capable of generating a genome equivalent of the genetic element.

[0223] 183. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the previous embodiments, wherein the proteinaceous exterior is provided in cis relative to the genetic element.

[0224] 184. The Anelloviridae vector (e.g., anellovector), ORF1 molecule, ORF2 molecule, VP1 molecule, VP2 molecule, nucleic acid molecule, or method of any of the previous embodiments, wherein the proteinaceous exterior is provided in trans to the genetic element.

[0225] 185. A method of delivering an exogenous effector to the posterior eye cup (PEC) of a subject, the method comprising: (i) a genetic element comprising a nucleic acid encoding an exogenous effector; (ii) a proteinaceous exterior that encapsulates the genetic element; The method includes administering to the subject's PEC an Anelloviridae vector comprising:

[0226] 186. A method for delivering an exogenous effector to the retinal pigment epithelium (RPE) of a subject, the method comprising: (i) a genetic element comprising a nucleic acid encoding an exogenous effector; (ii) a proteinaceous exterior that encapsulates the genetic element; The method includes administering to the RPE of a subject an Anelloviridae vector comprising:

[0227] 187. A method for delivering an exogenous effector to the retina of a subject, the method comprising: (i) a genetic element comprising a nucleic acid encoding an exogenous effector; (ii) a proteinaceous exterior that encapsulates the genetic element; 20. A method comprising administering to the retina of a subject an Anelloviridae vector comprising:

[0228] 188. The method of any of embodiments 185 to 187, wherein the genetic element comprises a nucleic acid sequence of nucleotides 1 to 71 of SEQ ID NO:1, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0229] 189. A genetic element comprising: (i) a nucleic acid sequence of nucleotides 1 to 100 of SEQ ID NO:1, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and / or (ii) a nucleic acid sequence of nucleotides 2463 to 2876 of SEQ ID NO:1, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. The method of any one of embodiments 185 to 188, comprising:

[0230] 190. The method of any of embodiments 185-189, wherein the proteinaceous exterior comprises an ORF1 molecule having an amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0231] 191. The method of any of embodiments 185-187, wherein the genetic element comprises a nucleic acid sequence of nucleotides 323-393 of SEQ ID NO:54, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0232] 192. A genetic element comprising: (i) a nucleic acid sequence of nucleotides 1 to 423 of SEQ ID NO: 54, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and / or (ii) a nucleic acid sequence from nucleotides 2813 to 2979 of SEQ ID NO: 54, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. The method of any one of embodiments 185 to 187 or 191, comprising:

[0233] 193. The method of any of embodiments 185-187, 191, or 192, wherein the proteinaceous exterior comprises an ORF1 molecule having an amino acid sequence of SEQ ID NO:58, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0234] 194. The method of any of embodiments 185-187, wherein the genetic element comprises a nucleic acid sequence of nucleotides 1-374 of SEQ ID NO:5, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0235] 195. A genetic element comprising: (i) a nucleic acid sequence of nucleotides 1 to 374 of SEQ ID NO:5, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and / or (ii) a nucleic acid sequence of nucleotides 2197 to 2313 of SEQ ID NO:5, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. The method of any one of embodiments 185 to 187 or 194, comprising:

[0236] 196. The method of any of embodiments 185-187, or 195, wherein the proteinaceous exterior comprises VP1 having an amino acid sequence of SEQ ID NO: 251, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0237] 197. The method of any of embodiments 185 to 196, wherein the Anelloviridae vector is substantially free of wild-type Anellovirus genome.

[0238] 198. The method of any of embodiments 185 to 197, wherein the anellovirus vector genetic element, or DNA comprising the nucleic acid sequence thereof, is detectable at least 21 or 49 days after administration.

[0239] 199. The method of any of embodiments 185 to 198, resulting in a level of at least 100 copies of an RNA molecule encoding an exogenous effector per 50 ng of RNA.

[0240] 200. The method of any of embodiments 185-199, which provides a level of at least 100 copies of an RNA molecule encoding an exogenous effector per 50 ng of RNA to the posterior eye cup.

[0241] 201. The method of any of embodiments 185-200, which provides a level of at least 10 copies of an RNA molecule encoding an exogenous effector per 50 ng of RNA to the retina.

[0242] 202. The method of any of embodiments 185-201, which results in a level of at least 100 copies of an RNA molecule encoding an exogenous effector per 50 ng of RNA in the posterior eye cup and less than 10 copies of an RNA molecule encoding an exogenous effector per 50 ng of RNA in the retina.

[0243] 203. The method of any of embodiments 185-202, wherein the subject has a monogenic or polygenic disease.

[0244] 204. The method of any of embodiments 185-203, wherein the subject has macular degeneration (e.g., age-related macular degeneration (AMD), e.g., wet AMD or dry AMD).

[0245] 205. The method of any of embodiments 185-204, wherein the subject has a retinal disease or a VEGF-related disorder, e.g., as described herein.

[0246] 206. A method of delivering an effector to a subject, the method comprising subretinal administration of an Anelloviridae vector (e.g., as described herein) to the subject.

[0247] 207. A method of delivering an effector to a subject, the method comprising intravitreally administering to the subject an Anelloviridae vector (e.g., as described herein).

[0248] 208. The method of embodiment 206 or 207, which results in transduction of retinal cells and / or PEC cells.

[0249] 209. The method of embodiment 206 or 207, which results in the transduction of RPE cells and / or PEC cells.

[0250] 210. A method of treating a disease or disorder selected from: a monogenic disease, a polygenic disease, a macular degeneration (e.g., AMD, e.g., wet AMD or dry AMD), a retinal disease, or a VEGF-related disorder (e.g., as described herein), the method comprising administering to a subject an Anelloviridae vector (e.g., as described herein).

[0251] 211. The method of any of embodiments 185-210, wherein the Anelloviridae vector is administered in an amount effective to result in a concentration of 0.330 μg / mL of the exogenous effector in the vitreous humor of the subject, for example, maintained for at least 3 months after administration.

[0252] 212. The method of embodiment 211, wherein the concentration of exogenous effector in the vitreous humor of the subject after 3 months is between 1.70 and 6.60 μg / mL.

[0253] 213. The method of any of embodiments 185-212, wherein the Anelloviridae vector is administered in an amount effective to result in a concentration of the exogenous effector of 0.110 μg / mL in the aqueous humor of the subject, for example, maintained for at least 3 months after administration.

[0254] 214. The method of embodiment 213, wherein the concentration of exogenous effector in the vitreous humor of the subject after 3 months is 0.567 to 2.20 μg / mL.

[0255] 215. The method of any of embodiments 185-214, wherein the Anelloviridae vector is administered in a volume of 0.1 mL to 0.5 mL.

[0256] 216. The method of any of embodiments 185-215, wherein the Anelloviridae vector is administered at a dosage of 1.2e+10 viral genomes (vg) / mL to 1.2e+11 vg / mL.

[0257] 217. The method of any of embodiments 185-216, wherein the Anelloviridae vector is administered at a dose of 1.2e+7vg / eye to 1.2e+8vg / eye.

[0258] 218. The following: (i) a genetic element comprising a nucleic acid sequence encoding an exogenous effector; (ii) a proteinaceous exterior that encapsulates the genetic element; A preparation comprising an Anelloviridae vector at a concentration of at least 2.48E+08 copies of a genetic element per mL, comprising (a) the genetic element comprises a nucleic acid sequence of nucleotides 1 to 71 of SEQ ID NO:1, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and / or (b) A preparation, wherein the proteinaceous exterior comprises an ORF1 molecule having an amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0259] 219. The preparation of embodiment 218, which is substantially free of wild-type Anellovirus.

[0260] 220. An ocular delivery device comprising an Anelloviridae vector (e.g., an anellovector, e.g., as described herein).

[0261] 221. The ocular delivery device of embodiment 220, configured for suprachoroidal injection.

[0262] 222. The ocular delivery device of embodiment 220, configured for subretinal administration.

[0263] 223. An ocular delivery device as described in embodiment 222, comprising a catheter and a needle passing through the catheter (e.g., into the subretinal space of the subject).

[0264] 224. The ocular delivery device of embodiment 220, configured for intravitreal administration.

[0265] 225. An ocular delivery device according to any of embodiments 220-224, comprising a microinjector (e.g., including a microneedle), a cannula (e.g., a fine cannula), and / or a syringe.

[0266] Other features, objects, and advantages of the invention will become apparent from the specification, the drawings, and from the claims.

[0267] 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.

[0268] 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, there are shown in the drawings embodiments which are illustrated herein. It should be understood, however, that the invention is not limited to the specific arrangements and instrumentalities of the embodiments shown in the drawings. [Brief description of the drawings]

[0269] [Figure 1] (FIG. 1A) Percentage of sequence similarity of amino acid regions of capsid protein sequences. (FIG. 1B) Percentage of sequence similarity of capsid protein sequences. [Diagram 2] FIG. 1 shows one embodiment of an anellovector. [Diagram 3] A schematic diagram of the kanamycin vector encoding the LY1 strain of TTMiniV ("anellovector1") is shown. [Figure 4] A schematic diagram of the kanamycin vector encoding the LY2 strain of TTMiniV ("anellovector2") is shown. [Diagram 5] 1 shows the transfection efficiency of synthetic anellovectors in 293T cells and A549 cells. [Figure 6A] Quantitative PCR results showing successful infection of 293T cells with synthetic anellovectors are presented. [Figure 6B] Quantitative PCR results showing successful infection of 293T cells with synthetic anellovectors are presented. [Figure 7A] Quantitative PCR results showing successful infection of A549 cells with synthetic anellovectors are presented. [Figure 7B] Quantitative PCR results showing successful infection of A549 cells with synthetic anellovectors are presented. [Figure 8A] Quantitative PCR results demonstrating successful infection of Raji cells with synthetic anellovectors are presented. [Figure 8B] Quantitative PCR results demonstrating successful infection of Raji cells with synthetic anellovectors are presented. [Figure 9A] Quantitative PCR results showing successful infection of Jurkat cells with synthetic anellovectors are presented. [Figure 9B] Quantitative PCR results showing successful infection of Jurkat cells with synthetic anellovectors are presented. [Figure 10A] Quantitative PCR results demonstrating successful infection of Chang cells with the synthetic anellovectors are shown. [Figure 10B] Quantitative PCR results demonstrating successful infection of Chang cells with the synthetic anellovectors are shown. [Figure 11AB]

[0023] Figure 1 is a series of graphs showing luciferase expression from cells transfected or infected with TTMV-LY2 A574-1371, A1432-2210, 2610::nLuc. Luminescence was observed in infected cells, indicating successful replication and packaging. [Figure 11C]Diagram depicting the phylogenetic tree of Alphatorquevirus (Torque Teno Virus; TTV) with clades highlighted. At least 100 Anellovirus strains are represented. Exemplary sequences from several clades are provided herein. [Figure 12] FIG. 1 is a schematic diagram showing an exemplary workflow for the production of anellovectors (e.g., replication-competent or replication-deficient anaellovectors described herein). [Figure 13] 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 clear peak for each amplification product with TTMV or TTV specific primer sets, as indicated, on plasmids encoding the respective genomes. [Figure 14] A series of graphs showing PCR efficiency for quantification of TTV genome equivalents by qPCR. Increasing concentrations of primers and a constant concentration of hydrolysis probe (250 nM) were used together with two commercially available qPCR master mixes. Efficiencies ranging from 90 to 110% ensured minimal error propagation during quantification. [Figure 15] Graph showing exemplary amplification plots of linear amplification of TTMV (target 1) or TTV (target 2) across 7 log10 genome equivalent concentrations. Genome equivalents were quantified across seven 10-fold dilutions with high PCR efficiency and linearity (R2 TTMV: 0.996; R2 TTV: 0.997). [Figure 16A] FIG. 1 is a series of graphs showing quantification of TTMV genome equivalents in anellovector stocks. (A) Amplification plots of two stocks, each diluted 1:10 and run in duplicate. (B) The same two samples shown in panel A, in this case shown relative to the linear range. Upper and lower limits of two representative samples are shown. PCR efficiency: 99.58%, R2:0988. [Figure 16B]FIG. 1 is a series of graphs showing quantification of TTMV genome equivalents in anellovector stocks. (A) Amplification plots of two stocks, each diluted 1:10 and run in duplicate. (B) The same two samples shown in panel A, in this case shown relative to the linear range. Upper and lower limits of two representative samples are shown. PCR efficiency: 99.58%, R2:0988. [Figure 17] Graph showing the fold change in miR-625 expression in HEK293T cells transfected with the indicated plasmids. [Figure 18] Figure 1 shows pairwise identity for an alignment of representative sequences from each Alphatorquevirus clade. DNA sequences of TTV-CT30F, TTV-P13-1, TTV-tth8, TTV-HD20a, TTV-16, TTV-TJN02, and TTV-HD16d were aligned. Percent pairwise identity over a 50 bp sliding window along the length of the alignment is shown. Upper brackets indicate noncoding and coding regions with pairwise identity indicated. Lower brackets indicate regions of high or low sequence conservation. [Figure 19] Figure 1 shows pairwise identity of amino acid alignments of putative proteins across seven Alphatorquevirus clades. Putative protein amino acid sequences from TTV-CT30F, TTV-P13-1, TTV-tth8, TTV-HD20a, TTV-16, TTV-TJN02, and TTV-HD16d were aligned. Percentage of pairwise identity over a sliding window of 15 aa along the length of each alignment is shown. Pairwise identity of open reading frame DNA sequence and protein amino acid sequence is shown. (*) Putative ORF2t / 3 amino acid sequences were aligned for TTV-CT30F, TTV-tth8, TTV-16, and TTV-TJN02. [Figure 20]FIG. 1 shows that domains within the 5'UTR are highly conserved across the seven Alphatorquevirus clades (SEQ ID NOs: 810-817, respectively, in order of appearance). The 71 bp 5'UTR conserved domain sequences of each representative Alphatorquevirus were aligned. The sequences have 95.2% pairwise identity across the seven clades. [Figure 21] Figure 1 shows an alignment of GC-rich domains from seven Alphatorquevirus clades. Each anellovirus has a region downstream of the ORF with a GC content of over 70%. Alignment of the GC-rich regions of TTV-CT30F, TTV-P13-1, TTV-tth8, TTV-HD20a, TTV-16, TTV-TJN02, and TTV-HD16d is shown. These regions vary in length, but share 75.4% pairwise identity where they match. [Figure 22] FIG. 1 shows infection of Raji B cells with anellovectors encoding miRNAs targeting n-myc interacting protein (NMI), and quantification of the genome equivalent of anellovector detected following infection of Raji B cells (arrow) or control cells with anellovectors encoding NMI miRNAs. [Diagram 23] Figure 1 shows infection of Raji B cells with anellovectors encoding miRNAs targeting n-myc interacting protein (NMI). Western blots revealed that anellovectors encoding miRNAs against NMI reduced NMI protein expression in Raji B cells, whereas Raji B cells infected with anellovectors lacking miRNA showed NMI protein expression equivalent to controls. [Figure 24] 1 is a series of graphs showing quantification of anaerovector particles produced in host cells following infection with anaerovectors containing endogenous miRNA coding sequences and corresponding anaerovectors lacking the endogenous miRNA coding sequences. [Figure 25A]A series of figures showing the subcellular localization of ORFs from TTMV-LY2 fused with nano-luciferase. (A) In Vero cells, ORF2 (top) was found to be localized in the cytoplasm, and ORF1 / 1 (bottom) were found to be localized in the nucleus. (B) In HEK293 cells, ORF2 (top) was found to be localized in the cytoplasm, and ORF1 / 1 (bottom) were found to be localized in the nucleus. (C) Localization patterns of ORF1 / 2 and ORF2 / 2 in cells. [Figure 25B] A series of figures showing the subcellular localization of ORFs from TTMV-LY2 fused with nano-luciferase. (A) In Vero cells, ORF2 (top) was found to be localized in the cytoplasm, and ORF1 / 1 (bottom) were found to be localized in the nucleus. (B) In HEK293 cells, ORF2 (top) was found to be localized in the cytoplasm, and ORF1 / 1 (bottom) were found to be localized in the nucleus. (C) Localization patterns of ORF1 / 2 and ORF2 / 2 in cells. [Figure 25C] A series of figures showing the subcellular localization of ORFs from TTMV-LY2 fused with nano-luciferase. (A) In Vero cells, ORF2 (top) was found to be localized in the cytoplasm, and ORF1 / 1 (bottom) were found to be localized in the nucleus. (B) In HEK293 cells, ORF2 (top) was found to be localized in the cytoplasm, and ORF1 / 1 (bottom) were found to be localized in the nucleus. (C) Localization patterns of ORF1 / 2 and ORF2 / 2 in cells. [Figure 26] A series of diagrams showing sequential deletion control in the 3' non-coding region (NCR) of TTV-tth8. The top row shows the structure of wild-type TTV-tth8 anellovirus. The second row shows TTV-th8 with a 36 nucleotide deletion in the GC-rich region of the 3'NCR (Δ36nt(GC)). The third row shows TTV-tth8 with a 36 nucleotide deletion and an additional deletion of the miRNA sequence resulting in a total deletion of 78 nucleotides (Δ36nt(GC)ΔmiR). The fourth row shows TTV-tth8 with a 171 nucleotide deletion from the 3'NCR, which includes both the 36 nucleotide deletion region and the miRNA sequence (Δ3'NCR). [Figure 27]FIG. 1 is a series of figures showing that sequential deletions in the 3′NCR of TTV-tth8 significantly affect Anellovirus ORF transcript levels. Expression of ORF1 and ORF2 at day 2 (A), ORF1 / 1 and ORF2 / 2 at day 2 (B), ORF1 / 2 and ORF2 / 3 at day 2 (C), and ORF2t3 at day 2 (D) are shown. [Figure 28A] FIG. 1 is a series of diagrams showing the constructs used to produce anaerovectors expressing nano-luciferase (A) and a series of anaerovector / plasmid combinations used to transfect cells (B). [Figure 28B] FIG. 1 is a series of diagrams showing the constructs used to produce anaerovectors expressing nano-luciferase (A) and a series of anaerovector / plasmid combinations used to transfect cells (B). [Figure 29A] A series of figures showing nano-luciferase expression in mice injected with anaerovectors. (A) Nano-luciferase expression in mice 0-9 days post-injection. (B) Nano-luciferase expression in mice injected with various anaerovector / plasmid construct combinations. (C) Quantification of nano-luciferase luminescence detected in mice post-injection. Group A received TTMV-LY2 vector ± nano-luciferase. Group B received nano-luciferase protein and TTMV-LY2 ORF. [Figure 29B] A series of figures showing nano-luciferase expression in mice injected with anaerovectors. (A) Nano-luciferase expression in mice 0-9 days post-injection. (B) Nano-luciferase expression in mice injected with various anaerovector / plasmid construct combinations. (C) Quantification of nano-luciferase luminescence detected in mice post-injection. Group A received TTMV-LY2 vector ± nano-luciferase. Group B received nano-luciferase protein and TTMV-LY2 ORF. [Figure 29C]A series of figures showing nano-luciferase expression in mice injected with anaerovectors. (A) Nano-luciferase expression in mice 0-9 days post-injection. (B) Nano-luciferase expression in mice injected with various anaerovector / plasmid construct combinations. (C) Quantification of nano-luciferase luminescence detected in mice post-injection. Group A received TTMV-LY2 vector ± nano-luciferase. Group B received nano-luciferase protein and TTMV-LY2 ORF. [Figure 29D-1] Schematic diagram of the genomic organization of representative anellopes from seven different Alphatorquevirus clades. Sequences of TTV-CT30F, TTV-P13-1, TTV-tth8, TTV-HD20a, TTV-16, TTV-TJN02, and TTV-HD16d were aligned and key regions were annotated. Putative open reading frames (ORFs) are represented in light grey, TATA boxes in dark grey, and key putative regulatory regions in medium grey, including initiator elements, 5'UTR conserved domains, and GC-rich regions (e.g., as indicated). [Figure 29D-2] Schematic diagram of the genomic organization of representative anellopes from seven different Alphatorquevirus clades. Sequences of TTV-CT30F, TTV-P13-1, TTV-tth8, TTV-HD20a, TTV-16, TTV-TJN02, and TTV-HD16d were aligned and key regions were annotated. Putative open reading frames (ORFs) are represented in light grey, TATA boxes in dark grey, and key putative regulatory regions in medium grey, including initiator elements, 5'UTR conserved domains, and GC-rich regions (e.g., as indicated). [Diagram 30] FIG. 1 is a schematic showing an exemplary workflow for determining endogenous targets of anellovirus pre-miRNAs. [Fig. 31AB]

[0023] Figure 1 is a series of figures showing that tandem anelloviral plasmids can increase anellovirus or annelovectin production. (A) Plasmid maps of exemplary tandem anelloviral plasmids. (B) Transfection of HEK293T cells with tandem anelloviral plasmids produced four-fold more viral genomes compared to single copy carrying plasmids. [Figure 31C] 1 is an image of a gel electrophoresis showing circularization of TTMV-LY2 plasmids pVL46-063 and pVL46-240. [Figure 31D] Chromatograms showing copy numbers of linear and circular TTMV-LY2 constructs as determined by size exclusion chromatography (SEC). [Diagram 32] FIG. 1 shows an alignment of 36-nucleotide GC-rich regions from nine anellovirus genome sequences, and the consensus sequences based thereon (SEQ ID NOs: 818 to 827, in order of appearance, respectively). [Diagram 33] A series of diagrams showing the ORF1 structures of 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). [Diagram 34] FIG. 1 shows the ORF1 structure from Betatorquevirus strain CBS203. Residues showing high similarity among the 110 Betatorqueviruses are displayed. Among all strains evaluated, residues showing 60-79.9% similarity, residues showing 80-99.9% similarity, and residues showing 100% similarity are displayed. [Figure 35-1]FIG. 828 shows the consensus sequence (SEQ ID NO: 828) obtained from the alignment of 258 sequences of Alphatorquevirus, with residues with high similarity scores highlighted in dark grey (100%), medium grey (80-99.9%) and light grey (60-80%). Predicted domains are shown in boxes. Percent identity is also indicated by a box graph below the consensus sequence, with medium grey boxes indicating 100% identity, light grey boxes indicating 30-99% identity and dark grey boxes indicating less than 30% identity. [Figure 35-2] FIG. 828 shows the consensus sequence (SEQ ID NO: 828) obtained from the alignment of 258 sequences of Alphatorquevirus, with residues with high similarity scores highlighted in dark grey (100%), medium grey (80-99.9%) and light grey (60-80%). Predicted domains are shown in boxes. Percent identity is also indicated by a box graph below the consensus sequence, with medium grey boxes indicating 100% identity, light grey boxes indicating 30-99% identity and dark grey boxes indicating less than 30% identity. [Diagram 36] FIG. 1 is a schematic showing the domains of anellovirus ORF1 molecules and the hypervariable regions that will be replaced with hypervariable domains from different anelloviruses. [Figure 37] FIG. 1 is a schematic diagram showing the domains of ORF1 and the hypervariable regions to be replaced with a protein or peptide of interest (POI) of non-anellovirus origin. [Figure 38]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 the anellovirus non-coding region (NCR) containing the viral promoter, 5'UTR conserved domain (5CD), and GC-rich region. Payload DNA was inserted into the non-coding region of the protein-encoding locus (right). The resulting anaerovector retained the payload DNA (including open reading frames, genes, non-coding RNA, etc.) and essential anaerovirus cis-replication and packaging elements, but deleted the protein elements essential for replication and packaging. [Figure 39] 1 is a bar graph showing that anellovectors containing genetic elements encoding exogenous human immunoadhesins successfully transduced the human lung-derived cell line EKVX. [Diagram 40] FIG. 1 is a graph showing that tth8 or LY2-based anellovectors engineered to contain sequences encoding human erythropoietin (hEpo) were able to deliver functional transgenes to mammalian cells. [Fig. 41AB] 1 is a series of graphs showing that engineered anellovectors administered to mice were detectable 7 days after intravenous injection. [Diagram 42] FIG. 1 is a graph showing that hGH mRNA was detected in the cellular fraction of whole blood 7 days after intravenous administration of an engineered anellovector encoding hGH. [Fig. 43A-D] FIG 43 is a series of diagrams showing highly conserved motifs in anellovirus ORF2. [Fig. 44A-44B] FIG. 1 is a series of figures showing evidence of full-length ORF1 mRNA expression in human tissues. [Diagram 45]FIG. 1 is a graph showing the ability of in vitro circularized (IVC) TTV-tth8 genome (IVC TTV-tth8) to produce TTV-tth8 genome copies at the expected density in HEK293T cells compared to the TTV-tth8 genome in a plasmid. [Diagram 46] 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 produce LY2 genome copies at the expected density in Jurkat cells. [Figure 47-1] FIG. 1 shows an alignment of the secondary structure of the jelly-roll domain of anellovirus ORF1 proteins (SEQ ID NOs: 950-975) from Alphatorquevirus, Betatorquevirus, and Gammatorquevirus. These secondary structure elements are highly conserved. [Figure 47-2] FIG. 1 shows an alignment of the secondary structure of the jelly-roll domain of anellovirus ORF1 proteins (SEQ ID NOs: 950-975) from Alphatorquevirus, Betatorquevirus, and Gammatorquevirus. These secondary structure elements are highly conserved. [Figure 48] FIG. 1 shows conserved sequences and secondary structures of ORF1 motifs located in the N22 domain (SEQ ID NOs: 976-1000 and 851, respectively, in order of appearance). The conserved YNPXXDXGXXN (SEQ ID NO: 829) motif of human TTV ORF1 has a conserved secondary structure. Notably, a tyrosine of the motif cleaves a β-strand and the second β-strand starts from the terminal asparagine of the motif. [Figure 49] FIG. 1 shows production of Ring19 anaerovectors in human cells. [Figure 50A]FIG. 1 Schematic representation of the anellovirus single-stranded circular DNA genome, which is alternatively spliced ​​to generate three different mRNAs encoding seven putative proteins of various molecular weights. [Figure 50B] RT-qPCR data from MOLT-4 cells transfected with a plasmid encoding two copies of the RING2 genome in tandem are shown. Untransfected MOLT4 cells (control) were used as a negative control and normalized using GAPDH mRNA as a housekeeping gene. [Figure 50C] Western blotting data are shown, performed at the indicated time points after transfection of a plasmid encoding two copies of the RING2 genome in tandem to examine the dynamics of anellovirus proteins ORF1 and ORF2 over time. GAPDH protein was used as a loading control. [Figure 51] Southern blots of digested samples from MOLT-4 cells transfected with either a plasmid encoding a single copy of the RING2 genome (sample #4) or a plasmid encoding two copies of the RING2 genome in tandem (sample #5). Samples #1, 2, and 3 are the in vitro circularized RING2 genome, the plasmid containing a single copy of the RING2 genome, and the plasmid containing two copies of the RING2 genome in tandem, respectively, which served as controls. [Figure 52] FIG. 1 is a graph plotting density (plotted in grey) versus virus titer (plotted in black) of clarified lysates subjected to isopycnic centrifugation using a linear CsCl gradient. [Figure 53]Graph showing DNase protection qPCR results from MOLT-4 cell samples transfected with a plasmid encoding two copies of the RING2 genome in tandem (WT RING2 tandem), an in vitro circularized genome of RING2 in which expression of all ORF1 mutants has been knocked out (ORF1 KO IVC), an in vitro circularized genome of RING2 in which expression of all ORF2 mutants has been knocked out (ORF2 KO IVC), or co-transfected with both ORF1 KO IVC and ORF2 KO IVC. [Fig. 54AB] (FIG. 54A) A schematic of the production and purification of RING2 particles from MOLT-4 cells. (FIG. 54B) A pair of graphs depicting the density and viral titer of each fraction following a CsCl gradient. [Fig. 54C] Graph showing virus titers in pooled material (input), concentrated material, and flow-through (FT). [Fig. 54D] Western blot analysis to detect capsid protein ORF1 in pooled material (input), concentrated material and flow-through (FT). [Figure 54E] 1 is a set of representative transmission electron microscopy images of enriched RING2 particles. [Figure 55A] Schematic diagram of the fully annotated circularized genome RING19 recovered from dissected RPE tissue. ORF1 and ORF2 were fully annotated by computation, and ORF2 / 2 and ORF2 / 3 were manually curated. [Figure 55B] FIG. 1 is a schematic diagram of the production and purification of RING19 particles from MOLT-4 cells. [Figure 55C] 13 is a graph showing DNase protection qPCR assay of fractions from SEC of purified RING19. [Fig. 55D] Representative transmission electron microscopy images of enriched RING19 particles. [Figure 55E] Representative transmission electron microscopy images of enriched RING19 particles. [Figure 56A]A series of figures showing infectivity of RING19 in the mouse retina and posterior eye cup. A) Table listing the various groups, treatments, virus / vector doses, route of administration, number of animals per group, and time points for the in vivo study. The bottom panel shows the anatomy of the mouse eye and study design. (B) Vector / viral genome copies present in the neural retina or posterior eye cup (PEC) assessed by qPCR in harvested DNA from eyes of mice injected once intravitreally (IVT) or subretinally (SR) with either PBS, 6.6E+5vg of Ring 19, or dose-matched AAV2.mCherry. N=5-6 eyes / group. Abbreviations: AAV=adeno-associated virus, DNA=deoxyribonucleic acid, IVT=intravitreous, PBS=phosphate-buffered saline, PEC=posterior eye cup, SR=subretinal. [Figure 56B] A series of figures showing infectivity of RING19 in the mouse retina and posterior eye cup. A) Table listing the various groups, treatments, virus / vector doses, route of administration, number of animals per group, and time points for the in vivo study. The bottom panel shows the anatomy of the mouse eye and study design. (B) Vector / viral genome copies present in the neural retina or posterior eye cup (PEC) assessed by qPCR in harvested DNA from eyes of mice injected once intravitreally (IVT) or subretinally (SR) with either PBS, 6.6E+5vg of Ring 19, or dose-matched AAV2.mCherry. N=5-6 eyes / group. Abbreviations: AAV=adeno-associated virus, DNA=deoxyribonucleic acid, IVT=intravitreous, PBS=phosphate-buffered saline, PEC=posterior eye cup, SR=subretinal. [Figure 57]FIG. 1 is a series of graphs showing infectivity of Ring2 in mouse retinas and PECs after subretinal and intravitreal injection of anellovirus. Vector genome (vg) copies present in eyes of mice injected either intraretinally or subretinally once with PBS, 1.6E6vg of Ring2, or the corresponding dose of AAV2.mCherry. On day 7 or day 21, three eyes from each group were enucleated and retinas and PECs were analyzed separately by qPCR analysis with probes targeting the Ring2 genome or the mCherry transgene. Abbreviations: AAV=adeno-associated virus, DNA=deoxyribonucleic acid, IVT=intravitreal, PBS=phosphate buffered saline, PEC=posterior eye cup, SR=subretinal, VG=vector genome. [Figure 58] A series of graphs showing CAV infectivity in mouse retinas and PECs after subretinal and intravitreal injections. DNA vector genome copies or mRNA transgene copies detected in eyes of mice injected once subretinal with PBS, 9.4E5vg of CAV, dose-matched AAV2.nLuc, or 1E+9vg of AAV2.nLuc. On day 14, 5-6 eyes from each group were enucleated and retinas and PECs were analyzed separately by qPCR (DNA) or RT-qPCR (mRNA) using a probe for the nLuc transgene. Abbreviations: AAV=adeno-associated virus, DNA=deoxyribonucleic acid, IVT=intravitreal, PBS=phosphate-buffered saline, PEC=posterior eye cup, SR=subretinal, nLuc=nanoluc luciferase, mRNA=messenger ribonucleic acid. [Figure 59A]Schematic diagram showing three exemplary Ring19 tandem vector constructs. In each construct, a CMV_nLuc cassette is inserted into the second copy of the Ring19 genome in the tandem construct at the indicated position to replace the corresponding nucleotide of the Ring19 genome sequence. In the first exemplary construct (herein referred to as the CMV_nLuc3 construct), the CMV_nLuc cassette replaces the C-terminal portion of the ORF2 gene as well as the N-terminal portion of the ORF1 gene. In the second exemplary construct (herein referred to as the CMV_nLuc4 construct), the CMV_nLuc cassette replaces an internal portion of the ORF1 gene. In the third exemplary construct (herein referred to as the CMV_nLuc5 construct), the CMV_nLuc cassette replaces an internal portion of the ORF1 gene that is C-terminal to the position replaced in the CMV_nLuc4 construct. [Figure 59B] FIG. 1 shows an exemplary workflow for producing Ring19 anellovector particles. [Figure 60] 59B is a graph showing recovery of the indicated Ring19 anaerovectors using the tandem vector-based workflow shown in FIG. 59B. Levels of DNase-protected nLuc-containing viral genomes following production of the indicated anaerovectors are shown. [Figure 61] 1 shows an exemplary tandem nucleic acid construct for producing a Ring19 anaerovector. The tandem construct comprises a first region (or first copy) comprising the Ring19 anellovirus genome (including the 5'UTR, ORF2 coding sequence, ORF1 coding sequence, ORF3 coding sequence, and GC-rich region of Ring19, as described herein), and a second region (or second copy) comprising the Ring19-based anaerovector genome (including the 5'UTR, at least a portion of the ORF2 nucleic acid sequence, a transgene sequence encoding a payload polypeptide of interest, a C-terminal portion of the ORF1 nucleic acid sequence, at least a portion of the ORF3 nucleic acid sequence, and a GC-rich region). [Fig.62AB]1 is a series of graphs showing qPCR titers of eGFP or mCherry amplicons following production of Ring19 anellovectors carrying the indicated transgenes under the control of various promoters (as listed on the x-axis). [Fig. 63A-D] 1 is a series of graphs showing qPCR titers of hGH, gLuc, iCre, or hEpo amplicons following production of Ring19 anellovectors carrying the indicated transgenes under the control of various promoters (as listed on the x-axis). [Fig.64AB] 1 is a series of graphs showing qPCR titers of wild-type Ring19 amplicons following production of Ring19 anellovectors under the control of various promoters (as listed on the x-axis). [Fig. 65A-D] 1 is a series of graphs showing qPCR titers of wild-type Ring19 amplicons following production of Ring19 anellovectors under the control of various promoters (as listed on the x-axis). [Figure 66] FIG. 1 is a schematic diagram of an exemplary Cre-loxp-based vector system. In this exemplary system, the genetic element sequence includes, from 5' to 3', a 3'UTR containing a GC-rich region, a 5'UTR, and a transgene sequence. The genetic element sequence is flanked by lox71 and lox66 sites. Introduction of Cre recombinase results in cleavage and circularization of the loxP-flanked sequences, forming a double-stranded minicircle containing the genetic element sequence (shown as "vector" in the diagram). After conversion of this minicircle to single-stranded circular DNA, anellovirus proteins supplied in trans form a proteinaceous exosporium containing the ORF1 molecule around the single-stranded circular DNA, thereby generating a packaged anaerovector. [Figure 67] FIG. 1 shows the results of a DNase protection assay after iodixanol on batch 1 anellovector Ring19-fCMV-eGFP material. [Figure 68]DNase protection assay results before and after concentration of batch 1 anellovector Ring19-fCMV-eGFP material are shown, revealing the absence of Ring19 WT viral genome. [Figure 69] Coomassie (left panel) and silver staining (right panel) of batch 1 anellovector Ring19-fCMV-eGFP material are shown. [Figure 70] Four plots of qPCR assays for Ring19-eGFP genetic material in the posterior eye cup (PEC) (top left) and retina (top right), and WT Ring19 genetic material in PEC (bottom left) and retina (bottom right) are depicted, revealing the presence of eGFP DNA in the retina 21 days after viral transduction. [Figure 71] FIG. 13 shows the results of a DNase protection assay after iodixanol for batch 2 anellovector Ring19-fCMV-eGFP material. [Figure 72] DNase protection assay results before and after concentration of batch 2 anellovector Ring19-fCMV-eGFP material are shown, revealing the absence of Ring19 WT viral genome. [Figure 73] 13 shows Coomassie staining of batch 2 anellovector Ring19-fCMV-eGFP material. [Fig. 74A] qPCR data are depicted in plots showing the presence of Ring19-eGFP DNA in PECs 21 days (left panel) and 49 days (right panel) after viral transduction. [Fig. 74B] Plots depicting qPCR data showing Ring19-eGFP DNA in retinas 21 days (left panel) and 49 days (right panel) after viral transduction, demonstrating the persistence of viral infection. [Fig. 75A] qPCR data for WT Ring19 DNA in PECs 21 days (left panel) and 49 days (right panel) after virus transduction are depicted, revealing the absence of WT Ring19 DNA. [Fig. 75B]qPCR data for WT Ring19 DNA in retinas 21 days (left panel) and 49 days (right panel) after viral transduction are depicted, revealing the absence of WT Ring19 DNA. [Figure 76] FIG. 13 is a plot showing eGFP RNA copies detected by RT-qPCR in PECs (left panel) and retina (right panel), demonstrating successful eGFP transfer by Ring19-eGFP in experiment 1. [Figure 77] FIG. 13 is a plot showing eGFP RNA copies detected by RT-ddPCR in PECs (left panel) and retinas (right panel), demonstrating successful transduction with Ring19-eGFP in experiment 1. [Figure 78] Results from experiment 2 are shown showing eGFP mRNA expression in PECs by RT-qPCR (top left panel) and RT-ddPCR (top right panel), and in the retina by RT-qPCR (bottom left panel) and RT-ddPCR (bottom right panel), 21 days after viral transduction. These data demonstrate successful infection of ocular tissues with Ring19-eGFP. [Figure 79] RT-qPCR (left panel) and RT-ddPCR (right panel) data showing eGFP mRNA expression in PECs 49 days after virus transduction are depicted, demonstrating that infection with Ring19-eGFP persists for at least 49 days after transduction. [Figure 80] RT-qPCR (left panel) and RT-ddPCR (right panel) data showing eGFP mRNA expression in the retina 49 days after viral transduction are depicted. [Fig. 81A-L]Fluorescence imaging of flat-mount preparations of mouse PECs. The top row shows GFP expression of PBS-treated negative control cells (Figure 81A), red blood cell autofluorescence in the Texas Red channel (Figure 81B), and merge (Figure 81C). The second row shows GFP expression of Ring19-eGFP-infected cells (Figure 81D), red blood cell autofluorescence in the Texas Red channel (Figure 81E), and merge (Figure 81F). The third row shows GFP expression of AAV2-eGFP-infected cells at the corresponding dose (Figure 81G), red blood cell autofluorescence in the Texas Red channel (Figure 81H), and merge (Figure 81I). The bottom row shows GFP expression of high-dose AAV2-eGFP-infected cells (Figure 81J), red blood cell autofluorescence in the Texas Red channel (Figure 81K), and merge (Figure 81L). Arrows indicate GFP-expressing cells. [Figure 82] FIG. 13 is a transmission electron microscope (TEM) image of Ring19-eGFP virus showing successful virus assembly. [Figure 83] 1 shows a retinal pigment epithelial (RPE) cell culture at day 5 demonstrating cell proliferation and confluency. [Figure 84] Shown are RPE cell cultures and cells spun down on day 28, showing visible melanin granule formation within the cells. [Figure 85] Shown is an RPE cell culture showing nuclear and ZO-1 staining around the cell membrane, demonstrating tight junction formation in the cultured cells. [Figure 86] A schematic diagram of the ELISA assay for VEGF (left panel) and a bar graph showing VEGF protein expression in RPE cell cultures (right panel) are shown. [Figure 87A] Phase imaging of cells showing human RPE cells transduced with Ring19-eGFP (left panel), a single GFP-positive cell in the center of the field of view (middle panel), and a merged phase of the phase and GFP images (right panel). [Fig. 87B]Phase imaging of cells showing AAV2-eGFP transduced human RPE cells (left panel), multiple GFP positive cells (middle panel), and a merged phase of phase and GFP images (right panel). [Fig. 88A] Fluorescence microscopy images of RPE cells transduced with Ring19-eGFP showing GFP expression (top left panel), immunostaining for GFP (top right panel), Hoechst DNA staining (bottom left panel), and a merged image of all three channels (bottom right panel) at 20x magnification. GFP expression overlaps with GFP staining in the cell at the bottom left of the image and in the cell at the top left of the image, indicating successful transduction of eGFP. [Fig. 88B] Fluorescence microscopy images of Hoechst, DNA staining (far left panel), GFP expression (middle left panel), GFP immunostaining (middle right panel), and a merge of all three channels (far right panel) in Ring19eGFP-infected RPE cells are shown at 40x magnification. [Fig. 88C] Fluorescence microscopy images of RPE cells transduced with AAV2-eGFP, showing GFP expression (top left panel), immunostaining for GFP (top right panel), Hoechst DNA staining (bottom left panel), and a merged image of all three channels (bottom right panel) at 20x magnification. GFP expression overlaps with GFP staining in some cells, indicating successful transduction of eGFP. [Fig. 88D] Fluorescence microscopy images of Hoechst, DNA staining (left panel), GFP expression (left center panel), GFP immunostaining (right center panel), and the merge of all three channels (right panel) in AAV2-eGFP infected RPE cells at the corresponding doses are shown at 40x magnification. [Fig. 88E] Fluorescence microscopy images of RPE cells not treated with virus showing GFP expression (top left), immunostaining for GFP (top right), Hoechst DNA staining (bottom left), and a merged image of all three channels (bottom right). As expected for this negative control, there are no cells with GFP expression overlapping with the GFP staining. [Fig. 89A-D]Shown are four plots of qPCR assays for eGFP DNA in the posterior eye cup (PEC) (Figure 89A) and retina (Figure 89B), and WT Ring19 DNA in the PEC (Figure 89C) and retina (Figure 89D), as well as a PBS negative control, 21 days after viral transduction with R19-eGFP(LD), R19-eGFP(HD), AAV2-eGFP(LD), and AAV2-eGFP(HD). Each dot represents one eye. N=3 / group. [Fig. 90A-D] Plots showing eGFP mRNA expression in PECs by RT-qPCR (FIG. 90A) and RT-ddPCR (FIG. 90B), and in the retina by RT-qPCR (FIG. 90C) and RT-ddPCR (FIG. 90D), as well as PBS negative control, 21 days after viral transduction with R19-eGFP(LD), R19-eGFP(HD), AAV2-eGFP(LD), and AAV2-eGFP(HD). To confirm eGFP expression, the two highest eGFP expressing samples as determined by RT-qPCR were subjected to RT-ddPCR again. For RT-qPCR, N=5; for RT-ddPCR, n=5. [Fig. 91A-O] Fluorescence imaging of flat-mount preparations of mouse PECs. The top row shows GFP expression of PBS-treated negative control cells (Figure 91A), red blood cell autofluorescence in the Texas Red channel (Figure 91B), and merge (Figure 91C). The second row shows GFP expression of low-dose (LD) Ring19-eGFP-infected cells (Figure 91D), red blood cell autofluorescence in the Texas Red channel (Figure 91E), and merge (Figure 91F). The third row shows GFP expression of high-dose (HD) Ring19-eGFP-infected cells (Figure 91G), red blood cell autofluorescence in the Texas Red channel (Figure 91H), and merge (Figure 91I). The fourth row shows GFP expression of low-dose (LD) AAV2-eGFP-infected cells (Figure 91J), red blood cell autofluorescence in the Texas Red channel (Figure 91K), and merge (Figure 91L). The bottom row shows GFP expression in high dose (HD) AAV2-eGFP infected cells (Figure 91M), red blood cell autofluorescence in the Texas Red channel (Figure 91N), and merge (Figure 91O). Arrows indicate GFP expressing cells. N=3 / group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0270] 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.

[0271] 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.

[0272] 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.

[0273] 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.

[0274] 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.

[0275] 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 an embodiment, a nucleic acid molecule comprises a nucleic acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anellovirus ORF1 encoding a 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 nucleotides 571-2613 of the nucleic acid sequence of Table 1.

[0276] As used herein, the term "Anelloviridae vector" refers to a vehicle derived from or similar to a virus of the Anelloviridae family (e.g., Alphatorquevirus, Betatorquevirus, or Gammatorquevirus, or Chicken Anemia Virus), where the vehicle comprises genetic elements enclosed in a proteinaceous exterior (e.g., the genetic elements are substantially protected from digestion by DNAse I by the proteinaceous exterior). In some embodiments, the Anelloviridae vector comprises genetic elements derived from or highly similar (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to Alphatorquevirus, Betatorquevirus, or Gammatorquevirus, or Chicken Anemia Virus (CAV).In some embodiments, the Anelloviridae vector comprises a proteinaceous outer layer comprising a protein derived from or similar to (e.g., at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to) an Alphatorquevirus, Betatorquevirus, or Gammatorquevirus, or Chicken Anemia Virus (CAV), an Alphatorquevirus, Betatorquevirus, Gammatorquevirus, or Chicken Anemia Virus (CAV) capsid protein (e.g., Alphatorquevirus ORF1, Betatorquevirus ORF1, Gammatorquevirus ORF1, or CAV VP1). In some embodiments, containment within the proteinaceous exterior encompasses 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, gaps or discontinuities may be present in the proteinaceous exterior (e.g., rendering the proteinaceous exterior permeable to water, ions, peptides, or small molecules), so long as the genetic elements are retained within the proteinaceous exterior or protected from digestion, e.g., by DNAse I, prior to entering the host cell. In some embodiments, the Anelloviridae vector is purified, e.g., separated from the original source, and / or substantially free (>50%, >60%, >70%, >80%, >90%) of other components. In some embodiments, the Anelloviridae vector is capable of introducing genetic elements into a target cell (e.g., by infection). In some embodiments, the Anelloviridae vector is an infectious synthetic viral particle.

[0277] As used herein, the term "anellovector" refers to a vehicle that includes genetic elements, e.g., episomes, e.g., circular DNA, that are encapsulated in a proteinaceous exterior. "Synthetic anaellovector" as used herein generally refers to an anaellovector that does not occur in nature, e.g., has a sequence that differs from a wild-type virus (e.g., a wild-type Anellovirus described herein). In some embodiments, a synthetic anaellovector is engineered or recombinant, e.g., includes genetic elements that include differences or modifications relative to a wild-type viral genome (e.g., a wild-type Anellovirus genome described herein). In some embodiments, encapsulated in a 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, 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, so long as the genetic elements are retained within the proteinaceous exterior, e.g., prior to entry into the host cell. 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%) from other constituents.

[0278] Anellovectors, in some embodiments, contain sufficient nucleic acid sequences 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 to permit packaging into a proteinaceous exterior (e.g., capsid), and may further include nucleic acid vectors that contain heterologous sequences. In some embodiments, the nucleic acid vector is a viral vector or a naked nucleic acid. In some embodiments, the nucleic acid vector comprises at least about 50, 60, 70, 71, 72, 73, 74, 75, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, or 3500 contiguous nucleotides of a native Anellovirus sequence, or a sequence highly similar thereto (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical). In some embodiments, the annelovector 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 some embodiments, the annelovector comprises the genetic elements described herein, for example, a genetic element having a promoter, a sequence encoding a therapeutic effector, and a capsid binding sequence.

[0279] 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 embodiments, a multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibody molecules are 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.

[0280] As used herein, a "nucleic acid that encodes" refers to a nucleic acid sequence that encodes an amino acid sequence or a functional polynucleotide (e.g., a non-coding RNA, such as an siRNA or miRNA).

[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 present 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 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" generally refers to a nucleic acid sequence in an anaerovector. It is understood that the genetic element is produced as naked DNA and is optionally further assembled into a proteinaceous exosomal. It is also understood that the anaerovector can insert its genetic element into a cell, such that the genetic element resides within the cell, and therefore the proteinaceous exosomal does not necessarily enter 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 described herein, e.g., an anellovirus ORF1 protein described in Table A1 or A2), 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 annellovector 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 (as listed in any of Tables N1-N2). 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. As used herein, an "anellovirus ORF1 protein" refers to an ORF1 protein encoded by an anellovirus genome (e.g., a wild-type anellovirus genome, e.g., as listed herein), e.g., an ORF1 protein having an amino acid sequence listed in Table A1 or A2, or an ORF1 protein encoded by an ORF1 gene listed in any of Tables N1-N2.

[0285] As used herein, the term "ORF2 molecule" refers to a polypeptide having an activity and / or structural characteristics of an anellovirus ORF2 protein (e.g., an anellovirus ORF2 protein described herein, e.g., an anellovirus ORF2 protein listed in Table A1 or A2), or a functional fragment thereof. As used herein, "anellovirus ORF2 protein" refers to an ORF2 protein encoded by an anellovirus genome (e.g., a wild-type anellovirus genome, e.g., described herein), e.g., an ORF2 protein having an amino acid sequence listed in Table A1 or A2, or an ORF2 protein encoded by an ORF2 gene as listed in any of Tables N1-N2.

[0286] As used herein, the term "VP1 molecule" refers to a polypeptide having activity and / or structural characteristics of a CAV VP1 protein (e.g., a CAV VP1 protein described herein, or a functional fragment thereof). A VP1 molecule, in some embodiments, can comprise a polypeptide encoded by a CAV VP1 nucleic acid. A VP1 molecule, in some embodiments, can further comprise a heterologous sequence, e.g., from a CAV VP1 protein, e.g., as described herein. In some embodiments, a VP1 molecule is encoded by a CAV genome (e.g., a wild-type CAV genome, e.g., as described herein). In some embodiments, a VP1 molecule is a polypeptide encoded by a CAV VP1 nucleic acid (e.g., a VP1 gene, e.g., as described herein). In some embodiments, a VP1 molecule is a splice variant or comprises a post-translational modification.

[0287] As used herein, the term "VP2 molecule" refers to a polypeptide having activity and / or structural characteristics of a CAV VP2 protein (e.g., a CAV VP2 protein described herein, or a functional fragment thereof). In some embodiments, a VP2 molecule is encoded by a CAV genome (e.g., a wild-type CAV genome, e.g., described herein). In some embodiments, a VP2 molecule is a polypeptide encoded by a CAV VP2 nucleic acid (e.g., a VP2 gene, e.g., described herein). In some embodiments, a VP2 molecule is a splice variant or includes a post-translational modification.

[0288] As used herein, the terms "apoptin molecule" and "VP3 molecule" are used interchangeably and refer to a polypeptide having activity and / or structural characteristics of a CAV apoptin protein (e.g., a CAV apoptin protein described herein, or a functional fragment thereof. In some embodiments, the apoptin molecule is encoded by a CAV genome (e.g., a wild-type CAV genome described herein). In some embodiments, the apoptin molecule is a polypeptide encoded by a CAV apoptin nucleic acid (e.g., an apoptin gene). In some embodiments, the apoptin molecule is a splice variant or includes a post-translational modification.

[0289] As used herein, the term "CAV capsid polypeptide" refers to a polypeptide present in the capsid of a wild-type CAV or a polypeptide having activity and / or structural characteristics of said polypeptide. In some embodiments, the CAV capsid polypeptide is a VP1 molecule.

[0290] As used herein, the term "VP1 nucleic acid" refers to a nucleic acid encoding a VP1 molecule or its reverse complement. The nucleic acid may be either single-stranded or double-stranded. In some embodiments, the VP1 nucleic acid comprises, for example, a CAV VP1 gene as described herein. "VP1 gene" generally refers to a nucleic acid sequence encoding a wild-type VP1 molecule or its reverse complement. In some embodiments, the VP1 gene comprises a sense strand. In some embodiments, the VP1 gene comprises an antisense strand. In some embodiments, the VP1 gene is double-stranded.

[0291] As used herein, the term "VP2 nucleic acid" refers to a nucleic acid encoding a VP2 molecule, or its reverse complement. The nucleic acid may be either single-stranded or double-stranded. In some embodiments, the VP2 nucleic acid comprises, for example, a CAV VP2 gene as described herein. "VP2 gene" generally refers to a nucleic acid sequence encoding a wild-type VP2 molecule, or its reverse complement. In some embodiments, the VP2 gene comprises a sense strand. In some embodiments, the VP2 gene comprises an antisense strand. In some embodiments, the VP2 gene is double-stranded.

[0292] As used herein, the terms "apoptin nucleic acid" and "VP3 nucleic acid" are used interchangeably and refer to a nucleic acid encoding an apoptin molecule or its reverse complement. The nucleic acid may be single-stranded or double-stranded. In some embodiments, the apoptin nucleic acid comprises, for example, a CAV apoptin gene as described herein. An "apoptin gene" or a "VP3 gene" generally refers to a nucleic acid sequence encoding a wild-type apoptin molecule or its reverse complement. In some embodiments, the apoptin gene comprises a sense strand. In some embodiments, the apoptin gene comprises an antisense strand. In some embodiments, the apoptin gene is double-stranded.

[0293] As used herein, the term "CAV genomic sequence" refers to a nucleic acid sequence that includes a full-length genomic sequence from a wild-type CAV, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto, e.g., as described herein. In some embodiments, the CAV genome consists of a CAV genomic sequence described herein (e.g., a wild-type CAV genomic sequence, e.g., as listed in any of Tables N3-N4).

[0294] As used herein, the term "CAV UTR" refers to a nucleic acid sequence that includes an untranslated region (UTR) sequence (e.g., a sequence of the 5'UTR or 3'UTR) from a CAV (e.g., a wild-type CAV, e.g., as described herein, e.g., listed in Tables N3-N4), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity thereto.

[0295] As used herein, the term "proteinaceous exterior" refers to exterior components that are predominantly (eg, >50%, >60%, >70%, >80%, >90%) protein.

[0296] 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 that encodes an expression product. In embodiments, the expression product comprises an RNA or a protein.

[0297] 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.

[0298] As used herein, the term "replication protein" refers to a protein, e.g., a viral protein, used during infection, replication / expression of the viral genome, synthesis of viral proteins, and / or assembly of viral components.

[0299] As used herein, a "substantially non-pathogenic" organism, particle, or construct refers to, for example, an organism, particle (e.g., a virus or anellovector, as described herein, for example), or construct thereof, that does not induce or cause an undetectable disease or pathology in a host organism, e.g., a mammal, e.g., a human. In some embodiments, administration of an anellovector to a subject may result in some reactions or side effects that are acceptable as part of standard treatment.

[0300] As used herein, "non-pathogenic" refers to an organism or component thereof that does not induce or induce undetectable disease or pathology in a host organism, e.g., a mammal, e.g., a human.

[0301] 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 into, for example, 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.

[0302] As used herein, a "substantially non-immunogenic" 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 elicit or induce an unwanted or non-targeted 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 construct does not generate a detectable immune response. In some embodiments, a substantially non-immunogenic anelovector does not generate a detectable immune response against a protein that includes an amino acid sequence encoded by a nucleic acid sequence as set forth in any of Tables N1-N2. In embodiments, an immune response (e.g., an unwanted or non-targeted immune response) is determined by assaying the presence or level of antibodies in a subject (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 method described in Tsuda et al. (1999; J. Virol. Methods 77:199-206; incorporated herein by reference) and / or the method for 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).

[0303] 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).

[0304] 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).

[0305] As used herein, the term "virome" refers to a virus in a particular environment, e.g., a part of the body, e.g., an organism, e.g., within a cell, e.g., a tissue.

[0306] The present invention relates generally to Anelloviridae vectors (e.g., anellovectors), e.g., synthetic Anelloviridae vectors (e.g., anellovectors), and uses thereof. The present disclosure provides Anelloviridae vectors (e.g., anellovectors), compositions comprising Anelloviridae vectors (e.g., anellovectors), and methods of making or using Anelloviridae vectors (e.g., anellovectors). Anelloviridae vectors (e.g., anellovectors) are generally useful as delivery vehicles, for example, for delivering therapeutic agents to eukaryotic cells. In general, Anelloviridae vectors (e.g., anellovectors) comprise a genetic element that includes a nucleic acid sequence (e.g., encoding an effector, e.g., an exogenous effector or an endogenous effector) encapsulated within a proteinaceous exterior. Anelloviridae vectors (e.g., anellovectors) can include one or more deletions of sequences (e.g., regions or domains described herein) compared to Anellovirus sequences (e.g., as described herein). Anelloviridae vectors (e.g., anellovectors) can be used as substantially non-immunogenic vehicles to deliver genetic elements, or effectors encoded therein (e.g., polypeptide or nucleic acid effectors described herein), to eukaryotic cells, e.g., to treat a disease or disorder in a subject comprising the cells.

[0307] table of contents I. Anelloviridae Vectors (e.g., Anellovectors) A. Anelloviridae viruses (e.g., anelloviruses and CAV) B. Capsid proteins (e.g., ORF1 and VP1 molecules) C.ORF2 molecule D. Genetic elements E. Protein Binding Sequences F.5'UTR region G.GC-rich region H. Effector I. Proteinaceous external Pr II. Compositions and Methods for Producing Anelloviridae Vectors A. Components and Assembly of Anelloviridae Vectors i. Capsid proteins for assembly of the annelovector (e.g., ORF1 and VP1 molecules) ii. ORF2 molecule for assembly of annelovector iii. Production of protein components 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 for providing proteins in cis or trans iii. Exemplary Cell Types E.Culture conditions F. Recovery G. In Vitro Assembly Method H. Concentration and Purification III. Vector IV. Composition V. Host cells VI.How to use VII. Production Method VIII. Administration / Delivery

[0308] I. Anelloviridae Vectors (e.g., Anellovectors) In some aspects, the invention described herein includes compositions of Anelloviridae vectors (e.g., anellovectors) and methods of making and using Anelloviridae vectors (e.g., anellovectors), Anelloviridae vector (e.g., anellovector) preparations, and therapeutic compositions. In some embodiments, anellovectors have a sequence, structure, and / or function based on an Anelloviridae virus (e.g., an Anellovirus or CAV described herein). It is understood that applicable embodiments described herein with respect to anellovectors can also be applied to Anelloviridae vectors (e.g., vectors based on or derived from Chicken Anemia Virus (CAV) described herein). In some embodiments, an Anelloviridae vector (e.g., an Anellovector) comprises a nucleic acid or polypeptide comprising a sequence as set forth in Tables A1-A3 (e.g., Tables A1, A1.1, A2, or A3); or Tables N1-N4 (e.g., Tables N1, N1.1, N2, N3, or N4), or a fragment or portion thereof, or another substantially non-pathogenic virus, e.g., a commensal virus, a commensal virus, a native virus. In some embodiments, a vector based on an Anelloviridae virus comprises at least one element exogenous to the Anelloviridae virus, e.g., an exogenous effector or a nucleic acid sequence encoding an exogenous effector, disposed within a genetic element of the vector. In some embodiments, an Anelloviridae virus-based vector includes at least one element that is heterologous to another element from that Anelloviridae virus, e.g., an effector-encoding nucleic acid sequence that is heterologous to another linked nucleic acid sequence, such as a promoter element.In some embodiments, the Anelloviridae vector comprises a genetic element (e.g., circular DNA, e.g., single-stranded DNA) that comprises at least one element that is heterologous to the remainder of the genetic element and / or to a proteinaceous exogenous element (e.g., an exogenous element encoding an effector, e.g., as described herein). The Anelloviridae vector can be a delivery vehicle (e.g., a substantially non-pathogenic delivery vehicle) for a payload to a host, e.g., a human. In some embodiments, the Anelloviridae vector is replicable in a eukaryotic cell, e.g., a mammalian cell, e.g., a human cell. In some embodiments, the Anelloviridae vector is substantially non-pathogenic and / or substantially non-integrating in a mammalian (e.g., human) cell. In some embodiments, the Anelloviridae vector is substantially non-immunogenic in a mammal, e.g., a human. In some embodiments, the Anelloviridae vector is replication-deficient. In some embodiments, the Anelloviridae vector is replication-competent.

[0309] In some embodiments, the Anelloviridae vector comprises a curon, or components thereof (e.g., genetic elements, including, e.g., sequences encoding effectors, and / or proteinaceous exterior), for example, as described in PCT Application No. PCT / US2018 / 037379, which is incorporated by reference in its entirety.

[0310] 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 Anelloviridae family vector (e.g. anellovector) comprising a proteinaceous exterior, wherein the genetic element is enclosed within the proteinaceous exterior; and the Anelloviridae family vector (e.g. anellovector) is capable of delivering the genetic element into a eukaryotic cell.

[0311] In some embodiments of the anellovectors described herein, 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 Anelloviridae family vectors (e.g., anellovectors) 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, genetic elements of a population of Anelloviridae family vectors (e.g., anellovectors) 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.

[0312] In one aspect, the invention provides a method for the production of a polypeptide comprising: (i) a genetic element comprising a promoter element and a sequence encoding an effector (e.g., an endogenous effector or an exogenous effector, e.g., a payload), and a protein binding sequence (e.g., an exogenous protein binding sequence), wherein the genetic element is a wild-type Anelloviridae virus (e.g., anellovirus or CAV) sequence (e.g., wild-type Torque Teno virus (TTV), Torque Teno minivirus (TTV), or a wild-type Anelloviridae virus (TTV), and / or ... a) a nucleotide sequence that has at least 75% (e.g., at least 75, 76, 77, 78, 79, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100) sequence identity to a wild-type Anelloviridae virus (e.g., an Anellovirus or CAV) sequence listed in any of Tables N1-N4, e.g., Tables N1, N1.1, N2, N3, or N4); and (ii) an Anelloviridae vector (e.g., an Anellovector) comprising a proteinaceous exterior (wherein the genetic elements are enclosed within the proteinaceous exterior; the Anelloviridae vector is capable of delivering the genetic elements to a eukaryotic cell).

[0313] 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 present invention also includes Anelloviridae vectors (e.g., anellovectors) comprising:

[0314] In some embodiments, the Anelloviridae family vector (e.g., 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 circular genomes. 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 the linear ssDNA viruses (ie, Parvoviridae) that infect non-plant eukaryotic cells.

[0315] In some embodiments, the Anelloviridae vector (e.g., 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.

[0316] 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 Anelloviridae family vector (e.g., Anellovirus or CAV) described herein.

[0317] In some embodiments the genetic element comprises one or more of the following characteristics: single stranded, circular, negative stranded, and / or DNA. In 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).

[0318] The Anelloviridae vectors (e.g., Anellovectors), compositions comprising Anelloviridae vectors (e.g., Anellovectors), methods of using such Anelloviridae vectors (e.g., Anellovectors), and the like, described herein are based in part on examples illustrating how different effectors, e.g., miRNAs (e.g., for IFN or miR-625), shRNAs, and the like, and protein binding sequences, e.g., DNA sequences that bind to capsid proteins such as Q99153, can be combined with a proteinaceous exoskeleton, e.g., the capsids disclosed in Arch Virol (2007) 152:1961-1975, to produce Anelloviridae vectors that can then be used to deliver effectors to cells (e.g., animal cells, e.g., human cells or non-human animal cells such as porcine or murine cells). In some embodiments, the effector can suppress the expression of a factor such as interferon. The above examples further explain how Anelloviridae vectors can be made, for example, by inserting an effector into a sequence from an Anelloviridae virus (e.g., Anellovirus or CAV). Based on these examples, the following description contemplates various variations of the specific findings and combinations considered in the examples. For example, those skilled in the art will understand from the examples that a specific miRNA is used only as an example of an effector, and other effectors may be, for example, other regulatory nucleic acids or therapeutic peptides. Similarly, the specific capsid used in the examples may be replaced by a substantially non-pathogenic protein as described below. Certain Anelloviridae virus (e.g., Anellovirus or CAV) sequences described in the Examples may also be replaced with the Anelloviridae virus (e.g., Anellovirus or CAV) sequences described below.These considerations apply equally to protein binding sequences, regulatory sequences such as promoters, etc. Regardless, those skilled in the art will specifically consider such embodiments to be closely related to the examples.

[0319] In some embodiments, an Anelloviridae vector (e.g., an anellovector), or a genetic element contained within an Anelloviridae vector (e.g., an anellovector), is introduced into a cell (e.g., a human cell). In some embodiments, an effector (e.g., an RNA, e.g., an miRNA), e.g., an effector encoded by a genetic element of an Anelloviridae vector (e.g., an anellovector), is expressed in a cell (e.g., a human cell), e.g., upon introduction of the Anelloviridae vector (e.g., an anellovector) or genetic element into the cell. In some embodiments, introduction of an Anelloviridae vector (e.g., an anellovector), 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, e.g., by altering the level of expression of the target molecule by the cell. In some embodiments, introduction of an Anelloviridae vector (e.g., anellovector) or a genetic element contained therein into a cell reduces the level of interferon produced by the cell. In some embodiments, introduction of an Anelloviridae vector (e.g., anellovector) or a genetic element contained therein into a cell modulates (e.g., increases or decreases) a function of the cell. In some embodiments, introduction of an Anelloviridae vector (e.g., anellovector) 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 an Anelloviridae vector (e.g., anellovector) or a genetic element contained therein into a cell reduces the viability of the cell (e.g., a cancer cell).

[0320] In some embodiments, the Anelloviridae vectors (e.g., anellovectors) (e.g., synthetic anellovectors) described herein 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, antibody prevalence is determined according to methods known in the art. In some embodiments, antibody prevalence is determined by detecting antibodies to an Anelloviridae virus (e.g., an Anellovirus or CAV) (e.g., as described herein), or an Anelloviridae vector based thereon, in a biological sample, e.g., according to the anti-TTV antibody detection method described in Tsuda et al. (1999; J. Virol. Methods 77:199-206; incorporated herein by reference), and / or the method for determining anti-TTV IgG seropositivity described in Kakkola et al. (2008; Virology 382:182-189; incorporated herein by reference). Antibodies to Anelloviridae viruses (e.g., anelloviruses or CAV) or Anelloviridae vectors based thereon can also be detected by methods in the art for detecting anti-viral antibodies, e.g., methods for detecting anti-AV antibodies, e.g., those described in Calcedo et al. (2013; Front. Immunol. 4(341):1-7; incorporated herein by reference).

[0321] 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, ORF2t / 3, VP1, VP2, and / or VP3, 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.

[0322] In some embodiments, the packaging-deficient, packaging-defective, or packaging-disabled genetic element cannot be packaged into a proteinaceous exterior (e.g., where the proteinaceous exterior comprises a capsid or a portion thereof, including, e.g., a polypeptide encoded by an ORF1 or VP1 nucleic acid described herein). In some embodiments, the packaging-deficient genetic element is packaged into a proteinaceous exterior 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 Anelloviridae virus (e.g., Anellovirus or CAV) (e.g., as described herein). In some embodiments, the packaging defective genetic element cannot be packaged into a proteinaceous exosomal even in the presence of factors (e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, VP1, VP2, or VP3) that allow packaging of the genetic element of a wild-type Anelloviridae virus (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 Anelloviridae virus (e.g., an Anellovirus or CAV) (e.g., as described herein), even in the presence of factors (e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, VP1, VP2, or VP3) that enable packaging of the genetic element of a wild-type Anelloviridae virus (e.g., as described herein).

[0323] In some embodiments, the packagable genetic element may be packaged into a proteinaceous exterior (e.g., the proteinaceous exterior comprises a capsid or a portion thereof that includes a polypeptide encoded by an ORF1 or VP1 nucleic acid, e.g., as described herein). In some embodiments, the packagable genetic element is packaged into the 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 Anelloviridae virus (e.g., Anellovirus or CAV) (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, ORF2t / 3, VP1, VP2, or VP3) that allow packaging of the genetic elements of a wild-type Anelloviridae virus (e.g., anellovirus or CAV) (e.g., as described herein). In some embodiments, the packagable genetic elements are packaged into the proteinaceous exterior in the presence of factors (e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, VP1, VP2, or VP3) that enable packaging of the genetic elements of a wild-type Anelloviridae virus (e.g., a wild-type Anelloviridae virus (e.g., anellovirus or CAV) (e.g., as described herein) 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 Anelloviridae virus (e.g., anellovirus or CAV) (e.g., as described herein).

[0324] Anelloviridae viruses (Anellovirus and CAV) In some embodiments, Anelloviridae vectors (e.g., those described herein) comprise sequences or expression products from an Anellovirus. In some embodiments, Anelloviridae vectors comprise one or more sequences or expression products exogenous to an Anellovirus. In some embodiments, Anelloviridae vectors comprise one or more sequences or expression products endogenous to an Anellovirus. In some embodiments, Anelloviridae vectors comprise one or more sequences or expression products that are heterologous to one or more other sequences or expression products in the Anelloviridae vector. Anelloviridae viruses (e.g., anelloviruses or CAVs) 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).

[0325] 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).

[0326] 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.

[0327] In some embodiments, the Anelloviridae vectors described herein include one or more nucleic acid molecules (e.g., genetic elements described herein) that include a sequence having at least about 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 some embodiments, the Anelloviridae vectors include a nucleic acid sequence selected from a sequence as set forth in any of Tables N1-N4, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto. In embodiments, the Anelloviridae vector comprises a polypeptide comprising a sequence set forth in Tables A1-A3, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0328] In some embodiments, the Anelloviridae vectors described herein comprise a vector that contains a TATA box, cap site, initiation element, transcription start site, 5'UTR conserved domain, ORF sequence of an Anelloviridae virus (e.g., anellovirus or CAV) (e.g., an Anelloviridae virus (e.g., anellovirus or CAV) sequence annotated or encoded by a sequence listed in any of Tables N1-N4). 1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, VP1, VP2, VP3 (apoptin), 3 open reading frame region, poly(A) signal, GC-rich region, or any combination thereof. In some embodiments, the nucleic acid molecule includes a sequence encoding a capsid protein, e.g., an ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, or VP1 sequence of any of the Anellovirus described herein (e.g., an Anelloviridae virus (e.g., anellovirus or CAV) sequences annotated or encoded by a sequence listed in any of Tables N1-N4).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 Anelloviridae virus (e.g., anellovirus or CAV) ORF1 ORF2, VP1, VP2, or apoptin protein (e.g., an ORF1, ORF2, VP1, VP2, or apoptin amino acid sequence as set forth in Tables A1-A3, or an ORF1, ORF2, VP1, VP2, or apoptin amino acid sequence encoded by a nucleic acid sequence as set forth in any of Tables N1-N4). In embodiments, the nucleic acid molecule comprises a sequence encoding a capsid protein comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to an Anelloviridae virus (e.g., anellovirus or CAV) ORF1 or VP1 protein (e.g., an ORF1 or VP1 amino acid sequence set forth in Tables A1-A3, or an ORF1 or VP1 amino acid sequence encoded by a nucleic acid sequence set forth in any of Tables N1-N4).

[0329] Nucleic acid sequence In some embodiments, a 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 Anelloviridae virus (e.g., anellovirus or CAV) ORF1 or VP1 nucleotide sequence of any of Tables N1-N4. In some embodiments, a 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 Anelloviridae virus (e.g., anellovirus or CAV) ORF2 or VP2 nucleotide sequence of any of Tables N1-N4. In some embodiments, a 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 Anelloviridae virus (e.g., anellovirus or CAV) ORF3 or VP3 nucleotide sequence of any of Tables N1-N4. In some embodiments, a 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 Anelloviridae virus (e.g., anellovirus or CAV) GC-rich region nucleotide sequence of any of Tables N1-N4. In some embodiments, a 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 Anelloviridae virus (e.g., anellovirus or CAV) 5'UTR conserved domain nucleotide sequence of any of Tables N1-N4.

[0330] 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 Anelloviridae virus (e.g., anellovirus or CAV) ORF1 or VP1 amino acid sequence of Table A1 or A2. 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 Anelloviridae virus (e.g., anellovirus or CAV) ORF2 or VP2 amino acid sequence of Table A1 or A2. 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 Anelloviridae virus (e.g., anellovirus or CAV) ORF3 or VP3 amino acid sequence of Table A1 or A2.

[0331] Proteins containing amino acid sequences In embodiments, the Anelloviridae vectors described herein comprise a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anelloviridae virus (e.g., anellovirus or CAV) ORF1 or VP1 amino acid sequence of Tables A1-A3. In embodiments, the Anelloviridae vectors described herein comprise a protein having an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anelloviridae virus (e.g., anellovirus or CAV) ORF2 or VP2 amino acid sequence of Table A1 or A2. In embodiments, an Anelloviridae vector 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 Anelloviridae virus (e.g., anellovirus or CAV) ORF3 or VP3 amino acid sequence of Table A1 or A2. In some embodiments, an ORF1 or VP1 molecule (e.g., included in an Anelloviridae vector) comprises a polypeptide encoded by an Anelloviridae virus (e.g., anellovirus or CAV) ORF1 or VP1 nucleic acid sequence of any of Tables N1-N4. In some embodiments, the ORF1 or VP1 molecule (e.g., included in an Anelloviridae vector) comprises an Anelloviridae virus (e.g., anellovirus or CAV) ORF1 or VP1 protein of Tables A1-A3, or a splice variant or post-translationally processed (e.g., proteolytically processed) variant thereof.In some embodiments, an ORF2 or VP2 molecule (e.g., included in an Anelloviridae vector) comprises a polypeptide encoded by an Anelloviridae virus (e.g., anellovirus or CAV) ORF2 or VP2 nucleic acid sequence of any of Tables N1-N4. In some embodiments, an ORF2 or VP2 molecule (e.g., included in an Anelloviridae vector) comprises an Anelloviridae virus (e.g., anellovirus or CAV) ORF2 or VP2 protein of Tables A1-A3, or a splice variant or post-translationally processed (e.g., proteolytically processed) variant thereof. In some embodiments, an ORF3 or VP3 molecule (e.g., included in an Anelloviridae vector) comprises a polypeptide encoded by an Anelloviridae virus (e.g., anellovirus or CAV) ORF3 or VP3 nucleic acid sequence of any of Tables N1-N4. In some embodiments, an ORF3 or VP3 molecule (e.g., included in an Anelloviridae vector) comprises an Anelloviridae virus (e.g., anellovirus or CAV) ORF3 or VP3 protein of Tables A1-A3, or a splice variant or post-translationally processed (e.g., proteolytically processed) variant thereof.

[0332] Polypeptides containing amino acid sequences 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 Anelloviridae virus (e.g., anellovirus or CAV) ORF1 or VP1 amino acid sequence 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 Anelloviridae virus (e.g., anellovirus or CAV) ORF1 or VP1 amino acid sequence of Tables A1-A3.

[0333] 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 ORF1 or VP1 molecule encoded by an Anelloviridae virus (e.g., anellovirus or CAV) ORF1 or VP1 nucleic acid described herein. 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 ORF1 or VP1 molecule encoded by an Anelloviridae virus (e.g., anellovirus or CAV) ORF1 or VP1 nucleic acid listed in Tables N1-N4.

[0334] 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 Anelloviridae virus (e.g., anellovirus or CAV) ORF2 or VP2 amino acid sequence described herein. 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 Anelloviridae virus (e.g., anellovirus or CAV) ORF2 or VP2 amino acid sequence of Table A1 or A2.

[0335] 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 ORF2 or VP2 molecule encoded by an Anelloviridae virus (e.g., anellovirus or CAV) ORF2 or VP2 nucleic acid described herein. 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 ORF2 or VP2 molecule encoded by an Anelloviridae virus (e.g., anellovirus or CAV) ORF2 or VP2 nucleic acid listed in Tables N1-N4.

[0336] 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 Anelloviridae virus (e.g., anellovirus or CAV) ORF3 or VP3 amino acid sequence described herein. 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 Anelloviridae virus (e.g., anellovirus or CAV) ORF3 or VP3 amino acid sequence of Table A1 or A2.

[0337] 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 ORF3 or VP3 molecule encoded by an Anelloviridae virus (e.g., anellovirus or CAV) ORF3 or VP3 nucleic acid described herein. 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 ORF3 or VP3 molecule encoded by an Anelloviridae virus (e.g., anellovirus or CAV) ORF3 or VP3 nucleic acid listed in Tables N1-N4.

[0338] In some embodiments, the polypeptide comprises an amino acid sequence set forth in Tables A1-A3 (e.g., an ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, VP1, VP2, VP3 sequence), or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0339]

Table N1-1

[0340]

Table N1-2

[0341]

Table A1-1

[0342]

Table N1-3

[0343]

Table N1-4

[0344]

Table N1-5

[0345]

Table A1-2

[0346]

Table N2-1

[0347]

Table N2-2

[0348]

Table N2-3

[0349]

Table A2

[0350]

Table N3-1

[0351]

Table N3-2

[0352]

Table N3-3

[0353]

Table N4-1

[0354]

Table N4-2

[0355]

Table N4-3

[0356]

Table A3

[0357]

Table N5-1

[0358]

Table N5-2

[0359]

Table N5-3

[0360]

Table N5-4

[0361]

Table N5-5

[0362]

Table N6-1

[0363]

Table N6-2

[0364]

Table N6-3

[0365]

Table N7-1

[0366]

Table N7-2

[0367]

Table N7-3

[0368]

Table N7-4

[0369]

Table N8-1

[0370] [Table N8-2]

[0371] [Table N8-3]

[0372] [Table N8-4]

[0373] [Table N8-5]

[0374] [Table N8-6]

[0375] [Table N8-7]

[0376] [Table N8-8]

[0377] An exemplary SV40 large T antigen amino acid sequence (e.g., encoded by nucleotides 4790-6916 of Table N8): [ka]

[0378] In some embodiments, an Anelloviridae vector (e.g., anellovector) described herein is a chimeric Anelloviridae vector (e.g., a chimeric anellovector). In some embodiments, a chimeric Anelloviridae vector further comprises one or more elements, polypeptides, or nucleic acids derived from a virus other than an Anelloviridae virus.

[0379] In some embodiments, a chimeric Anelloviridae vector comprises multiple polypeptides (e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, VP1, VP2, and / or VP3) that comprise sequences from multiple different Anelloviridae viruses (e.g., as described herein).

[0380] In some embodiments, an Anelloviridae vector comprises a chimeric polypeptide (e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, VP1, VP2, and / or VP3), e.g., that includes at least one portion from an Anelloviridae virus (e.g., as described herein) and at least one portion from a different virus (e.g., as described herein).

[0381] In some embodiments, the Anelloviridae vector comprises a chimeric polypeptide (e.g., ORF1, ORF1 / 1, ORF1 / 2, ORF2, ORF2 / 2, ORF2 / 3, ORF2t / 3, VP1, VP2, and / or VP3), e.g., comprising at least one portion from an Anelloviridae virus (e.g., as described herein) and at least one portion from a different Anelloviridae virus (e.g., as described herein). In some embodiments, an Anelloviridae vector comprises a chimeric ORF1 or VP1 molecule comprising at least a portion of, or an ORF or VP1 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, from an Anelloviridae virus (e.g., as described herein) and at least a portion of, or an ORF or VP1 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity thereto, from a different Anelloviridae virus (e.g., as described herein). In some embodiments, a chimeric ORF1 or VP1 molecule comprises an ORF1 or VP1 jellyroll domain from one Anelloviridae virus, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and an ORF1 or VP1 amino acid subsequence (e.g., as described herein) from a different Anelloviridae virus, 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 or VP1 molecule comprises an ORF1 or VP1 arginine-rich region from one Anelloviridae virus, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and an ORF1 or VP1 amino acid subsequence (e.g., as described herein) from a different Anelloviridae virus, 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 or VP1 molecule comprises an ORF1 or VP1 hypervariable domain from one Anelloviridae virus, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and an ORF1 or VP1 amino acid subsequence (e.g., as described herein) from a different Anelloviridae virus, 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 Anelloviridae virus, 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 Anelloviridae virus, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.In some embodiments, the chimeric ORF1 or VP1 molecule is an ORF1 or VP1 C-terminal domain from one Anelloviridae virus, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and an ORF1 or VP1 amino acid subsequence (e.g., as described herein) from a different Anelloviridae virus, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0382] In some embodiments, an Anelloviridae vector comprises a chimeric ORF1 / 1 molecule comprising at least a portion of, or an ORF1 / 1 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, from one Anelloviridae virus (e.g., as described herein) and at least a portion of, or an ORF1 / 1 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, from a different Anelloviridae virus (e.g., as described herein). In some embodiments, an Anelloviridae vector comprises a chimeric ORF1 / 2 molecule comprising at least a portion of, or an ORF1 / 2 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, from one Anelloviridae virus (e.g., as described herein) and at least a portion of, or an ORF1 / 2 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, from a different Anelloviridae virus (e.g., as described herein).In some embodiments, an Anelloviridae vector comprises a chimeric ORF2 or VP2 molecule comprising at least a portion of an ORF2 or VP2 molecule from one Anelloviridae virus (e.g., as described herein), or an ORF2 or VP2 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 or VP2 molecule from a different Anelloviridae virus, or an ORF2 or VP2 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto. In some embodiments, an Anelloviridae vector comprises a chimeric ORF2 / 2 molecule comprising at least a portion of, or an ORF2 / 2 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, from one Anelloviridae virus (e.g., as described herein) and at least a portion of, or an ORF2 / 2 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, from a different Anelloviridae virus (e.g., as described herein).In some embodiments, an Anelloviridae vector comprises a chimeric ORF2 / 3 molecule comprising at least a portion of, or an ORF2 / 3 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, from one Anelloviridae virus (e.g., as described herein) and at least a portion of, or an ORF2 / 3 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, from a different Anelloviridae virus (e.g., as described herein). In some embodiments, an Anelloviridae vector comprises a chimeric ORF2T / 3 molecule comprising at least a portion of, or an ORF2T / 3 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, from one Anelloviridae virus (e.g., as described herein) and at least a portion of, or an ORF2T / 3 molecule having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity thereto, from a different Anelloviridae virus (e.g., as described herein).

[0383] In some embodiments, the Anelloviridae vector comprises a nucleic acid having a sequence as described in PCT Application No. PCT / US2018 / 037379, which is incorporated by reference in its entirety. In some embodiments, the Anelloviridae vector comprises a polypeptide having a sequence as described in PCT Application No. PCT / US2018 / 037379, which is incorporated by reference in its entirety.

[0384] In some embodiments, the Anelloviridae vector comprises an Anelloviridae viral genome, for example, as identified according to the methods described in Example 9. In some embodiments, the Anelloviridae vector comprises an Anelloviridae viral sequence, or a portion thereof, as described in Example 13.

[0385] 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 as shown in Table 19 represents any amino acid. In some embodiments, Z as shown in Table 19 represents glutamic acid or glutamine. In some embodiments, B as shown in Table 19 represents aspartic acid or asparagine. In some embodiments, J as shown in Table 19 represents leucine or isoleucine.

[0386] [Table 19-1]

[0387] [Table 19-2]

[0388] Capsid proteins (e.g., ORF1 and VP1 molecules) In some embodiments, the annelovector comprises an ORF1 molecule or a VP1 molecule, and / or a nucleic acid encoding an ORF1 molecule or a VP1 molecule.

[0389] Generally, an ORF1 molecule comprises a polypeptide having the structural features and / or activity of an anellovirus ORF1 protein (e.g., an anellovirus ORF1 protein as described herein, e.g., listed in Table A1 or A2), or a functional fragment thereof. In some embodiments, an ORF1 molecule comprises a truncation relative to an anellovirus ORF1 protein (e.g., an anellovirus ORF1 protein as described herein, e.g., listed in Table A1 or A2). In some embodiments, an 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 of an 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 an anellovirus ORF1 protein sequence as shown in Table A1 or A2. 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 to a Betatorquevirus ORF1 protein, for example, as described herein. The ORF1 molecule may generally bind 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 a cell. In certain embodiments, the ORF1 molecule is localized to the nucleolus of a cell. In some embodiments, the ORF1 molecule is encoded by an ORF1 nucleic acid. In some embodiments, the ORF1 nucleic acid comprises an antisense strand, which can be directly transcribed to produce an mRNA encoding the ORF1 molecule. In some embodiments, the ORF1 nucleic acid comprises a sense strand.

[0390] Generally, a VP1 molecule comprises a polypeptide having structural features and / or activity of a CAV VP1 protein (e.g., a CAV VP1 protein as described herein, e.g., listed in Table A3), or a functional fragment thereof. In some embodiments, the VP1 molecule comprises a truncation relative to a CAV VP1 protein (e.g., a CAV VP1 protein as described herein, e.g., listed in Table A3). In some embodiments, the VP1 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 of a CAV VP1 protein. In some embodiments, the VP1 molecule comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a CAV VP1 protein sequence as shown in Table A3. The VP1 molecule is generally capable of binding to a nucleic acid molecule such as DNA (e.g., a genetic element described herein). In some embodiments, the VP1 molecule is localized to the nucleus of the cell. In certain embodiments, the VP1 molecule is localized to the nucleolus of the cell. In some embodiments, the VP1 molecule is encoded by a VP1 nucleic acid. In some embodiments, the VP1 nucleic acid comprises an antisense strand, which can be directly transcribed to produce an mRNA encoding the VP1 molecule. In some embodiments, the VP1 nucleic acid comprises a sense strand.

[0391] In some embodiments, the ORF1 molecule described herein comprises an amino acid sequence (e.g., an ORF1 sequence, or an arginine-rich region, jelly-roll domain, HVR, N22, or C-terminal domain sequence) as listed in any of Tables A2, A4, A6, A8, A10, A12, C1-C5, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20-37, or D1-D10 of WO2020 / 123816, which is 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.

[0392] Without wishing to be bound by theory, an ORF1 molecule or VP1 molecule may be capable of combining with other ORF1 molecules or VP1 molecules to form, for example, a proteinaceous exoskeleton (e.g., as described herein). Such an ORF1 molecule or VP1 molecule may be described as capable of forming a capsid. In some embodiments, a proteinaceous exoskeleton may encapsidate a nucleic acid molecule (e.g., a genetic element described herein). In some embodiments, multiple ORF1 molecules or VP1 molecules may form multimers, for example, to produce a proteinaceous exoskeleton. 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 or VP1 molecules). It is also contemplated that an ORF1 molecule or VP1 molecule may have replicase activity.

[0393] An ORF1 molecule or a VP1 molecule, in some embodiments, can 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). In some embodiments, a VP1 molecule may include one or more of the following: 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 jelly-roll domain.

[0394] Arginine-rich region An arginine-rich region (e.g., including an ORF1 molecule or a VP1 molecule described herein) 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 to 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).

[0395] Jellyroll Domain A jelly-roll domain or region (e.g., contained in an ORF1 molecule or a VP1 molecule described herein) has 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:

[0396] In certain embodiments, the jellyroll domain comprises two beta sheets.

[0397] 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.

[0398] 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 embodiments, the first and second β-sheets comprise, in total, about eight (e.g., 6, 7, 8, 9, 10, 11, or 12) β-strands.

[0399] 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.

[0400] The ORF1 molecule may also include a third region that includes the structure or activity of an Anellovirus N22 domain (e.g., an N22 domain described herein, e.g., 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., a CTD described herein, e.g., from an Anellovirus ORF1 protein described herein). In some embodiments, the ORF1 molecule includes, in order from N-terminus to C-terminus, the first region, the second region, the third region, and the fourth region.

[0401] 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).

[0402] 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.

[0403] 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., a capsid) or portion thereof.

[0404] In some embodiments, the fourth region is exposed on the surface of a proteinaceous exterior (eg, a proteinaceous exterior that comprises a multimer of ORF1 molecules, as described herein).

[0405] In some embodiments, the first region, the second region, the third region, the fourth region, and / or the HVR each comprise three or fewer (e.g., 0, 1, 2, or 3) beta sheets.

[0406] 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.

[0407] 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, or more amino acids) (e.g., as shown in Figure 34). 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 each have a length of 1-1000 (e.g., 5-10, 5-15, 5-20, 10-15, 10-20, 15-20, 5-50, 5-100, 10-50, 10-100, 10-1000, 50-100, 50-1000, or 100-1000) amino acids. In certain embodiments, the conserved motifs comprise about 2-4% (e.g., about 1-8%, 1-6%, 1-5%, 1-4%, 2-8%, 2-6%, 2-5%, or 2-4%) 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.

[0408] In some embodiments, the ORF1 molecule includes at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration), e.g., compared to a wild-type ORF1 protein described herein (e.g., as shown in Table A1 or A2).

[0409] 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).

[0410] In some embodiments, a polypeptide (e.g., an ORF1 molecule) can have, e.g., a YNPX, YN, or YN2 sequence, 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 (SEQ ID NO: 829) motif contains a tyrosine (Y) residue at the N-terminus. 2 DXGX 2The 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 comprises the motif YNPX 2 DXGX 2 The N (SEQ ID NO: 829) motif contains an asparagine (N) residue at its C-terminus.

[0411] Exemplary YNPX 2 DXGX 2 N (SEQ ID NO: 829) motif flanking secondary structures are described in Example 47 and in Figure 48. 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., beta strands) shown ... 2 DXGX 2 The present invention relates to a method for producing a nucleotide sequence comprising the steps of: (a) providing a nucleotide sequence for a nucleotide sequence comprising: a ...

[0412] 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.

[0413] 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.

[0414] 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.

[0415] Exemplary jellyroll domain secondary structures are described in Example 47 and herein in Figure 25. 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 jellyroll domain secondary structures shown in Figure 47 herein.

[0416] Exemplary ORF1 and VP1 Sequences In some embodiments, a polypeptide described herein (e.g., an ORF1 or VP1 molecule) 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 or CAV VP1 subsequences, e.g., as described herein. In some embodiments, an Anelloviridae vector described herein (e.g., an Anellovector) comprises an ORF1 or VP1 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 or CAV VP1 subsequences, e.g., as described herein. In some embodiments, the anellovectors described herein include a nucleic acid molecule (e.g., a genetic element) encoding an ORF1 or VP1 molecule that includes 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 or CAV VP1 subsequences described herein, for example.

[0417] In some embodiments, one or more Anellovirus ORF1 or CAV VP1 subsequences comprise 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 enumerated 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 comprises multiple subsequences from different Anelloviruses. In some embodiments, an ORF1 or VP1 molecule comprises one or more of an Arg-rich domain, a jelly-roll domain, an N22 domain, and a CTD from one Anelloviridae virus (e.g., Anellovirus) and an HVR from another Anelloviridae virus. In some embodiments, an ORF1 or VP1 molecule comprises one or more of a jelly-roll domain, an HVR, an N22 domain, and a CTD from one Anelloviridae virus (e.g., Anellovirus) and an Arg-rich domain from another Anelloviridae virus. In some embodiments, an ORF1 or VP1 molecule comprises one or more of an Arg-rich domain, an HVR, an N22 domain, and a CTD from one Anelloviridae virus (e.g., Anellovirus) and a jelly-roll domain from another Anelloviridae virus. In some embodiments, an ORF1 or VP1 molecule comprises one or more of an Arg-rich domain, an jelly-roll domain, an HVR, and a CTD from one Anelloviridae virus (e.g., Anellovirus) and an N22 domain from another Anelloviridae virus.In some embodiments, the ORF1 or VP1 molecule comprises one or more of the Arg-rich domain, the jellyroll domain, the HVR, and the N22 domain from one Anelloviridae virus (e.g., an Anellovirus) and the CTD from another Anelloviridae virus.

[0418] Exemplary anellovirus ORF1 amino acid sequences, and sequences of exemplary ORF1 domains, are provided in the Tables below. In some embodiments, a polypeptide (e.g., 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 subsequences, e.g., as set forth in any of Tables P-Q. In some embodiments, an annelovector 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 Anellovirus ORF1 subsequences, e.g., as set forth in any of Tables P-Q. In some embodiments, the anaerovectors 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 Anellovirus ORF1 subsequences, e.g., as set forth in any of Tables P-Q.

[0419] In some embodiments, one or more Anellovirus ORF1 subsequences comprise 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 in any of Tables P-Q), 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 Anelloviruses (e.g., any combination of ORF1 subsequences selected from the subsequences of Alphatorquevirus clades 1-7 listed in Tables P-Q). In some embodiments, 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 Anellovirus. In some embodiments, 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 Anellovirus. In some embodiments, 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 Anellovirus. In some embodiments, 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 Anellovirus. In some embodiments, the ORF1 molecule comprises one or more of the Arg-rich domain, the jellyroll domain, the HVR, and the N22 domain from one Anellovirus and the CTD from another Anellovirus.

[0420] In some embodiments, the one or more anellovirus ORF1 partial sequences 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), as described in WO 2020 / 123816, which is incorporated by reference in its entirety. In some embodiments, the one or more CAV VP1 partial sequences comprise one or more of an arginine (Arg)-rich domain or a jelly-roll domain, as described in PCT Application No. PCT / US2021 / 057292, which is incorporated by reference in its entirety.

[0421] [Table P]

[0422] [Table Q]

[0423] 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-bAmino 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.

[0424] [Table 37A]

[0425] [Table 37B]

[0426] [Table 37C]

[0427] In some embodiments, the jellyroll domain comprises a jellyroll domain amino acid sequence 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. In some embodiments, the N22 domain comprises a N22 domain amino acid sequence 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. In some embodiments, the CTD domain comprises a CTD domain amino acid sequence 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.

[0428] Identification of ORF1 or VP1 protein sequences In some embodiments, the ORF1 or VP1 protein sequence, or a nucleic acid sequence encoding the ORF1 or VP1 protein, can be identified from the genome of an Anelloviridae virus, e.g., an Anellovirus (e.g., a putative Anelloviridae virus genome identified by a nucleic acid sequencing technique, e.g., a deep sequencing technique). In some embodiments, the ORF1 or VP1 protein sequence is identified by one or more (e.g., one, two, or all three) of the following selection criteria:

[0429] (i) Length Selection: Protein sequences (e.g., putative ORF1 or VP1 sequences that pass the criteria set forth in (ii) or (iii) below) may be size-selected for greater than about 600 amino acid residues to identify putative ORF1 or VP1 proteins. In some embodiments, ORF1 or VP1 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 ORF1 or VP1 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.

[0430] (ii) Presence of an ORF1 motif: Protein sequences (e.g., putative ORF1 or VP1 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].

[0431] (iii) Presence of Arginine-Rich Regions: Protein sequences (e.g., putative ORF1 or VP1 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 ORF1 or VP1 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 ORF1 or VP1 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 ORF1 or VP1 protein. In some embodiments, the arginine-rich region is located at least about 50 amino acids downstream of the start codon of the putative ORF1 or VP1 protein.

[0432] In some embodiments, the ORF1 protein is identified in an anellovirus genomic sequence as described in Example 36 of WO 2020 / 123816, which is incorporated by reference in its entirety.

[0433] ORF2 or VP2 molecule In some embodiments, the annellovector comprises an ORF2 or VP2 molecule, and / or a nucleic acid encoding the ORF2 or VP2 molecule. Generally, the ORF2 or VP2 molecule comprises a polypeptide having structural features and / or activity of an anellovirus ORF2 protein (e.g., an anellovirus ORF2 protein as described herein, e.g., listed in Table A1 or A2) or a CAV VP2 protein (e.g., a CAV VP2 protein as described herein, e.g., listed in Table A3), or a functional fragment thereof. In some embodiments, the ORF2 or VP2 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 or CAV protein sequence as shown in Tables A1-A3. In some embodiments, the ORF2 molecule is encoded by an ORF2 nucleic acid. In some embodiments, the ORF2 nucleic acid comprises an antisense strand, which can be directly transcribed to produce an mRNA encoding the ORF2 molecule. In some embodiments, the ORF2 nucleic acid comprises a sense strand.

[0434] 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 or VP2 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 or VP2 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 or VP2 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 or VP2 molecule comprises the amino acid sequence of the ORF2 protein of TTV isolate TA278 or TTV isolate SANBAN. In some embodiments, the ORF2 or VP2 molecule has protein phosphatase activity.In some embodiments, the ORF2 or VP2 molecule includes at least one difference (e.g., a mutation, chemical modification, or epigenetic alteration) compared to a wild-type ORF2 or CAV protein described herein (e.g., as shown in Tables A1-A3).

[0435] 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 (e.g., a nucleic acid sequence encoding an ORF2 molecule as described herein).

[0436] Genetic elements In some embodiments, an Anelloviridae vector (e.g., anellovector) comprises a genetic element. In some embodiments, the genetic element has one or more of the following characteristics: does not substantially interact with the genome of a host cell, is an episomal nucleic acid, is single stranded DNA, is circular, is about 1-10 kb, is present in the nucleus of a cell, can be bound by an endogenous protein, produces an effector such as a polypeptide or nucleic acid (e.g., RNA, iRNA, microRNA) that targets a gene, activity, or function of a 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 of the packaging or capsid binding sequences, the genetic elements have less than 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% sequence identity to wild-type Anellovirus or CAV nucleic acid sequences, e.g., less than 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% sequence identity to the Anellovirus or CAV nucleic acid sequences described herein. In some embodiments, outside of the packaging or capsid binding sequences, the genetic element has 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 or CAV nucleic acid sequence. In certain embodiments, the genetic element is a circular single-stranded DNA comprising a promoter sequence, a sequence encoding a therapeutic effector, and a capsid binding protein.

[0437] 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, for example, an Anellovirus or CAV nucleic acid sequence described herein (e.g., as described in any of Tables N1-N4), 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 or CAV amino acid sequence (e.g., as described in any of Tables A1-A3), 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).

[0438] In some embodiments the genetic elements have a length of less than 20kb (e.g. less than about 19kb, 18kb, 17kb, 16kb, 15kb, 14kb, 13kb, 12kb, 11kb, 10kb, 9kb, 8kb, 7kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1kb). In some embodiments the genetic elements independently or in addition have a length of more than 1000b (e.g. at least about 1.1kb, 1.2kb, 1.3kb, 1.4kb, 1.5kb, 1.6kb, 1.7kb, 1.8kb, 1.9kb, 2kb, 2.1kb, 2.2kb, 2.3kb, 2.4kb, 2.5kb, 2.6kb, 2.7kb). kb, 2.8 kb, 2.9 kb, 3 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, 3.9 kb, 4 kb, 4.1 kb, 4.2 kb, 4.3 kb, 4.4 kb, 4.5 kb, 4.6 kb, 4.7 kb, 4.8 kb, 4.9 kb, 5 kb or more) in length). 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.

[0439] 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 a regulatory nucleic acid, one or more regulatory sequences, one or more sequences encoding a replication protein, 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 an amino acid sequence described herein, e.g., any one of the Anellovirus or CAV amino acid sequences as listed in any of Tables A1-A3.

[0440] 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.

[0441] In some embodiments, the double stranded circular DNA and / or genetic elements do not comprise 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 comprise a bacterial plasmid backbone.

[0442] 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 or CAV sequence, e.g., as described herein).

[0443] In some embodiments, the genetic elements described herein include a sequence (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or multiple GC-rich region sequences) listed in any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17 of WO 2020 / 123816, which is 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.

[0444] 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 or CAV 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, or more nucleotides) from an anellovirus or CAV genomic sequence. In some embodiments, the effector coding sequence may include any of the following: a TATA box, cap site, transcription start site, 5' UTR, open reading frame (ORF), poly(A) signal, or a nucleotide sequence as listed in, for example, any of Tables A1, A3, A5, A7, A9, A11, B1-B5, 1, 3, 5, 7, 9, 11, 13, 15, or 17 of WO 2020 / 123816, which is incorporated by reference in its entirety. or a GC-rich region sequence, or a portion thereof (e.g., a portion comprising at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides), or a sequence having at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% nucleotide sequence identity thereto.

[0445] In some embodiments, the sequence of a first nucleic acid element comprised in a genetic element (e.g., the TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region) overlaps with the sequence of a second nucleic acid element (e.g., the 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 comprised in a genetic element (e.g., a TATA box, cap site, transcription start site, 5'UTR, open reading frame (ORF), poly(A) signal, or GC-rich region) 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).

[0446] 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.

[0447] 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. 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 or CAV sequence (e.g. as shown in any of Tables N1-N4).

[0448] 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 or CAV 5'UTR conserved domain nucleotide sequence of any of Tables N1-N4.

[0449] 5'UTR region 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 nucleic acid sequence shown, for example, in Table 38 and / or Figure 20. In some embodiments, the genetic element (e.g., the protein binding sequence of the genetic element) comprises a nucleic acid sequence of a consensus 5'UTR sequence shown in Table 38, where X 1 , X 2 , X 3 , X 4 , and X 5 are each independently any nucleotide, for example, X 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 sequences shown in Table 38.

[0450] 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 consensus 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 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., 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 Alphatorquevirus Clade 4 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 Alphatorquevirus Clade 5 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 Alphatorquevirus Clade 6 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 Alphatorquevirus Clade 7 5'UTR sequence shown in Table 38.

[0451] 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 or CAV 5'UTR conserved domain nucleotide sequence of any of Tables N1-N4.

[0452] [Table 38-1]

[0453] [Table 38-2]

[0454] Identification of 5'UTR sequences In some embodiments, Anelloviridae (e.g., Anellovirus or CAV) 5'UTR sequences may be identified within an Anelloviridae (e.g., Anellovirus or CAV) genome (e.g., a putative Anelloviridae genome identified by a nucleic acid sequencing technique, e.g., a deep sequencing technique). In some embodiments, Anelloviridae (e.g., Anellovirus or CAV) 5'UTR sequences are identified by one or both of the following steps:

[0455] (i) Identification of circularization junction: In some embodiments, the 5'UTR will be located near the circularization junction of the full-length circularized Anelloviridae (e.g., Anellovirus or CAV) 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 Anelloviridae (e.g., Anellovirus or CAV) 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 sequence to be non-biologically oriented. Landmarks within the sequence can be used to re-orient the sequence in the appropriate direction. For example, the landmark sequence may include a sequence having substantial homology to one or more elements within an Anelloviridae (e.g., Anellovirus or CAV) genome described herein (e.g., one or more of the 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 of an Anelloviridae (e.g., Anellovirus or CAV) described herein).

[0456] (ii) Identification of 5' UTR Sequences: Once a putative Anelloviridae (e.g., Anellovirus or CAV) 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 Anelloviridae (e.g., Anellovirus or CAV) 5' UTR sequences (e.g., as described herein) to identify sequences having substantial homology thereto. In some embodiments, the predicted Anelloviridae (e.g., Anellovirus or CAV) 5'UTR region has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an Anelloviridae (e.g., Anellovirus or CAV) 5'UTR sequence described herein.

[0457] GC-rich regions 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 nucleic acid sequence shown in Table 39 and / or Figures 20 and 32. 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 GC rich sequence shown in Table 39.

[0458] 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).

[0459] 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, e.g., selected from TTV-CT30F, TTV-P13-1, TTV-tth8, TTV-HD20a, TTV-16, TTV-TJN02, or TTV-HD16d, e.g., as listed in Table 39. In some embodiments, the genetic element (e.g. a protein binding sequence of the genetic element) comprises a nucleic acid sequence selecte...

Claims

1. 1. A method of delivering an exogenous effector to the posterior eye cup (PEC) of a subject, the method comprising: (i) a genetic element comprising a nucleic acid encoding an exogenous effector; (ii) a proteinaceous exterior that encapsulates the genetic element; and administering to the PEC of the subject an Anelloviridae vector comprising:

2. 1. A method of delivering an exogenous effector to a retinal pigment epithelium (RPE) of a subject, the method comprising: (i) a genetic element comprising a nucleic acid encoding an exogenous effector; (ii) a proteinaceous exterior that encapsulates the genetic element; and administering to the RPE of the subject an Anelloviridae vector comprising:

3. 3. The method of claim 1 or 2, wherein the genetic element comprises a nucleic acid sequence of nucleotides 1 to 71 of SEQ ID NO:1, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

4. The genetic element comprises: (i) a nucleic acid sequence of nucleotides 1 to 100 of SEQ ID NO:1, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and / or (ii) a nucleic acid sequence from nucleotides 2463 to 2876 of SEQ ID NO:1, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. The method according to any one of claims 1 to 3, comprising:

5. The method of any one of claims 1 to 4, wherein the proteinaceous exterior comprises an ORF1 molecule having an amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

6. 3. The method of claim 1 or 2, wherein the genetic element comprises a nucleic acid sequence of nucleotides 323 to 393 of SEQ ID NO:54, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

7. The genetic element comprises: (i) a nucleic acid sequence of nucleotides 1 to 423 of SEQ ID NO:54, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and / or (ii) a nucleic acid sequence from nucleotide 2813 to 2979 of SEQ ID NO:54, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

7. The method of claim 1, 2, or 6, comprising:

8. The method of any one of claims 1, 2, 6, or 7, wherein the proteinaceous exterior comprises an ORF1 molecule having an amino acid sequence of SEQ ID NO:58, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

9. 3. The method of claim 1 or 2, wherein the genetic element comprises a nucleic acid sequence of nucleotides 1 to 374 of SEQ ID NO:5, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

10. The genetic element comprises: (i) a nucleic acid sequence of nucleotides 1 to 374 of SEQ ID NO:5, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and / or (ii) a nucleic acid sequence from nucleotides 2197 to 2313 of SEQ ID NO:5, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

10. The method of claim 1, 2, or 9, comprising:

11. The method of any one of claims 1, 2, 9, or 10, wherein the proteinaceous exterior comprises VP1 having an amino acid sequence of SEQ ID NO: 251, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

12. 12. The method of any one of claims 1 to 11, wherein the Anelloviridae vector is substantially free of a wild-type Anellovirus genome.

13. 13. The method of any one of claims 1 to 12, wherein the anellovirus vector genetic element, or DNA comprising the nucleic acid sequence thereof, is detectable at least 21 or 49 days after administration.

14. The method of any one of claims 1 to 13, wherein the subject has a monogenic or polygenic disease.

15. The method of any one of claims 1 to 14, wherein the subject has macular degeneration (e.g., age-related macular degeneration (AMD), e.g., wet AMD or dry AMD).

16. The method of any one of claims 1 to 15, wherein the subject has a retinal disease or a VEGF-associated disorder, for example as described herein.

17. A method of delivering an effector to a subject, the method comprising subretinal administration of an Anelloviridae vector (e.g., as described herein) to the subject.

18. A method of delivering an effector to a subject, the method comprising intravitreally administering to the subject an Anelloviridae vector (e.g., as described herein).

19. 19. The method of claim 17 or 18, which results in the transduction of retinal cells and / or PEC cells.

20. 19. The method of claim 17 or 18, which results in the transduction of RPE and / or PEC cells.

21. 1. A method of treating a disease or disorder selected from a monogenic disease, a polygenic disease, a macular degeneration (e.g., AMD, e.g., wet AMD or dry AMD), a retinal disease, or a VEGF-related disorder (e.g., as described herein), the method comprising subretinal administration of an Anelloviridae vector (e.g., as described herein) to a subject.

22. 22. The method of any one of claims 1 to 21, wherein the Anelloviridae vector is administered in an amount effective to result in a concentration of 0.330 μg / mL of exogenous effector in the vitreous humor of the subject, e.g., maintained for at least 3 months after administration.

23. 23. The method of claim 22, wherein the concentration of exogenous effector in the vitreous humor of the subject after three months is between 1.70 and 6.60 μg / mL.

24. 24. The method of any one of claims 1 to 23, wherein the Anelloviridae vector is administered in an amount effective to result in a concentration of exogenous effector of 0.110 μg / mL in the aqueous humor of the subject, e.g., maintained for at least 3 months after administration.

25. 25. The method of claim 24, wherein the concentration of exogenous effector in the vitreous humor of the subject after three months is between 0.567 and 2.20 μg / mL.

26. 26. The method of any one of claims 1 to 25, wherein the Anelloviridae vector is administered in a volume of 0.1 mL to 0.5 mL.

27. (i) a genetic element comprising a nucleic acid encoding an exogenous effector; (ii) a proteinaceous exterior that encapsulates the genetic element; at a concentration of at least 2.48E+08 copies of said genetic element per mL, (a) the genetic element comprises a nucleic acid sequence of nucleotides 1 to 71 of SEQ ID NO:1, or a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and / or (b) the preparation, wherein the proteinaceous exterior comprises an ORF1 molecule having an amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

28. 28. The preparation of claim 27, which is substantially free of wild-type Anellovirus.

29. An ocular delivery device comprising an Anelloviridae vector (eg, an anellovector, for example, as described herein).

30. 30. The ocular delivery device of claim 29 configured for suprachoroidal injection.

31. 30. The ocular delivery device of claim 29 configured for subretinal administration.

32. 32. The ocular delivery device of claim 31, comprising a catheter and a needle passing through the catheter (e.g., into the subretinal space of the subject).

33. 30. The ocular delivery device of claim 29 configured for intravitreal administration.

34. 34. An ocular delivery device according to any one of claims 29 to 33, comprising a microinjector (eg including a microneedle), a cannula (eg a fine cannula), and / or a syringe.

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